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Sample records for proteins primary radicals

  1. Oxidative stress, free radicals and protein peroxides.

    PubMed

    Gebicki, Janusz M

    2016-04-01

    Primary free radicals generated under oxidative stress in cells and tissues produce a cascade of reactive secondary radicals, which attack biomolecules with efficiency determined by the reaction rate constants and target concentration. Proteins are prominent targets because they constitute the bulk of the organic content of cells and tissues and react readily with many of the secondary radicals. The reactions commonly lead to the formation of carbon-centered radicals, which generally convert in vivo to peroxyl radicals and finally to semistable hydroperoxides. All of these intermediates can initiate biological damage. This article outlines the advantages of the application of ionizing radiations to studies of radicals, with particular reference to the generation of desired radicals, studies of the kinetics of their reactions and correlating the results with events in biological systems. In one such application, formation of protein hydroperoxides in irradiated cells was inhibited by the intracellular ascorbate and glutathione. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Primary radical yields in pulse irradiated alkaline aqueous solution

    NASA Technical Reports Server (NTRS)

    Fielden, E. M.; Hart, E. J.

    1969-01-01

    Primary radical yields of hydrated electrons, H atoms, and OH radicals are determined by measuring hydrated electron formation following a 4 microsecond pulse of X rays. The pH dependence of free radical yields beyond pH 12 is determined by observation of the hydrated electrons.

  3. Biochemistry and pathology of radical-mediated protein oxidation.

    PubMed Central

    Dean, R T; Fu, S; Stocker, R; Davies, M J

    1997-01-01

    Radical-mediated damage to proteins may be initiated by electron leakage, metal-ion-dependent reactions and autoxidation of lipids and sugars. The consequent protein oxidation is O2-dependent, and involves several propagating radicals, notably alkoxyl radicals. Its products include several categories of reactive species, and a range of stable products whose chemistry is currently being elucidated. Among the reactive products, protein hydroperoxides can generate further radical fluxes on reaction with transition-metal ions; protein-bound reductants (notably dopa) can reduce transition-metal ions and thereby facilitate their reaction with hydroperoxides; and aldehydes may participate in Schiff-base formation and other reactions. Cells can detoxify some of the reactive species, e.g. by reducing protein hydroperoxides to unreactive hydroxides. Oxidized proteins are often functionally inactive and their unfolding is associated with enhanced susceptibility to proteinases. Thus cells can generally remove oxidized proteins by proteolysis. However, certain oxidized proteins are poorly handled by cells, and together with possible alterations in the rate of production of oxidized proteins, this may contribute to the observed accumulation and damaging actions of oxidized proteins during aging and in pathologies such as diabetes, atherosclerosis and neurodegenerative diseases. Protein oxidation may also sometimes play controlling roles in cellular remodelling and cell growth. Proteins are also key targets in defensive cytolysis and in inflammatory self-damage. The possibility of selective protection against protein oxidation (antioxidation) is raised. PMID:9164834

  4. Hydroxyl Radical Dosimetry for High Flux Hydroxyl Radical Protein Footprinting Applications Using a Simple Optical Detection Method

    PubMed Central

    Xie, Boer; Sharp, Joshua S.

    2015-01-01

    Hydroxyl radical protein footprinting (HRPF) by Fast Photochemical Oxidation of Proteins (FPOP) is a powerful benchtop tool used to probe protein structure, interactions, and conformational changes in solution. However, the reproducibility of all HRPF techniques is limited by the ability to deliver a defined concentration of hydroxyl radicals to the protein. This ability is impacted by both the amount of radical generated and the presence of radical scavengers in solution. In order to compare HRPF data from sample to sample, a hydroxyl radical dosimeter is needed that can measure the effective concentration of radical that is delivered to the protein, after accounting for both differences in hydroxyl radical generation and non-analyte radical consumption. Here, we test three radical dosimeters (Alexa Fluor 488, terepthalic acid, and adenine) for their ability to quantitatively measure the effective radical dose under the high radical concentration conditions of FPOP. Adenine has a quantitative relationship between UV spectrophotometric response, effective hydroxyl radical dose delivered, and peptide and protein oxidation levels over the range of radical concentrations typically encountered in FPOP. The simplicity of an adenine-based dosimeter allows for convenient and flexible incorporation into FPOP applications, and the ability to accurately measure the delivered radical dose will enable reproducible and reliable FPOP across a variety of platforms and applications. PMID:26455423

  5. Hydroxyl Radical Dosimetry for High Flux Hydroxyl Radical Protein Footprinting Applications Using a Simple Optical Detection Method.

    PubMed

    Xie, Boer; Sharp, Joshua S

    2015-11-03

    Hydroxyl radical protein footprinting (HRPF) by fast photochemical oxidation of proteins (FPOP) is a powerful benchtop tool used to probe protein structure, interactions, and conformational changes in solution. However, the reproducibility of all HRPF techniques is limited by the ability to deliver a defined concentration of hydroxyl radicals to the protein. This ability is impacted by both the amount of radical generated and the presence of radical scavengers in solution. In order to compare HRPF data from sample to sample, a hydroxyl radical dosimeter is needed that can measure the effective concentration of radical that is delivered to the protein, after accounting for both differences in hydroxyl radical generation and nonanalyte radical consumption. Here, we test three radical dosimeters (Alexa Fluor 488, terepthalic acid, and adenine) for their ability to quantitatively measure the effective radical dose under the high radical concentration conditions of FPOP. Adenine has a quantitative relationship between UV spectrophotometric response, effective hydroxyl radical dose delivered, and peptide and protein oxidation levels over the range of radical concentrations typically encountered in FPOP. The simplicity of an adenine-based dosimeter allows for convenient and flexible incorporation into FPOP applications, and the ability to accurately measure the delivered radical dose will enable reproducible and reliable FPOP across a variety of platforms and applications.

  6. Cu, Zn-Superoxide Dismutase-driven Free Radical Modifications: Copper- and Carbonate Radical Anion-initiated Protein Radical Chemistry

    PubMed Central

    Ramirez, Dario C.; Gomez-Mejiba, Sandra E.; Corbett, Jean T.; Deterding, Leesa J.; Tomer, Kenneth B.; Mason, Ronald P.

    2009-01-01

    SYNOPSIS The understanding of the mechanism, oxidant(s) involved, and how/what protein radicals are produced during the reaction of wild type Cu, Zn-superoxide dismutase (SOD1) with H2O2 and their fate is incomplete, but a better understanding of the role of this reaction is needed. We used immuno-spin trapping and mass spectrometry analysis to study the protein oxidations driven by human (h) and bovine (b) SOD1 when reacting with H2O2 using human serum albumin (HSA) and mouse brain homogenate (mBH) as target models. In order to gain mechanistic information about this reaction, we considered both copper- and carbonate radical anion-initiated protein oxidation. We chose experimental conditions that clearly separated SOD1-driven oxidation via CO3 •− from that initiated by copper released from the SOD1 active site. In the absence of (bi)carbonate, site-specific radical-mediated fragmentation is produced by SOD1 active-site copper. In the presence of (bi)carbonate and DTPA (to suppress copper chemistry), CO3 •− radical produced distinct radical sites in both SOD1 and HSA, which caused protein aggregation without causing protein fragmentation. The CO3 •− produced by reaction of hSOD1 with H2O2 also produced distinctive DMPO nitrone adduct positive protein bands in the mBH. Finally, we propose a biochemical mechanism to explain CO3 •− production from carbon dioxide, enhanced protein radical formation, and protection by (bi)carbonate against H2O2-induced fragmentation of the SOD1 active site. Our study is important for establishing experimental conditions for studying the molecular mechanism and targets of oxidation during the reverse reaction of SOD1 with H2O2; these results are the first step in analyzing the critical targets of SOD1-driven oxidation during such pathological processes as neuroinflammation. PMID:18764780

  7. Mapping RNA-protein interactions using hydroxyl-radical footprinting.

    PubMed

    Nilsen, Timothy W

    2014-12-01

    The binding of a protein to an RNA sequence protects the region of the RNA from cleavage by chemicals or RNases; this protected region is known as the protein's "footprint." In the footprinting protocol presented here, end-labeled RNAs with and without bound protein are cleaved using chemical methods. Fe(II)-EDTA is used to generate hydroxyl radicals in the presence of a reducing agent. These hydroxyl radicals indiscriminately cleave ribose groups in regions of the ribose-phosphate backbone that are exposed to solvent. After termination of cleavage, the resulting RNA fragments are analyzed by gel electrophoresis on denaturing polyacrylamide gels. Because hydroxyl radicals are smaller and cleave less specifically than RNases, this approach, if feasible, is often the method of choice for monitoring sites of RNA-protein interactions. © 2014 Cold Spring Harbor Laboratory Press.

  8. Sulfite-radical anions in isolated soy proteins.

    PubMed

    Lei, Q; Boatright, W L

    2007-06-01

    Aqueous mixtures of manganese and sulfite, at levels found in isolated soy proteins (ISP) and defatted soy flakes, spontaneously react in the presence of oxygen to produce methanethiol from the 1-electron oxidation of methionine. The carbon and sulfur of methanethiol originate from the methyl-carbon and sulfur of methionine. Similar aqueous mixtures of sulfite, manganese, and oxygen also produce sufficient levels of free radicals to degrade fluorescein. The degradation of methionine by free radicals generated in the sulfite, manganese, and oxygen reaction mixture is inhibited by the free radical spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide. Processing ISP with either L-cystine or potassium iodate reduces the free sulfite content of ISP and reduces the headspace methanethiol from aqueous ISP slurries to nondetectable levels. ISP processed without additives contained sufficient levels of free radicals to generate methanethiol from the oxidation of added methionine. There were no detectable levels of methanethiol produced when methionine was added to ISP processed with iodate.

  9. Molecular architectures and functions of radical enzymes and their (re)activating proteins.

    PubMed

    Shibata, Naoki; Toraya, Tetsuo

    2015-10-01

    Certain proteins utilize the high reactivity of radicals for catalysing chemically challenging reactions. These proteins contain or form a radical and therefore named 'radical enzymes'. Radicals are introduced by enzymes themselves or by (re)activating proteins called (re)activases. The X-ray structures of radical enzymes and their (re)activases revealed some structural features of these molecular apparatuses which solved common enigmas of radical enzymes—i.e. how the enzymes form or introduce radicals at the active sites, how they use the high reactivity of radicals for catalysis, how they suppress undesired side reactions of highly reactive radicals and how they are (re)activated when inactivated by extinction of radicals. This review highlights molecular architectures of radical B12 enzymes, radical SAM enzymes, tyrosyl radical enzymes, glycyl radical enzymes and their (re)activating proteins that support their functions. For generalization, comparisons of the recently reported structures of radical enzymes with those of canonical radical enzymes are summarized here.

  10. Redox Regulation of Protein Tyrosine Phosphatase Activity by Hydroxyl Radical

    PubMed Central

    Meng, Fan-Guo; Zhang, Zhong-Yin

    2012-01-01

    Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H2O2 include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H2O2 abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. Unfortunately, H2O2 is poorly reactive in chemical terms and the second order rate constants for the H2O2-mediated PTP inactivation are ~10 M−1s−1, which is too slow to be compatible with the transient signaling events occurring at the physiological concentrations of H2O2. We find that hydroxyl radical is produced from H2O2 solutions in the absence of metal chelating agent by the Fenton reaction. We show that hydroxyl radical is capable of inactivating the PTPs and the inactivation is active site directed, through oxidation of the catalytic Cys to sulfenic acid, which can be reduced by low molecular weight thiols. We also show that hydroxyl radical is a kinetically more efficient oxidant than H2O2 for inactivating the PTPs. The second-order rate constants for the hydroxyl radical-mediated PTP inactivation are at least 2–3 orders of magnitude higher than those mediated by H2O2 under the same conditions. Thus, hydroxyl radical generated in vivo may serve as a more physiologically relevant oxidizing agent for PTP inactivation. PMID:22819876

  11. Redox regulation of protein tyrosine phosphatase activity by hydroxyl radical.

    PubMed

    Meng, Fan-Guo; Zhang, Zhong-Yin

    2013-01-01

    Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H(2)O(2) include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H(2)O(2) abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. Unfortunately, H(2)O(2) is poorly reactive in chemical terms and the second order rate constants for the H(2)O(2)-mediated PTP inactivation are ~10M(-1)s(-1), which is too slow to be compatible with the transient signaling events occurring at the physiological concentrations of H(2)O(2). We find that hydroxyl radical is produced from H(2)O(2) solutions in the absence of metal chelating agent by the Fenton reaction. We show that the hydroxyl radical is capable of inactivating the PTPs and the inactivation is active site directed, through oxidation of the catalytic Cys to sulfenic acid, which can be reduced by low molecular weight thiols. We also show that hydroxyl radical is a kinetically more efficient oxidant than H(2)O(2) for inactivating the PTPs. The second-order rate constants for the hydroxyl radical-mediated PTP inactivation are at least 2-3 orders of magnitude higher than those mediated by H(2)O(2) under the same conditions. Thus, hydroxyl radical generated in vivo may serve as a more physiologically relevant oxidizing agent for PTP inactivation. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.

  12. Structural Mass Spectrometry of Proteins Using Hydroxyl Radical Based Protein Footprinting

    PubMed Central

    Wang, Liwen; Chance, Mark R.

    2011-01-01

    Structural MS is a rapidly growing field with many applications in basic research and pharmaceutical drug development. In this feature article the overall technology is described and several examples of how hydroxyl radical based footprinting MS can be used to map interfaces, evaluate protein structure, and identify ligand dependent conformational changes in proteins are described. PMID:21770468

  13. (Bi)sulfite Oxidation by Copper,Zinc-Superoxide Dismutase: Sulfite-Derived, Radical-Initiated Protein Radical Formation

    PubMed Central

    Ranguelova, Kalina; Bonini, Marcelo G.; Mason, Ronald P.

    2010-01-01

    Background Sulfur dioxide, formed during the combustion of fossil fuels, is a major air pollutant near large cities. Its two ionized forms in aqueous solution, sulfite and (bi)sulfite, are widely used as preservatives and antioxidants to prevent food and beverage spoilage. (Bi)sulfite can be oxidized by peroxidases to form the very reactive sulfur trioxide anion radical (•SO3−). This free radical further reacts with oxygen to form the peroxymonosulfate anion radical (−O3SOO•) and sulfate anion radical (SO4• −). Objective To explore the critical role of these radical intermediates in further oxidizing biomolecules, we examined the ability of copper,zinc-superoxide dismutase (Cu,Zn-SOD) to initiate this radical chain reaction, using human serum albumin (HSA) as a model target. Methods We used electron paramagnetic resonance, optical spectroscopy, oxygen uptake, and immuno-spin trapping to study the protein oxidations driven by sulfite-derived radicals. Results We found that when Cu,Zn-SOD reacted with (bi)sulfite, •SO3− was produced, with the concomitant reduction of SOD-Cu(II) to SOD-Cu(I). Further, we demonstrated that sulfite oxidation mediated by Cu,Zn-SOD induced the formation of radical-derived 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin-trapped HSA radicals. Conclusions The present study suggests that protein oxidative damage resulting from (bi)sulfite oxidation promoted by Cu,Zn-SOD could be involved in oxidative damage and tissue injury in (bi)sulfite-exacerbated allergic reactions. PMID:20348042

  14. Mapping protein-protein interactions by localized oxidation: consequences of the reach of hydroxyl radical.

    PubMed

    Cheal, Sarah M; Ng, Mindy; Barrios, Brianda; Miao, Zheng; Kalani, Amir K; Meares, Claude F

    2009-06-02

    Hydroxyl radicals generated from a variety of methods, including not only synchrotron radiation but also Fenton reactions involving chelated iron, have become an accepted macromolecular footprinting tool. Hydroxyl radicals react with proteins via multiple mechanisms that lead to both polypeptide backbone cleavage events and side chain modifications (e.g., hydroxylation and carbonyl formation). The use of site-specifically tethered iron chelates can reveal protein-protein interactions, but the interpretation of such experiments will be strengthened by improving our understanding of how hydroxyl radicals produced at a point on a protein react with other protein sites. We have developed methods for monitoring carbonyl formation on proteins as a function of distance from a hydroxyl generator, iron-(S)-1-[p-(bromoacetamido)benzyl]EDTA (FeBABE), conjugated to an engineered cysteine residue. After activation of the chelated iron with ascorbate and peroxide produces new protein carbonyl groups, their positions can be identified using element-coded affinity tagging (ECAT), with carbonyl-specific tags {e.g., rare earth chelates of (S)-2-[4-(2-aminooxy)acetamidobenzyl]-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (AOD)} that allow for affinity purification, identification, and relative quantitation of oxidation sites using mass spectrometry. Intraprotein oxidation of single-cysteine mutants of Escherichia coli sigma(70) by tethered FeBABE was used to calibrate the reach of hydroxyl radical by comparison to the crystal structure; the application to protein-protein interactions was demonstrated using the same sigma(70) FeBABE conjugates in complexes with the RNA polymerase core enzyme. The results provide fundamental information for interpreting protein footprinting experiments in other systems.

  15. Radical sequestration by protein-bound 3,4-dihydroxyphenylalanine.

    PubMed

    Nelson, Michelle; Foxwell, A Ruth; Tyrer, Peter; Dean, Roger T

    2010-05-01

    Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA), a redox-active product of protein oxidation, is capable of functioning as both a pro- and antioxidant. A number of in vitro and in vivo studies have demonstrated a toxic, non-toxic or even beneficial effect of free DOPA, however little investigation has examined the physiological activity of PB-DOPA. Being the major treatment available for Parkinson's disease, most studies have focused on the effect of DOPA within neurological cells or tissues, although the presence of PB-DOPA in other locations, for example within atherosclerotic plaques, suggests that broader research is needed to fully understand the physiological effects of both free and PB-DOPA. We hypothesise that the generation of PB-DOPA can trigger an enhancement of the cellular antioxidant defence system, thus enabling PB-DOPA to restrict and potentially terminate the initiating oxidative stress, minimising the level of oxidative damage. Using luminol-enhanced chemiluminescence, we demonstrate that free DOPA is capable of direct peroxyl radical scavenging, even in the presence of competing scavengers, and has a different effect to that of the parent amino acid, tyrosine. Furthermore, we show that both free and PB-DOPA, in combination or individually, were able to protect monocytes and macrophages from peroxyl radical-induced oxidative stress in vitro. These results confirm a role for both free and PB-DOPA in cellular antioxidant defences and suggest the possibility of using DOPA as a potential therapeutic for the treatment of diseases involving oxidative stress or the accumulation of oxidative damage. 2010 Elsevier Ltd. All rights reserved.

  16. Participation of oxygen-free radicals in the oxido-reduction of proteins.

    PubMed

    Vilar-Rojas, C; Guzman-Grenfell, A M; Hicks, J J

    1996-01-01

    Recent studies have focused attention on the possible role of active oxygen species on protein damage and degradation. The reactions of free radicals on biomolecules are important in physiology and pathology. A number of systems that generate free radicals catalyze the oxidative modification of proteins in two species: protein peroxides, which can consume important antioxidants; and protein-bound reducing moieties, which can reduce transition metals, and may enhance their activity in radical reactions. Protein oxidation also contributes to the pool of damaged enzymes and accumulation of abnormal and damaged proteins, which increases during aging and in various pathological states, such as atherosclerosis, cancer, etc.

  17. Competitive reduction of perferrylmyoglobin radicals by protein thiols and plant phenols.

    PubMed

    Jongberg, Sisse; Lund, Marianne N; Skibsted, Leif H; Davies, Michael J

    2014-11-19

    Radical transfer from perferrylmyoglobin to other target species (myofibrillar proteins, MPI) and bovine serum albumin (BSA), extracts from green tea (GTE), maté (ME), and rosemary (RE), and three phenolic compounds, catechin, caffeic acid, and carnosic acid) was investigated by electron paramagnetic resonance (EPR) spectroscopy to determine the concentrations of plant extracts required to protect against protein oxidation. Blocking of MPI thiol groups by N-ethylmaleimide was found to reduce the rate of reaction of MPI with perferrylmyoglobin radicals, signifying the importance of protein thiols as radical scavengers. GTE had the highest phenolic content of the three extracts and was most effective as a radical scavenger. IC50 values indicated that the molar ratio between phenols in plant extract and MPI thiols needs to be >15 in order to obtain efficient protection against protein-to-protein radical transfer in meat. Caffeic acid was found most effective among the plant phenols.

  18. Protein Radical Formation Resulting from Eosinophil Peroxidase-catalyzed Oxidation of Sulfite*

    PubMed Central

    Ranguelova, Kalina; Chatterjee, Saurabh; Ehrenshaft, Marilyn; Ramirez, Dario C.; Summers, Fiona A.; Kadiiska, Maria B.; Mason, Ronald P.

    2010-01-01

    Eosinophil peroxidase (EPO) is an abundant heme protein in eosinophils that catalyzes the formation of cytotoxic oxidants implicated in asthma, allergic inflammatory disorders, and cancer. It is known that some proteins with peroxidase activity (horseradish peroxidase and prostaglandin hydroperoxidase) can catalyze oxidation of bisulfite (hydrated sulfur dioxide), leading to the formation of sulfur trioxide anion radical (·SO3−). This free radical further reacts with oxygen to form peroxymonosulfate anion radical (−O3SOO·) and the very reactive sulfate anion radical (SO4˙̄), which is nearly as strong an oxidant as the hydroxyl radical. However, the ability of EPO to generate reactive sulfur radicals has not yet been reported. Here we demonstrate that eosinophil peroxidase/H2O2 is able to oxidize bisulfite, ultimately forming the sulfate anion radical (SO4˙̄), and that these reactive intermediates can oxidize target proteins to protein radicals, thereby initiating protein oxidation. We used immuno-spin trapping and confocal microscopy to study protein oxidation by EPO/H2O2 in the presence of bisulfite in a pure enzymatic system and in human promyelocytic leukemia HL-60 clone 15 cells, maturated to eosinophils. Polyclonal antiserum raised against the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) detected the presence of DMPO covalently attached to the proteins resulting from the DMPO trapping of protein free radicals. We found that sulfite oxidation mediated by EPO/H2O2 induced the formation of radical-derived DMPO spin-trapped human serum albumin and, to a lesser extent, of DMPO-EPO. These studies suggest that EPO-dependent oxidative damage may play a role in tissue injury in bisulfite-exacerbated eosinophilic inflammatory disorders. PMID:20501663

  19. Primary quantum yields of ketyl radicals in photoreduction by amines. Abstraction of H from N

    SciTech Connect

    Inbar, S.; Linschitz, H.; Cohen, S.G.

    1980-02-13

    Results of laser flash photolysis studies of the primary reaction of benzophenone triplet with aliphatic amines in benzene solution are reported. Quantum yield of formation of benzophenone ketyl radical was 0.9 - 1.0. Quantum yields for reduction of ketone also were determined for various amines, and the effects of tert-butyl alcohol on radical formation was investigated. Data indicated that H is not abstracted from -CH/sub 3/ but is abstracted efficiently from -NH/sub 2/. The very high quantum yields observed with tertiary and secondary amines were thought to imply exciplex formation, but lower quantum yields with primary amines were conditionally attributed to higher ionization potentials. (BLM)

  20. Synthesis of unnatural amino acids from serine derivatives by beta-fragmentation of primary alkoxyl radicals.

    PubMed

    Boto, Alicia; Gallardo, Juan A; Hernández, Dacil; Hernández, Rosendo

    2007-09-14

    The fragmentation of primary alkoxyl radicals has been scarcely used in synthesis since other competing processes (such as oxidation or hydrogen abstraction) usually predominate. However, when serine derivatives were used as substrates, the scission took place in excellent yields. Tandem scission-allylation, -alkylation, or -arylation reactions were subsequently developed. This one-pot methodology was applied to the synthesis of unnatural amino acids, which are useful synthetic blocks or amino acid surrogates in peptidomimetics.

  1. Outcome of radical prostatectomy in primary circulating prostate cell negative prostate cancer

    PubMed Central

    Murray, Nigel P; Aedo, Sócrates; Reyes, Eduardo; Fuentealba, Cynthia; Jacob, Omar

    2016-01-01

    Introduction Around 90% of prostate cancers detected using the serum prostate specific antigen (PSA) as a screening test are considered to be localised. However, 20–30% of men treated by radical prostatectomy experience biochemical failure within two years of treatment. The presence of primary circulating prostate cells (CPCs) in the blood of these men implies a dissemination of the tumour and could indicate a greater risk of treatment failure. Objective To evaluate the use of the number of primary CPCs detected before surgery in the prediction of biochemical failure at ten years. Hypothesis The dissemination of cancer cells to distant sites will determine the patient’s prognosis. The absence of primary CPCs in men undergoing radical prostatectomy for prostate cancer may imply a less aggressive disease and therefore could be utilised as a prognostic factor to predict biochemical failure after surgery. Methods and patients A single-centre observational study of a cohort of 285 men who underwent radical prostatectomy as monotherapy for prostate cancer, in whom the number of CPCs prior to treatment was determined, and who were followed up for ten years to determine biochemical failure. A Cox proportional risks with polynomial fractions analysis was used to predict biochemical failure based on the number of primary CPCs detected. A decision curve analysis was performed for the model obtained. Results Kaplan–Meier curves for biochemical free survival at ten years was 47.34% (95% CI 38.71–55.48%). It is important to note that in CPC negative men, the ten years Kaplan–Meier biochemical-free survival was 90.35% (95% CI 75.0–96.27) whereas in men who were primary CPC positive, the biochemical free survival rate was 30.00% (95% CI 20.34–40.60%). The Coxs´model to predict biochemical failure using transformed data with a power of minus one for the number of primary CPCs detected, showed a Harrell´s C concordance index of 0.74 and a decision analysis curve

  2. Immunochemical detection of hemoglobin-derived radicals formed by reaction with hydrogen peroxide: involvement of a protein-tyrosyl radical.

    PubMed

    Ramirez, Dario C; Chen, Yeong-Renn; Mason, Ronald P

    2003-04-01

    To investigate the involvement of a hemoglobin radical in the human oxyhemoglobin (oxyHb) or metHb/H2O2 system, we have used a new approach called "immuno-spin trapping," which combines the specificity and sensitivity of both spin trapping and antigen:antibody interactions. Previously, a novel rabbit polyclonal anti-DMPO nitrone adduct antiserum, which specifically recognizes protein radical-derived nitrone adducts, was developed and validated in our laboratory. In the present study, the formation of nitrone adducts on hemoglobin was shown to depend on the oxidation state of the iron heme, the concentrations of H2O2 and DMPO, and time as determined by enzyme-linked immunosorbent assay (ELISA) and by Western blotting. The presence of reduced glutathione or L-ascorbate significantly decreased the level of nitrone adducts on metHb in a dose-dependent manner. To confirm the ELISA results, Western blotting analysis showed that only the complete system (oxy- or metHb/DMPO/H2O2) generates epitopes recognized by the antiserum. The specific modification of tyrosine residues on metHb by iodination nearly abolished antibody binding, while the thiylation of cysteine residues caused a small but reproducible decrease in the amount of nitrone adducts. These findings strongly suggest that tyrosine residues are the site of formation of the immunochemically detectable hemoglobin radical-derived nitrone adducts. In addition, we were able to demonstrate the presence of hemoglobin radical-derived nitrone adducts inside red blood cells exposed to H2O2 and DMPO. In conclusion, our new approach showed several advantages over EPR spin trapping with the anti-DMPO nitrone adduct antiserum by demonstrating the formation of tyrosyl radical-derived nitrone adduct(s) in human oxyHb/metHb at much lower concentrations than was possible with EPR and detecting radicals inside RBC exposed to H2O2.

  3. 4.2.2 Primary alkyl radicals, type R2CH-CH2•

    NASA Astrophysics Data System (ADS)

    Beckwith, A. L. J.

    This document is part of Subvolume A1 `Inorganic Radicals, Metal Complexes and Nonconjugated Carbon Centered Radicals' of Volume 26 `Magnetic Properties of Free Radicals' of Landolt-Börnstein Group II `Molecules and Radicals'.

  4. 4.2.4 Primary alkyl radicals, type R-CH2•

    NASA Astrophysics Data System (ADS)

    Beckwith, A. L. J.

    This document is part of Subvolume A1 `Inorganic Radicals, Metal Complexes and Nonconjugated Carbon Centered Radicals' of Volume 26 `Magnetic Properties of Free Radicals' of Landolt-Börnstein Group II `Molecules and Radicals'.

  5. 4.2.1 Primary alkyl radicals, type R-CH2-CH2•

    NASA Astrophysics Data System (ADS)

    Beckwith, A. L. J.

    This document is part of Subvolume A1 `Inorganic Radicals, Metal Complexes and Nonconjugated Carbon Centered Radicals' of Volume 26 `Magnetic Properties of Free Radicals' of Landolt-Börnstein Group II `Molecules and Radicals'.

  6. 4.2.3 Primary alkyl radicals, type R3C-CH2•

    NASA Astrophysics Data System (ADS)

    Beckwith, A. L. J.

    This document is part of Subvolume A1 `Inorganic Radicals, Metal Complexes and Nonconjugated Carbon Centered Radicals' of Volume 26 `Magnetic Properties of Free Radicals' of Landolt-Börnstein Group II `Molecules and Radicals'.

  7. Sulfur Radical-Induced Redox Modifications in Proteins: Analysis and Mechanistic Aspects.

    PubMed

    Schöneich, Christian

    2017-03-10

    The sulfur-containing amino acids cysteine (Cys) and methionine (Met) are prominent protein targets of redox modification during conditions of oxidative stress. Here, two-electron pathways have received widespread attention, in part due to their role in signaling processes. However, Cys and Met are equally prone to one-electron pathways, generating intermediary radicals and/or radial ions. These radicals/radical ions can generate various reaction products that are not commonly monitored in redox proteomic studies, but they may be relevant for the fate of proteins during oxidative stress. Recent Advances: Time-resolved kinetic studies and product analysis have expanded our mechanistic understanding of radical reaction pathways of sulfur-containing amino acids. These reactions are now studied in some detail for Met and Cys in proteins, and homocysteine (Hcy) chemically linked to proteins, and the role of protein radical reactions in physiological processes is evolving. Radical-derived products from Cys, Hcy, and Met can react with additional amino acids in proteins, leading to secondary protein modifications, which are potentially remote from initial points of radical attack. These products may contain intra- and intermolecular cross-links, which may lead to protein aggregation. Protein sequence and conformation will have a significant impact on the formation of such products, and a thorough understanding of reaction mechanisms and specifically how protein structure influences reaction pathways will be critical for identification and characterization of novel reaction products. Future studies must evaluate the biological significance of novel reaction products that are derived from radical reactions of sulfur-containing amino acids. Antioxid. Redox Signal. 26, 388-405.

  8. Protein, lipid, and DNA radicals to measure skin UVA damage and modulation by melanin.

    PubMed

    Haywood, Rachel; Rogge, Fabrice; Lee, Martin

    2008-03-15

    Afro-Caribbeans have a lower incidence of skin cancer than Caucasians, but the effectiveness of melanin as a photoprotective pigment is debated. We investigated the UVA and solar irradiation of ex vivo human skin and DMPO using electron spin resonance spectroscopy, to determine whether pigmented skin is protected by melanin against free radical damage. Initial ascorbate radicals in Caucasian skin were superseded by lipid and/or protein radical adducts with isotropic (a(H)=1.8 mT) and anisotropic spectra comparable to spectra in irradiated pig fat (a(H)=1.9 mT) and BSA. DNA carbon-centered radical adducts (a(H)=2.3 mT) and a broad singlet were detected in genomic DNA/melanin but were not distinguishable in irradiated Caucasian skin. Protein and lipid radicals (n=6 in Caucasian skin) were minimal in Afro-Caribbean skin (n=4) and intermediate skin pigmentations were variable (n=3). In irradiated Afro-Caribbean skin a shoulder to the melanin radical (also in UVA-irradiated pigmented melanoma cells and genomic DNA/melanin and intrinsic to pheomelanin) was detected. In this sample group, protein (but not lipid) radical adducts decreased directly with pigmentation. ESR/spin trapping methodology has potential for screening skin susceptibility to aging and cancer-related radical damage and for measuring protection afforded by melanin, sunscreens, and antiaging creams.

  9. Radicality of initial surgery for primary malignant melanoma of the vagina.

    PubMed

    Todo, Yukiharu; Okamoto, Kazuhira; Suzuki, Yoshihiro; Minobe, Shinichiro; Kato, Hidenori

    2016-04-01

    Radical surgery is considered not to improve the prognosis of primary malignant melanoma of the vagina (PMMV). This study was carried out to review the general consensus. A systematic review was performed on the basis of data from 10 patients in our cohort and 147 patients in the previous literature. The radicality of the initial surgery (RAINS) score was defined as the total number of points in terms of the resected organs. The target organs were the vagina, vulva, urethra, bladder, uterus, anus, rectum, pelvic lymph nodes, and inguinal lymph nodes. Overall survival (OS) according to the RAINS score was analyzed using the Kaplan-Meier method. Information on tumor stage, size, and depth of invasion was not obtained in 15, 47, and 43% of patients, respectively. The median follow-up period was 18 months. OS with a RAINS score of at least 7 was significantly longer than that with a RAINS score of up to 6 (median survival time, 41 vs. 19 months; log-rank test, P=0.037), despite the fact that the former group included significantly more patients with advanced-stage disease. A significant difference in OS was not found between patients with a RAINS score of at least 6 and up to 5. The therapeutic significance of radical surgery for PMMV has not been assessed appropriately in previous studies because of the lack of comparability among groups and differences in the definitions of surgical radicality. Patients with PMMV might benefit from initial surgery with appropriate surgical radicality, despite incomplete validation of the RAINS score.

  10. Self Sacrifice in Radical S-adenosylmethionine Proteins

    PubMed Central

    Booker, Squire J.; Cicchillo, Robert M.; Grove, Tyler L.

    2007-01-01

    The radical SAM superfamily of metalloproteins catalyze the reductive cleavage of S-adenosyl-l-methionine to generate a 5′-deoxyadenosyl radical (5′-dA•) intermediate that is obligate for turnover. The 5′-dA• acts as a potent oxidant, initiating turnover by abstracting a hydrogen atom from an appropriate substrate. A special class of these enzymes use this strategy to functionalize unactivated C–H bonds by insertion of sulfur atoms. This review will describe the characterization of three members of this class—biotin synthase, lipoyl synthase, and MiaB protein—each of which has been shown to cannibalize itself during turnover. PMID:17936058

  11. Unusually high reactivity of apolipoprotein B-100 among proteins to radical reactions induced in human plasma.

    PubMed

    Hashimoto, R; Narita, S; Yamada, Y; Tanaka, K; Kojo, S

    2000-01-17

    Relative reactivities of proteins to radical reactions caused in human plasma were studied for the first time utilizing an immunoblotting assay. When radical reactions were caused by Cu(2+), apolipoprotein B-100 (apoB) underwent extensive fragmentation concurrently with the decrease in alpha-tocopherol, while human serum albumin (HSA) and transferrin (TF) were not decreased at all. When radical reactions were initiated by Cu(2+) with hydrogen peroxide or 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), alpha-tocopherol and apoB were also decreased steadily but HSA and TF were not decreased. These observations indicate that apoB is extremely reactive, even comparable to alpha-tocopherol, towards radical reactions. These results also suggest that the radical reaction of apoB is a possible process in vivo and it is involved in atherogenesis along with low density lipoprotein lipid peroxidation, which has been studied extensively.

  12. Radical SAM, A Novel Protein Superfamily Linking Unresolved Steps in Familiar Biosynthetic Pathways with Radical Mechanisms: Functional Characterization Using New Analysis and Information Visualization Methods

    SciTech Connect

    Sofia, Heidi J.; Chen, Guang; Hetzler, Elizabeth G.; Reyes Spindola, Jorge F.; Miller, Nancy E.

    2001-03-01

    A large protein superfamily with over 500 members has been discovered and analyzed using powerful new bioinformatics and information visualization methods. Evidence exists that these proteins generate a 5?-deoxyadenosyl radical by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. Radical SAM superfamily proteins function in DNA precursor, vitamin, cofactor, antibiotic, and herbicide biosynthesis in a collection of basic and familiar pathways. One of the members is interferon-inducible and is considered a candidate drug target for osteoporosis. The identification of this superfamily suggests that radical-based catalysis is important in a number of previously well-studied but unresolved biochemical pathways.

  13. Artifacts Generated During Azoalkane Peroxy Radical Oxidative Stress Testing of Pharmaceuticals Containing Primary and Secondary Amines.

    PubMed

    Nefliu, Marcela; Zelesky, Todd; Jansen, Patrick; Sluggett, Gregory W; Foti, Christopher; Baertschi, Steven W; Harmon, Paul A

    2015-12-01

    We report artifactual degradation of pharmaceutical compounds containing primary and secondary amines during peroxy radical-mediated oxidative stress carried out using azoalkane initiators. Two degradation products were detected when model drug compounds dissolved in methanol/water were heated to 40°C with radical initiators such as 2,2'-azobis(2-methylpropionitrile) (AIBN). The primary artifact was identified as an α-aminonitrile generated from the reaction of the amine group of the model drug with formaldehyde and hydrogen cyanide, generated as byproducts of the stress reaction. A minor artifact was generated from the reaction between the amine group and isocyanic acid, also a byproduct of the stress reaction. We report the effects of pH, initiator/drug molar ratio, and type of azoalkane initiator on the formation of these artifacts. Mass spectrometry and nuclear magnetic resonance were used for structure elucidation, whereas mechanistic studies, including stable isotope labeling experiments, cyanide analysis, and experiments exploring the effects of butylated hydroxyanisole addition, were employed to support the degradation pathways. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  14. Concerning the production of free radicals in proteins by ultraviolet light.

    NASA Technical Reports Server (NTRS)

    Androes, G. M.; Gloria, H. R.; Reinisch, R. F.

    1972-01-01

    The response to UV light of several solid proteins and model compounds has been studied in vacuum and at low temperature, using electron paramagnetic resonance techniques. The results indicate that the details of amino acid composition and sequence, and the tertiary structure of a protein are important in determining both the rate of, and the mechanism for, the production of free radicals, and in determining the conditions under which sulfur-type radicals can be produced. The results presented are related to enzyme inactivation and to the UV stability of proteins generally.

  15. Concerning the production of free radicals in proteins by ultraviolet light.

    NASA Technical Reports Server (NTRS)

    Androes, G. M.; Gloria, H. R.; Reinisch, R. F.

    1972-01-01

    The response to UV light of several solid proteins and model compounds has been studied in vacuum and at low temperature, using electron paramagnetic resonance techniques. The results indicate that the details of amino acid composition and sequence, and the tertiary structure of a protein are important in determining both the rate of, and the mechanism for, the production of free radicals, and in determining the conditions under which sulfur-type radicals can be produced. The results presented are related to enzyme inactivation and to the UV stability of proteins generally.

  16. Beta-carotene encapsulated in food protein nanoparticles reduces peroxyl radical oxidation in Caco-2 cells

    USDA-ARS?s Scientific Manuscript database

    Beta-carotene (BC) was encapsulated by sodium caseinate (SC), whey protein isolate (WPI), and soybean protein isolate (SPI) by the homogenization-evaporation method forming nanoparticles of 78, 90 and 370 nm diameter. Indices of the chemical antioxidant assays, the reducing power, DPPH radical scave...

  17. Theories of biological aging: genes, proteins, and free radicals.

    PubMed

    Rattan, Suresh I S

    2006-12-01

    Traditional categorization of theories of aging into programmed and stochastic ones is outdated and obsolete. Biological aging is considered to occur mainly during the period of survival beyond the natural or essential lifespan (ELS) in Darwinian terms. Organisms survive to achieve ELS by virtue of genetically determined longevity assuring maintenance and repair systems (MRS). Aging at the molecular level is characterized by the progressive accumulation of molecular damage caused by environmental and metabolically generated free radicals, by spontaneous errors in biochemical reactions, and by nutritional components. Damages in the MRS and other pathways lead to age-related failure of MRS, molecular heterogeneity, cellular dysfunctioning, reduced stress tolerance, diseases and ultimate death. A unified theory of biological aging in terms of failure of homeodynamics comprising of MRS, and involving genes, milieu and chance, is acquiring a definitive shape and wider acceptance. Such a theory also establishes the basis for testing and developing effective means of intervention, prevention and modulation of aging.

  18. Pulsed electron beam water radiolysis for submicrosecond hydroxyl radical protein footprinting.

    PubMed

    Watson, Caroline; Janik, Ireneusz; Zhuang, Tiandi; Charvátová, Olga; Woods, Robert J; Sharp, Joshua S

    2009-04-01

    Hydroxyl radical footprinting is a valuable technique for studying protein structure, but care must be taken to ensure that the protein does not unfold during the labeling process due to oxidative damage. Footprinting methods based on submicrosecond laser photolysis of peroxide that complete the labeling process faster than the protein can unfold have been recently described; however, the mere presence of large amounts of hydrogen peroxide can also cause uncontrolled oxidation and minor conformational changes. We have developed a novel method for submicrosecond hydroxyl radical protein footprinting using a pulsed electron beam from a 2 MeV Van de Graaff electron accelerator to generate a high concentration of hydroxyl radicals by radiolysis of water. The amount of oxidation can be controlled by buffer composition, pulsewidth, dose, and dissolved nitrous oxide gas in the sample. Our results with ubiquitin and beta-lactoglobulin A demonstrate that one submicrosecond electron beam pulse produces extensive protein surface modifications. Highly reactive residues that are buried within the protein structure are not oxidized, indicating that the protein retains its folded structure during the labeling process. Time-resolved spectroscopy indicates that the major part of protein oxidation is complete in a time scale shorter than that of large scale protein motions.

  19. Pulsed Electron Beam Water Radiolysis for Sub-Microsecond Hydroxyl Radical Protein Footprinting

    PubMed Central

    Watson, Caroline; Janik, Ireneusz; Zhuang, Tiandi; Charvátová, Olga; Woods, Robert J.; Sharp, Joshua S.

    2009-01-01

    Hydroxyl radical footprinting is a valuable technique for studying protein structure, but care must be taken to ensure that the protein does not unfold during the labeling process due to oxidative damage. Footprinting methods based on sub-microsecond laser photolysis of peroxide that complete the labeling process faster than the protein can unfold have been recently described; however, the mere presence of large amounts of hydrogen peroxide can also cause uncontrolled oxidation and minor conformational changes. We have developed a novel method for sub-microsecond hydroxyl radical protein footprinting using a pulsed electron beam from a 2 MeV Van de Graaff electron accelerator to generate a high concentration of hydroxyl radicals by radiolysis of water. The amount of oxidation can be controlled by buffer composition, pulsewidth, dose, and dissolved nitrous oxide gas in the sample. Our results with ubiquitin and β-lactoglobulin A demonstrate that one sub-microsecond electron beam pulse produces extensive protein surface modifications. Highly reactive residues that are buried within the protein structure are not oxidized, indicating that the protein retains its folded structure during the labeling process. Time-resolved spectroscopy indicates that the major part of protein oxidation is complete in a timescale shorter than that of large scale protein motions. PMID:19265387

  20. Molecularly imprinted protein recognition thin films constructed by controlled/living radical polymerization.

    PubMed

    Sasaki, Shogo; Ooya, Tooru; Kitayama, Yukiya; Takeuchi, Toshifumi

    2015-02-01

    We demonstrated the synthesis of molecularly imprinted polymers (MIPs) with binding affinity toward a target protein, ribonuclease A (RNase) by atom transfer radical polymerization (ATRP) of acrylic acid, acrylamide, and N,N'-methylenebisacrylamide in the presence of RNase. The binding activity of the MIPs was evaluated by surface plasmon resonance (SPR) of the MIP thin layers prepared on the gold-coated sensor chips. The MIPs prepared by ATRP (MIP-ATRP) had a binding affinity toward RNase with larger binding amount compared to MIPs prepared by conventional free radical polymerization methods (MIP-RP). Moreover, protein selectivity was evaluated using reference proteins (cytochrome c, myoglobin, and α-lactalbumin) and was confirmed in MIP-ATRP of optimum film thickness determined experimentally to be 15-30 nm; however, protein selectivity was not achieved in all MIP-RP. We have shown that ATRP is powerful technique for preparing protein recognition materials by molecular imprinting.

  1. Dosimetry determines the initial OH radical concentration in fast photochemical oxidation of proteins (FPOP).

    PubMed

    Niu, Ben; Zhang, Hao; Giblin, Daryl; Rempel, Don L; Gross, Michael L

    2015-05-01

    Fast photochemical oxidation of proteins (FPOP) employs laser photolysis of hydrogen peroxide to give OH radicals that label amino acid side-chains of proteins on the microsecond time scale. A method for quantitation of hydroxyl radicals after laser photolysis is of importance to FPOP because it establishes a means to adjust the yield of •OH, offers the opportunity of tunable modifications, and provides a basis for kinetic measurements. The initial concentration of OH radicals has yet to be measured experimentally. We report here an approach using isotope dilution gas chromatography/mass spectrometry (GC/MS) to determine quantitatively the initial •OH concentration (we found ~0.95 mM from 15 mM H2O2) from laser photolysis and to investigate the quenching efficiencies for various •OH scavengers.

  2. Dosimetry Determines the Initial OH Radical Concentration in Fast Photochemical Oxidation of Proteins (FPOP)

    NASA Astrophysics Data System (ADS)

    Niu, Ben; Zhang, Hao; Giblin, Daryl; Rempel, Don L.; Gross, Michael L.

    2015-05-01

    Fast photochemical oxidation of proteins (FPOP) employs laser photolysis of hydrogen peroxide to give OH radicals that label amino acid side-chains of proteins on the microsecond time scale. A method for quantitation of hydroxyl radicals after laser photolysis is of importance to FPOP because it establishes a means to adjust the yield of •OH, offers the opportunity of tunable modifications, and provides a basis for kinetic measurements. The initial concentration of OH radicals has yet to be measured experimentally. We report here an approach using isotope dilution gas chromatography/mass spectrometry (GC/MS) to determine quantitatively the initial •OH concentration (we found ~0.95 mM from 15 mM H2O2) from laser photolysis and to investigate the quenching efficiencies for various •OH scavengers.

  3. Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals.

    PubMed

    Liu, Fobang; Lai, Senchao; Tong, Haijie; Lakey, Pascale S J; Shiraiwa, Manabu; Weller, Michael G; Pöschl, Ulrich; Kampf, Christopher J

    2017-03-01

    Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32-55% for BSA, ∼10-21% for OVA) were substantially higher than those for the other identified amino acids (∼5-12% for BSA, ∼4-6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals.

  4. Aliphatic peptidyl hydroperoxides as a source of secondary oxidation in hydroxyl radical protein footprinting.

    PubMed

    Saladino, Jessica; Liu, Mian; Live, David; Sharp, Joshua S

    2009-06-01

    Hydroxyl radical footprinting is a technique for studying protein structure and binding that entails oxidizing a protein system of interest with diffusing hydroxyl radicals, and then measuring the amount of oxidation of each amino acid. One important issue in hydroxyl radical footprinting is limiting amino acid oxidation by secondary oxidants to prevent uncontrolled oxidation, which can cause amino acids to appear more solvent accessible than they really are. Previous work suggested that hydrogen peroxide was the major secondary oxidant of concern in hydroxyl radical footprinting experiments; however, even after elimination of all hydrogen peroxide, some secondary oxidation was still detected. Evidence is presented for the formation of peptidyl hydroperoxides as the most abundant product upon oxidation of aliphatic amino acids. Both reverse phase liquid chromatography and catalase treatment were shown to be ineffective at eliminating peptidyl hydroperoxides. The ability of these peptidyl hydroperoxides to directly oxidize methionine is demonstrated, suggesting the value of methionine amide as an in situ protectant. Hydroxyl radical footprinting protocols require the use of an organic sulfide or similar peroxide scavenger in addition to removal of hydrogen peroxide to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues.

  5. The nitroxide TEMPO is an efficient scavenger of protein radicals: cellular and kinetic studies.

    PubMed

    Pattison, David I; Lam, Magdalena; Shinde, Sujata S; Anderson, Robert F; Davies, Michael J

    2012-11-01

    Protein oxidation occurs during multiple human pathologies, and protein radicals are known to induce damage to other cell components. Such damage may be modulated by agents that scavenge protein radicals. In this study, the potential protective reactions of the nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl radical) against Tyr- and Trp-derived radicals (TyrO./TrpN.) have been investigated. Pretreatment of macrophage cells with TEMPO provided protection against photo-oxidation-induced loss of cell viability and Tyr oxidation, with the nitroxide more effective than the hydroxylamine or parent amine. Pulse radiolysis was employed to determine rate constants, k, for the reaction of TEMPO with TyrO. and TrpN. generated on N-Ac-Tyr-amide and N-Ac-Trp-amide, with values of k~10(8) and 7×10(6)M(-1)s(-1), respectively, determined. Analogous studies with lysozyme, chymotrypsin, and pepsin yielded k for TEMPO reacting with TrpN. ranging from 1.5×10(7) (lysozyme) to 1.1×10(8) (pepsin)M(-1)s(-1). Pepsin-derived TyrO. reacted with TEMPO with k~4×10(7)M(-1)s(-1); analogous reactions for lysozyme and chymotrypsin TyrO. were much slower. These data indicate that TEMPO can inhibit secondary reactions of both TyrO. and TrpN., though this is protein dependent. Such protein radical scavenging may contribute to the positive biological effects of nitroxides.

  6. Ultrafast primary processes of the stable neutral organic radical, 1,3,5-triphenylverdazyl, in liquid solution.

    PubMed

    Weinert, Christoph; Wezisla, Boris; Lindner, Jörg; Vöhringer, Peter

    2015-05-28

    Femtosecond spectroscopy with hyperspectral white-light detection was used to elucidate the ultrafast primary processes of the thermodynamically stable organic radical, 1,3,5-triphenylverdazyl, in liquid acetonitrile solution at room temperature. The radical was excited with optical pulses having a duration of 39 fs and a center wavelength of 800 nm thereby accessing its energetically lowest electronically excited state (D1). The apparent spectrotemporal response is understood in terms of an ultrafast primary D1-to-D0 internal conversion that generates the electronic ground state of the radical in a highly vibrationally excited fashion within a few hundred femtoseconds. The replenished electronic ground state subsequently undergoes vibrational cooling on a time scale of a few picoseconds. The instantaneous absorption spectra of the radical derived from the femtosecond pump-probe data are analyzed within the Sulzer-Wieland formalism for calculating the electronic spectra of "hot" polyatomic molecules. The pump-probe spectra together with transient anisotropy data in the region of the D0 → D1 ground-state bleach gives evidence for an additional transient absorption that arises from a dark excited state, which gains oscillator strength with increasing vibrational excitation of the radical by virtue of vibronic coupling.

  7. Stable free-radical forms of plasma proteins or simpler related structures which induce brain excitatory effects.

    PubMed

    Polis, B D; Wyeth, J; Goldstein, L; Graedon, J

    1969-10-01

    Stable free radicals have been prepared from purified plasma proteins, pituitary peptides, and simpler related structures like 5-OH tryptophan and melatonin by oxidation with the free-radical nitrosyl disulfonate in alkaline solution under controlled conditions. The presence of tyrosine or trytophan amino acid residues in the protein was found essential for free-radical formation. These red-colored, stable free radicals showed electron spin resonance spectra in aqueous solutions at room temperature and maintained this characteristic for weeks when stored at 5 degrees C. Illumination, by visible light, of the free-radical proteins and peptides separated from excess nitrosyl disulfonate by salt fractionation or chromatography enhanced the free-radical concentration in the light. The increased signal decayed in the dark. Intravenous administration of the free-radical proteins or peptides into rabbits equipped with chronic cranial electrodes and sedated with a small dose of pentobarbital caused a sudden EEG arousal accompanied by behavioral changes indicative of brain excitation. Illumination of the free-radical compounds prior to administration enhanced the effects. Untreated control proteins or peptides had no effects. The observations are interpreted to suggest the involvement of free-radical structures in the transfer of energy in nervous tissue.

  8. A computational study of radical initiated protein backbone homolytic dissociation on all natural amino acids.

    PubMed

    Uranga, Jon; Lakuntza, Oier; Ramos-Cordoba, Eloy; Matxain, Jon M; Mujika, Jon I

    2016-11-16

    Hydroxyl radical (˙OH) is known to be one of the most reactive species. In this work, the hydrogen abstraction by ˙OH from Cα and Cβ atoms of all amino acids is studied in the framework of density functional theory as this is the most favorable reaction mechanism when this kind of radical attacks a protein. From the myriad routes that the oxidation of a protein by a ˙OH radical may follow, fragmentation of the protein is one of the most damaging ones as it hampers the normal function of the protein. Therefore, cleavages of the Cα-C and Cα-N backbone bonds have been investigated as the second step of the mechanism. To the best of our knowledge, this is the first time that this reaction pathway has been systematically studied for all natural amino acids. The study includes the effects that the solvent dielectrics or the conformation of the peptide model employed has on the reaction. Interestingly, the results indicate that the nature of the side chain has little effect on the H abstraction reaction, and that for most of amino acids the attack at the Cα atom is favored over the attack at the Cβ atom. The origin of this preference relies on the larger capability of the formed radical intermediate to delocalize the unpaired electron, thus maximizing the captodative effect. Moreover, the reaction is more favorable when the reactant presents a β-sheet conformation, with a completely planar peptide backbone. With respect to the homolytic splitting of the Cα-C and Cα-N bonds, the former is favorable for almost all amino acids, whereas Ser and Thr are the only amino acids favoring the latter. These results agree with previous investigations but an accurate description of the electronic density analysis performed indicates that the origin of the different reaction pathway preferences relies on a large stabilization of the product rather than bond weakening at the radical intermediate.

  9. Formation of peroxides in amino acids and proteins exposed to oxygen free radicals.

    PubMed Central

    Gebicki, S; Gebicki, J M

    1993-01-01

    Dilute aqueous solutions of BSA or lysozyme gave positive tests for peroxides after exposure to reactive oxygen species. The reactive species were generated by gamma-irradiation, reduction of H2O2 with Fe2+ ions or thermal decomposition of an azo compound. Peroxides were assayed by an iodometric method. Identification of the new groups as hydroperoxides was confirmed by their ability to oxidize a range of compounds and by the kinetics of their reaction with iodide. The hydroperoxide groups were bound to the proteins and their yields (G values) corresponded to 1.2 -OOH groups per 100 eV of radiation energy absorbed for BSA, and 0.8 for lysozyme. The oxygen free radicals effective in protein peroxidation were the hydroxyl and organic peroxyl, but not superoxide or its protonated form. The efficiency of BSA peroxidation initiated by the hydroxyl radicals was 40%. Protein peroxides decayed spontaneously with a half-life of about 1.5 days at 20 degrees C. Exposure of the common amino acids to hydroxyl free radicals showed that six of them (glutamate, isoleucine, leucine, lysine, proline and valine) were peroxidized with similar efficiency to the proteins, whereas the rest were inert or much less susceptible. These results suggest that some proteins may be peroxidized by a variety of agents in vivo and that their subsequent reactions with protective agents, such as ascorbate or glutathione, may decrease the antioxidant potential of cells and tissues. PMID:8435071

  10. EPR evidence of hydroxyl radical generation as an initiator of lipid peroxidation in amyloid beta-protein-stimulated PC12 cells.

    PubMed

    Hayashi, Yoshihito; Ueda, Yuto; Nakajima, Akira; Mitsuyama, Yoshio

    2004-10-29

    Recent data from several groups suggest that the primary mechanism of amyloid beta-protein (Abeta) neurotoxicity may be mediated by free radicals. To evaluate this hypothesis, our aim is to make the mechanism of Abeta neurotoxicity clear, especially in the formation of free radicals. In this study, rat pheochromocytoma (PC12) cells were exposed to Abeta25-35 and confirmed free radical generations using two kinds of spin trap agents, 5,5-dimethyl-1-pyrroline-N-oxide; DMPO and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone; POBN. DMPO spin adduct revealed that hydroxyl radical (OH), while POBN spin adduct identified a lipid radical (L) as electron paramagnetic resonance (EPR) evidence of lipid peroxidation in the process of cell damage by Abeta25-35 exposure. An Abeta cytotoxicity assay also was performed by using WST-8 reduction system and histochemical analysis. These analyses showed cell damage induced by Abeta. This study provides EPR evidence that Abeta neurotoxicity is derived from hydrogen abstraction from polyunsaturated lipid acid by hydroxyl radical as a cause of lipid peroxidation.

  11. Efficient DNP NMR of Membrane Proteins: Sample Preparation Protocols, Sensitivity, and Radical Location

    PubMed Central

    Liao, Shu Y.; Lee, Myungwoon; Wang, Tuo; Sergeyev, Ivan V.; Hong, Mei

    2016-01-01

    Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~4 fold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes. PMID:26873390

  12. Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location.

    PubMed

    Liao, Shu Y; Lee, Myungwoon; Wang, Tuo; Sergeyev, Ivan V; Hong, Mei

    2016-03-01

    Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105-160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes.

  13. Protein-Polymer Conjugates: Synthetic Approaches by Controlled Radical Polymerizations & Interesting Applications

    PubMed Central

    Grover, Gregory N.; Maynard, Heather D.

    2011-01-01

    Protein-polymer conjugates are of interest to researchers in diverse fields. Attachment of polymers to proteins results in improved pharmacokinetics, which is important in medicine. From an engineering standpoint, conjugates are exciting because they exhibit properties of both the biomolecules and synthetic polymers. This allows the activity of the protein to be altered or tuned, a key aspect in therapeutic design, anchoring conjugates to surfaces, and utilizing these materials for supramolecular self-assembly. Thus, there is broad interest in straightforward synthetic methods to make protein-polymer conjugates. Controlled radical polymerization (CRP) techniques have emerged as excellent strategies to make conjugates because the resulting polymers have narrow molecular weight distributions, targeted molecular weights, and attach to specific sites on proteins. Herein, recent advances in the synthesis and application of protein-polymer conjugates by CRP are highlighted. PMID:21071260

  14. Suppression of iron catalyzed free radical generation by iron tyrosinate protein models.

    PubMed

    Kitazawa, M; Iwasaki, K

    1996-03-07

    Novel antioxidants, N-(2-Hydroxy-1-naphthal)amino acids which mimic iron proteins, have been intensively studied for suppressive effect on iron-catalyzed free radical generation. These compounds exhibit inhibition of the Fenton reaction in electron spin resonance assessment. In addition, it is shown that the compounds inhibit iron-induced peroxidation by thiobarbituric acid test. Since these antioxidants form stable complexes with Fe3+, this antioxidative activity is expected to be derived from sequestration of catalytic iron.

  15. A Substrate Radical Intermediate in Catalysis by the Antibiotic Resistance Protein Cfr

    PubMed Central

    Grove, Tyler L.; Livada, Jovan; Schwalm, Erica L.; Green, Michael T.; Booker, Squire J.; Silakov, Alexey

    2013-01-01

    Cfr-dependent methylation of C8 of adenosine 2503 (A2503) in 23S rRNA confers bacterial resistance to an array of clinically important antibiotics that target the large subunit of the ribosome, including the synthetic oxazolidinone antibiotic linezolid. The key element of the proposed mechanism for Cfr, a radical S-adenosylmethionine (SAM) enzyme, is the addition of a methylene radical — generated by hydrogen-atom abstraction from the methyl group of an S-methylated cysteine residue (mCys) — onto C8 of A2503 to form a protein – nucleic acid cross-linked species containing an unpaired electron. Herein we use continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques to provide direct spectroscopic evidence for this intermediate, showing a spin-delocalized radical with maximum spin density at N7 of the adenine ring. In addition, we use rapid-freeze quench EPR to show that the radical forms and decays with rate constants that are consistent with the rate of formation of the methylated product. PMID:23644479

  16. Tyrosyl radical generated by myeloperoxidase catalyzes the oxidative cross-linking of proteins.

    PubMed

    Heinecke, J W; Li, W; Francis, G A; Goldstein, J A

    1993-06-01

    Phagocytes generate H2O2 for use by a secreted heme enzyme, myeloperoxidase, to kill invading bacteria, viruses, and fungi. We have explored the possibility that myeloperoxidase might also convert L-tyrosine to a radical catalyst that cross-links proteins. Protein-bound tyrosyl residues exposed to myeloperoxidase, H2O2, and L-tyrosine were oxidized to o,o'-dityrosine, a stable product of the tyrosyl radical. The cross-linking reaction required L-tyrosine but was independent of halide and free transition metal ions; the heme poisons azide and aminotriazole were inhibitory. Activated neutrophils likewise converted polypeptide tyrosines to dityrosine. The pathway for oxidation of peptide tyrosyl residues was dependent upon L-tyrosine and was inhibited by heme poisons and catalase. Dityrosine synthesis was little affected by plasma concentrations of Cl- and amino acids, suggesting that the reaction pathway might be physiologically relevant. The requirement for free L-tyrosine and H2O2 for dityrosine formation and the inhibition by heme poisons support the hypothesis that myeloperoxidase catalyzes the cross-linking of proteins by a peroxidative mechanism involving tyrosyl radical. In striking contrast to the pathways generally used to study protein oxidation in vitro, the reaction does not require free metal ions. We speculate that protein dityrosine cross-linking by myeloperoxidase may play a role in bacterial killing or injuring normal tissue. The intense fluorescence and stability of biphenolic compounds may allow dityrosine to act as a marker for proteins oxidatively damaged by myeloperoxidase in phagocyte-rich inflammatory lesions.

  17. Hypochlorite-induced damage to proteins: formation of nitrogen-centred radicals from lysine residues and their role in protein fragmentation.

    PubMed Central

    Hawkins, C L; Davies, M J

    1998-01-01

    Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl damages proteins by reaction with amino acid side-chains or backbone cleavage. Little information is available about the mechanisms and intermediates involved in these reactions. EPR spin trapping has been employed to identify radicals on proteins, peptides and amino acids after treatment with HOCl. Reaction with HOCl gives both high- and low-molecular-mass nitrogen-centred, protein-derived radicals; the yield of the latter increases with both higher HOCl:protein ratios and enzymic digestion. These radicals, which arise from lysine side-chain amino groups, react with ascorbate, glutathione and Trolox. Reaction of HOCl-treated proteins with excess methionine eliminates radical formation, which is consistent with lysine-derived chloramines (via homolysis of N-Cl bonds) being the radical source. Incubation of HOCl-treated proteins, after removal of excess oxidant, gives rise to both nitrogen-centred radicals, over a period of hours, and time-dependent fragmentation of the protein. Treatment with excess methionine or antioxidants (Trolox, ascorbate, glutathione) protects against fragmentation; urate and bilirubin do not. Chloramine formation and nitrogen-centred radicals are therefore key species in HOCl-induced protein fragmentation. PMID:9620862

  18. Resveratrol potently reduces prostaglandin E2 production and free radical formation in lipopolysaccharide-activated primary rat microglia

    PubMed Central

    Candelario-Jalil, Eduardo; de Oliveira, Antonio C Pinheiro; Gräf, Sybille; Bhatia, Harsharan S; Hüll, Michael; Muñoz, Eduardo; Fiebich, Bernd L

    2007-01-01

    Background Neuroinflammatory responses are triggered by diverse ethiologies and can provide either beneficial or harmful results. Microglial cells are the major cell type involved in neuroinflammation, releasing several mediators, which contribute to the neuronal demise in several diseases including cerebral ischemia and neurodegenerative disorders. Attenuation of microglial activation has been shown to confer protection against different types of brain injury. Recent evidence suggests that resveratrol has anti-inflammatory and potent antioxidant properties. It has been also shown that resveratrol is a potent inhibitor of cyclooxygenase (COX)-1 activity. Previous findings have demonstrated that this compound is able to reduce neuronal injury in different models, both in vitro and in vivo. The aim of this study was to examine whether resveratrol is able to reduce prostaglandin E2 (PGE2) and 8-iso-prostaglandin F2α (8-iso-PGF2α) production by lipopolysaccharide (LPS)-activated primary rat microglia. Methods Primary microglial cell cultures were prepared from cerebral cortices of neonatal rats. Microglial cells were stimulated with 10 ng/ml of LPS in the presence or absence of different concentrations of resveratrol (1–50 μM). After 24 h incubation, culture media were collected to measure the production of PGE2 and 8-iso-PGF2α using enzyme immunoassays. Protein levels of COX-1, COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) were studied by Western blotting after 24 h of incubation with LPS. Expression of mPGES-1 at the mRNA level was investigated using reverse transcription-polymerase chain reaction (RT-PCR) analysis. Results Our results indicate that resveratrol potently reduced LPS-induced PGE2 synthesis and the formation of 8-iso-PGF2α, a measure of free radical production. Interestingly, resveratrol dose-dependently reduced the expression (mRNA and protein) of mPGES-1, which is a key enzyme responsible for the synthesis of PGE2 by activated

  19. Effects of oxidative modification on gel properties of isolated porcine myofibrillar protein by peroxyl radicals.

    PubMed

    Zhou, Feibai; Zhao, Mouming; Zhao, Haifeng; Sun, Weizheng; Cui, Chun

    2014-04-01

    AAPH-derived (2,2'-azobis (2-amidinopropane) dihydrochloride) peroxyl radicals were selected as representative free radicals of lipid peroxidation to investigate the effects of oxidative modifications on isolated porcine myofibrillar protein structures as well as their rheological and gelling properties. Incubation of myofibrillar protein with increasing concentrations of AAPH resulted in a gradual increase (p<0.05) in carbonyl content and SH→S-S conversion. Results from SDS-PAGE indicated that medium (~1 mM) and relatively high (>3 mM) concentrations of AAPH induced aggregation of myosin and denaturation of myosin, troponin and tropomyosin, respectively. These structural changes resulted in changes on gelation of myofibrillar protein. Low level protein oxidation (AAPH≤0.5 mM) had no remarkable effect (p>0.05) on the viscoelastic pattern of myofibrillar protein gelation. Moderate oxidative modification (AAPH~1mM) enhanced the water-holding capacity (WHC) and texture properties of gels, while further oxidation (AAPH>3mM) significantly reduced the gel quality.

  20. Evidence for roles of radicals in protein oxidation in advanced human atherosclerotic plaque.

    PubMed Central

    Fu, S; Davies, M J; Stocker, R; Dean, R T

    1998-01-01

    Oxidative damage might be important in atherogenesis. Oxidized lipids are present at significant concentrations in advanced human plaque, although tissue antioxidants are mostly present at normal concentrations. Indirect evidence of protein modification (notably derivatization of lysine) or oxidation has been obtained by immunochemical methods; the specificities of these antibodies are unclear. Here we present chemical determinations of six protein-bound oxidation products: dopa, o-tyrosine, m-tyrosine, dityrosine, hydroxyleucine and hydroxyvaline, some of which reflect particularly oxy-radical-mediated reaction pathways, which seem to involve mainly the participation of transition- metal ions. We compared the relative abundance of these oxidation products in normal intima, and in human carotid plaque samples with that observed after radiolytically generated hydroxyl radical attack on BSA in vitro. The close similarities in relative abundances in the latter two circumstances indicate that hydroxyl radical damage might occur in plaque. The relatively higher level of dityrosine in plaque than that observed after radiolysis suggests the additional involvement of HOCl-mediated reactions in advanced plaque. PMID:9677308

  1. Effects of the protein environment on the spectral properties of tryptophan radicals in Pseudomonas aeruginosa azurin.

    PubMed

    Bernini, Caterina; Andruniów, Tadeusz; Olivucci, Massimo; Pogni, Rebecca; Basosi, Riccardo; Sinicropi, Adalgisa

    2013-03-27

    Many biological electron-transfer reactions involve short-lived tryptophan radicals as key reactive intermediates. While these species are difficult to investigate, the recent photogeneration of a long-lived neutral tryptophan radical in two Pseudomonas aeruginosa azurin mutants (Az48W and ReAz108W) made it possible to characterize the electronic, vibrational, and magnetic properties of such species and their sensitivity to the molecular environment. Indeed, in Az48W the radical is embedded in the hydrophobic core while, in ReAz108W it is solvent-exposed. Here we use density functional theory and multiconfigurational perturbation theory to construct quantum-mechanics/molecular-mechanics models of Az48W(•) and ReAz108W(•) capable of reproducing specific features of their observed UV-vis, resonance Raman, and electron paramagnetic resonance spectra. The results show that the models can correctly replicate the spectral changes imposed by the two contrasting hydrophobic and hydrophilic environments. Most importantly, the same models can be employed to disentangle the molecular-level interactions responsible for such changes. It is found that the control of the hydrogen bonding between the tryptophan radical and a single specific surface water molecule in ReAz108W(•) represents an effective means of spectral modulation. Similarly, a specific electrostatic interaction between the radical moiety and a Val residue is found to control the Az48W(•) excitation energy. These modulations appear to be mediated by the increase in nitrogen negative charge (and consequent increase in hydrogen bonding) of the spectroscopic D2 state with respect to the D0 state of the chromophore. Finally, the same protein models are used to predict the relaxed Az48W(•) and ReAz108W(•) D2 structures, showing that the effect of the environment on the corresponding fluorescence maxima must parallel that of D0 absorption spectra.

  2. RNAi Screen for NRF2 Inducers Identifies Targets That Rescue Primary Lung Epithelial Cells from Cigarette Smoke Induced Radical Stress

    PubMed Central

    Schumacher, Frances-Rose; Schubert, Steffen; Hannus, Michael; Sönnichsen, Birte; Ittrich, Carina; Kreideweiss, Stefan; Rippmann, Jörg F.

    2016-01-01

    Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the ‘druggable’ genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism. PMID:27832175

  3. Primary Cilia and Intraflagellar Transport Proteins in Bone and Cartilage.

    PubMed

    Yuan, X; Yang, S

    2016-11-01

    Primary cilia, present on most mammalian cells, function as a sensor to sense the environment change and transduce signaling. Loss of primary cilia causes a group of human pleiotropic syndromes called Ciliopathies. Some of the ciliopathies display skeletal dysplasias, implying the important role of primary cilia in skeletal development and homeostasis. Emerging evidence has shown that loss or malfunction of primary cilia or ciliary proteins in bone and cartilage is associated with developmental and function defects. Intraflagellar transport (IFT) proteins are essential for cilia formation and/or function. In this review, we discuss the role of primary cilia and IFT proteins in the development of bone and cartilage, as well as the differentiation and mechanotransduction of mesenchymal stem cells, osteoblasts, osteocytes, and chondrocytes. We also include the role of primary cilia in tooth development and highlight the current advance of primary cilia and IFT proteins in the pathogenesis of cartilage diseases, including osteoarthritis, osteosarcoma, and chondrosarcoma.

  4. Protein–DNA charge transport: Redox activation of a DNA repair protein by guanine radical

    PubMed Central

    Yavin, Eylon; Boal, Amie K.; Stemp, Eric D. A.; Boon, Elizabeth M.; Livingston, Alison L.; O'Shea, Valerie L.; David, Sheila S.; Barton, Jacqueline K.

    2005-01-01

    DNA charge transport (CT) chemistry provides a route to carry out oxidative DNA damage from a distance in a reaction that is sensitive to DNA mismatches and lesions. Here, DNA-mediated CT also leads to oxidation of a DNA-bound base excision repair enzyme, MutY. DNA-bound Ru(III), generated through a flash/quench technique, is found to promote oxidation of the [4Fe-4S]2+ cluster of MutY to [4Fe-4S]3+ and its decomposition product [3Fe-4S]1+. Flash/quench experiments monitored by EPR spectroscopy reveal spectra with g = 2.08, 2.06, and 2.02, characteristic of the oxidized clusters. Transient absorption spectra of poly(dGC) and [Ru(phen)2dppz]3+ (dppz = dipyridophenazine), generated in situ, show an absorption characteristic of the guanine radical that is depleted in the presence of MutY with formation instead of a long-lived species with an absorption at 405 nm; we attribute this absorption also to formation of the oxidized [4Fe-4S]3+ and [3Fe-4S]1+ clusters. In ruthenium-tethered DNA assemblies, oxidative damage to the 5′-G of a 5′-GG-3′ doublet is generated from a distance but this irreversible damage is inhibited by MutY and instead EPR experiments reveal cluster oxidation. With ruthenium-tethered assemblies containing duplex versus single-stranded regions, MutY oxidation is found to be mediated by the DNA duplex, with guanine radical as an intermediate oxidant; guanine radical formation facilitates MutY oxidation. A model is proposed for the redox activation of DNA repair proteins through DNA CT, with guanine radicals, the first product under oxidative stress, in oxidizing the DNA-bound repair proteins, providing the signal to stimulate DNA repair. PMID:15738421

  5. Differential Motion Between Mediastinal Lymph Nodes and Primary Tumor in Radically Irradiated Lung Cancer Patients

    SciTech Connect

    Schaake, Eva E.; Rossi, Maddalena M.G.; Buikhuisen, Wieneke A.; Burgers, Jacobus A.; Smit, Adrianus A.J.; Belderbos, José S.A.; Sonke, Jan-Jakob

    2014-11-15

    Purpose/Objective: In patients with locally advanced lung cancer, planning target volume margins for mediastinal lymph nodes and tumor after a correction protocol based on bony anatomy registration typically range from 1 to 1.5 cm. Detailed information about lymph node motion variability and differential motion with the primary tumor, however, is lacking from large series. In this study, lymph node and tumor position variability were analyzed in detail and correlated to the main carina to evaluate possible margin reduction. Methods and Materials: Small gold fiducial markers (0.35 × 5 mm) were placed in the mediastinal lymph nodes of 51 patients with non-small cell lung cancer during routine diagnostic esophageal or bronchial endoscopic ultrasonography. Four-dimensional (4D) planning computed tomographic (CT) and daily 4D cone beam (CB) CT scans were acquired before and during radical radiation therapy (66 Gy in 24 fractions). Each CBCT was registered in 3-dimensions (bony anatomy) and 4D (tumor, marker, and carina) to the planning CT scan. Subsequently, systematic and random residual misalignments of the time-averaged lymph node and tumor position relative to the bony anatomy and carina were determined. Additionally, tumor and lymph node respiratory amplitude variability was quantified. Finally, required margins were quantified by use of a recipe for dual targets. Results: Relative to the bony anatomy, systematic and random errors ranged from 0.16 to 0.32 cm for the markers and from 0.15 to 0.33 cm for the tumor, but despite similar ranges there was limited correlation (0.17-0.71) owing to differential motion. A large variability in lymph node amplitude between patients was observed, with an average motion of 0.56 cm in the cranial-caudal direction. Margins could be reduced by 10% (left-right), 27% (cranial-caudal), and 10% (anteroposterior) for the lymph nodes and −2%, 15%, and 7% for the tumor if an online carina registration protocol replaced a

  6. Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins.

    PubMed

    Medicherla, Balasubrahmanyam; Goldberg, Alfred L

    2008-08-25

    Accumulation of misfolded oxidant-damaged proteins is characteristic of many diseases and aging. To understand how cells handle postsynthetically damaged proteins, we studied in Saccharomyces cerevisiae the effects on overall protein degradation of shifting from 30 to 38 degrees C, exposure to reactive oxygen species generators (paraquat or cadmium), or lack of superoxide dismutases. Degradation rates of long-lived proteins (i.e., most cell proteins) were not affected by these insults, even when there was widespread oxidative damage to proteins. However, exposure to 38 degrees C, paraquat, cadmium, or deletion of SOD1 enhanced two- to threefold the degradation of newly synthesized proteins. By 1 h after synthesis, their degradation was not affected by these treatments. Degradation of these damaged cytosolic proteins requires the ubiquitin-proteasome pathway, including the E2s UBC4/UBC5, proteasomal subunit RPN10, and the CDC48-UfD1-NPL4 complex. In yeast lacking these components, the nondegraded polypeptides accumulate as aggregates. Thus, many cytosolic proteins proceed through a prolonged "fragile period" during which they are sensitive to degradation induced by superoxide radicals or increased temperatures.

  7. Reversible voltammograms and a Pourbaix diagram for a protein tyrosine radical

    PubMed Central

    Berry, Bruce W.; Martínez-Rivera, Melissa C.; Tommos, Cecilia

    2012-01-01

    Reversible voltammograms and a voltammetry half-wave potential versus solution pH diagram are described for a protein tyrosine radical. This work required a de novo designed tyrosine-radical protein displaying a unique combination of structural and electrochemical properties. The α3Y protein is structurally stable across a broad pH range. The redox-active tyrosine Y32 resides in a desolvated and well-structured environment. Y32 gives rise to reversible square-wave and differential pulse voltammograms at alkaline pH. The formal potential of the Y32-O•/Y32-OH redox couple is determined to 918 ± 2 mV versus the normal hydrogen electrode at pH 8.40 ± 0.01. The observation that Y32 gives rise to fully reversible voltammograms translates into an estimated lifetime of ≥30 ms for the Y32-O• state. This illustrates the range of tyrosine-radical stabilization that a structured protein can offer. Y32 gives rise to quasireversible square-wave and differential pulse voltammograms at acidic pH. These voltammograms represent the Y32 species at the upper edge of the quasirevesible range. The square-wave net potential closely approximates the formal potential of the Y32-O•/Y32-OH redox couple to 1,070 ± 1 mV versus the normal hydrogen electrode at pH 5.52 ± 0.01. The differential pulse voltammetry half-wave potential of the Y32-O•/Y32-OH redox pair is measured between pH 4.7 and 9.0. These results are described and analyzed. PMID:22675121

  8. Laser induced photoluminiscence studies of primary photochemical production processes of cometary radicals

    NASA Technical Reports Server (NTRS)

    Jackson, W. M.

    1977-01-01

    A tunable vacuum ultraviolet flash lamp was constructed. This unique flash lamp was coupled with a tunable dye laser detector and permits the experimenter to measure the production rates of ground state radicals as a function of wavelength. A new technique for producing fluorescent radicals was discovered. This technique called multiphoton ultraviolet photodissociation is currently being applied to several problems of both cometary and stratospheric interest. It was demonstrated that NO2 will dissociate to produce an excited fragment and the radiation can possibly be used for remote detection of this species.

  9. Oxidation of glyoxylic acid by cerium(IV): Oxygen-induced enhancement of the primary radical concentration

    SciTech Connect

    Neumann, B. ||; Steinbock, O.; Dalal, N.S. |; Mueller, S.C. |||

    1996-07-25

    In order to help understand the role of oxygen in Ce(IV)-induced oxidation of small carbonic acids, we investigated the reaction of glyoxylic acid (HCOCOOH) and Ce(IV) in 1 M sulfuric acid. Spectrophotometric data showed that in excess of glyoxylic acid the consumption of Ce(IV) obeys pseudo-first-order kinetics, with a rate constant of 8.8 L mol{sup -1} s{sup -1} at 25{degree}C and an activation energy of 80 kJ mol{sup -1}. Rapid-flow EPR measurements revealed an approximately 1:2:1 triplet with a g value of 2.0071{+-}0.0005 and a hyperfine splitting of 7.1{+-}0.2 G, assignable to the primary radical formed by abstraction of a hydrogen atom from hydrated glyoxylic acid. The rate constant for the anaerobic self-decay of the radical was measured as approximately 3.7x10{sup 9} L mol{sup -1} s{sup -1}. Surprisingly, oxygen had no effect on the Ce(IV) kinetics, while the radical decay was significantly inhibited under aerobic conditions (ratio of experimental rate constants = 6.3). Amperometric measurements revealed accompanying oxygen consumption. Analyses based on numerical simulations show that the observed oxygen-induced increase in radical concentration cannot be explained in the framework of standard autooxidation mechanisms. An alternative reaction scheme is suggested which reproduces the observed aerobic radical kinetics and which thus could be relevant to similar oxidation reactions. 30 refs., 7 figs., 2 tabs.

  10. Hypochlorite-induced oxidation of proteins in plasma: formation of chloramines and nitrogen-centred radicals and their role in protein fragmentation.

    PubMed

    Hawkins, C L; Davies, M J

    1999-06-01

    Activated phagocyte cells generate hypochlorite (HOCl) via the release of H2O2 and the enzyme myeloperoxidase. Plasma proteins are major targets for HOCl, although little information is available about the mechanism(s) of oxidation. In this study the reaction of HOCl (at least 50 microM) with diluted fresh human plasma has been shown to generate material that oxidizes 5-thio-2-nitrobenzoic acid; these oxidants are believed to be chloramines formed from the reaction of HOCl with protein amine groups. Chloramines have also been detected with isolated plasma proteins treated with HOCl. In both cases chloramine formation accounts for approx. 20-30% of the added HOCl. These chloramines decompose in a time-dependent manner when incubated at 20 or 37 degrees C but not at 4 degrees C. Ascorbate and urate remove these chloramines in a time- and concentration-dependent manner, with the former being more efficient. The reaction of fresh diluted plasma with HOCl also gives rise to protein-derived nitrogen-centred radicals in a time- and HOCl-concentration-dependent manner; these have been detected by EPR spin trapping. Identical radicals have been detected with isolated HOCl-treated plasma proteins. Radical formation was inhibited by excess methionine, implicating protein-derived chloramines (probably from lysine side chains) as the radical source. Plasma protein fragmentation occurs in a time- and HOCl-concentration-dependent manner, as evidenced by the increased mobility of the EPR spin adducts, the detection of further radical species believed to be intermediates in protein degradation and the loss of the parent protein bands on SDS/PAGE. Fragmentation can be inhibited by methionine and other agents (ascorbate, urate, Trolox C or GSH) capable of removing chloramines and reactive radicals. These results are consistent with protein-derived chloramines, and the radicals derived from them, as contributing agents in HOCl-induced plasma protein oxidation.

  11. Hypochlorite-induced oxidation of proteins in plasma: formation of chloramines and nitrogen-centred radicals and their role in protein fragmentation.

    PubMed Central

    Hawkins, C L; Davies, M J

    1999-01-01

    Activated phagocyte cells generate hypochlorite (HOCl) via the release of H2O2 and the enzyme myeloperoxidase. Plasma proteins are major targets for HOCl, although little information is available about the mechanism(s) of oxidation. In this study the reaction of HOCl (at least 50 microM) with diluted fresh human plasma has been shown to generate material that oxidizes 5-thio-2-nitrobenzoic acid; these oxidants are believed to be chloramines formed from the reaction of HOCl with protein amine groups. Chloramines have also been detected with isolated plasma proteins treated with HOCl. In both cases chloramine formation accounts for approx. 20-30% of the added HOCl. These chloramines decompose in a time-dependent manner when incubated at 20 or 37 degrees C but not at 4 degrees C. Ascorbate and urate remove these chloramines in a time- and concentration-dependent manner, with the former being more efficient. The reaction of fresh diluted plasma with HOCl also gives rise to protein-derived nitrogen-centred radicals in a time- and HOCl-concentration-dependent manner; these have been detected by EPR spin trapping. Identical radicals have been detected with isolated HOCl-treated plasma proteins. Radical formation was inhibited by excess methionine, implicating protein-derived chloramines (probably from lysine side chains) as the radical source. Plasma protein fragmentation occurs in a time- and HOCl-concentration-dependent manner, as evidenced by the increased mobility of the EPR spin adducts, the detection of further radical species believed to be intermediates in protein degradation and the loss of the parent protein bands on SDS/PAGE. Fragmentation can be inhibited by methionine and other agents (ascorbate, urate, Trolox C or GSH) capable of removing chloramines and reactive radicals. These results are consistent with protein-derived chloramines, and the radicals derived from them, as contributing agents in HOCl-induced plasma protein oxidation. PMID:10333500

  12. Laparoscopic radical hysterectomy with vaginectomy and reconstruction of vagina in patients with stage I of primary vaginal carcinoma.

    PubMed

    Ling, Bin; Gao, Zongxia; Sun, Minwen; Sun, Fanglin; Zhang, Aijun; Zhao, Weidong; Hu, Weiping

    2008-04-01

    The purpose of this study was to retrospectively evaluate the technique, feasibility and oncological safety of laparoscopic radical hysterectomy with vaginectomy and reconstruction of vagina in patients with stage I primary vaginal carcinomas. Between February 2003 and July 2004, four patients, that had needs of sexual life, aging from 41 to 61 years with stage I primary vaginal carcinoma located at the upper third or 2/3 of the vagina, were submitted to laparoscopic radical hysterectomy with vaginectomy and reconstruction of the vagina using the sigmoid colon. The average operative time was 305 min (range 260-350 min). The average estimated blood loss was 325 ml (range 250-400 ml), and the medial number of the lymph nodes removed was 16 (range 13-20). All surgical margins and nodes removed were negative histopathologically. There were no intra-operative and postoperative complications. The mean stay day after surgery was 7 days (range 6-8 days). The mean length of a neo-vagina was 13 cm (range 12-15 cm) and the introitus admitted two fingers in breadth. The mean follow-up was 46 months (range 40-54 months). All patients are clinically free of disease and have satisfactory sexual life. None require dilation of the introitus. During the first 6 months, all the patients had little complaints of excessive leucorrhoea. To our knowledge, this is the first reported laparoscopical radical surgery combined with reconstruction of the vagina in patients with early-stage primary vaginal cancer. Our results have demonstrated the oncological safety and feasibility of the laparoscopical procedure. Intermediate-term follow-up validates the adequacy of this procedure.

  13. Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis.

    PubMed

    Stadler, Krisztian; Bonini, Marcelo G; Dallas, Shannon; Duma, Danielle; Mason, Ronald P; Kadiiska, Maria B

    2008-08-01

    Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression.

  14. Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis

    PubMed Central

    Stadler, Krisztian; Bonini, Marcelo G.; Dallas, Shannon; Duma, Danielle; Mason, Ronald P.; Kadiiska, Maria B.

    2008-01-01

    Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the α-(4-pyridyl-1-oxide)-N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression. PMID:18559982

  15. Effect of Soy Protein Isolate Supplementation on Biochemical Recurrence of Prostate Cancer After Radical Prostatectomy

    PubMed Central

    Bosland, Maarten C; Kato, Ikuko; Zeleniuch-Jacquotte, Anne; Schmoll, Joanne; Rueter, Erika Enk; Melamed, Jonathan; Xiangtian Kong, Max; Macias, Virgilia; Kajdacsy-Balla, Andre; Lumey, L. H.; Xie, Hui; Gao, Weihua; Walden, Paul; Lepor, Herbert; Taneja, Samir S.; Randolph, Carla; Schlicht, Michael J.; Meserve-Watanabe, Hiroko; Deaton, Ryan J.; Davies, Joanne A.

    2013-01-01

    IMPORTANCE Soy consumption has been suggested to reduce risk or recurrence of prostate cancer, but this has not been tested in a randomized trial with prostate cancer as the end point. OBJECTIVE To determine whether daily consumption of a soy protein isolate supplement for 2 years reduces the rate of biochemical recurrence of prostate cancer after radical prostatectomy or delays such recurrence. DESIGN, SETTING, AND PARTICIPANTS Randomized, double-blind trial conducted from July 1997 to May 2010 at 7 US centers comparing daily consumption of a soy protein supplement vs placebo in 177 men at high risk of recurrence after radical prostatectomy for prostate cancer. Supplement intervention was started within 4 months after surgery and continued for up to 2 years, with prostate-specific antigen (PSA) measurements made at 2-month intervals in the first year and every 3 months thereafter. INTERVENTION Participants were randomized to receive a daily serving of a beverage powder containing 20 g of protein in the form of either soy protein isolate (n=87)or, as placebo, calcium caseinate (n=90). MAIN OUTCOMES AND MEASURES Biochemical recurrence rate of prostate cancer (defined as development of a PSA level of ≥0.07 ng/mL) over the first 2 years following randomization and time to recurrence. RESULTS The trial was stopped early for lack of treatment effects at a planned interim analysis with 81 evaluable participants in the intervention group and 78 in the placebo group. Overall, 28.3% of participants developed biochemical recurrence within 2 years of entering the trial (close to the a priori predicted recurrence rate of 30%). Among these, 22 (27.2%) occurred in the intervention group and 23 (29.5%) in the placebo group. The resulting hazard ratio for active treatment was 0.96 (95% CI, 0.53–1.72; log-rank P = .89). Adherence was greater than 90% and there were no apparent adverse events related to supplementation. CONCLUSION AND RELEVANCE Daily consumption of a beverage

  16. High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface*

    PubMed Central

    Li, Zixuan; Moniz, Heather; Wang, Shuo; Ramiah, Annapoorani; Zhang, Fuming; Moremen, Kelley W.; Linhardt, Robert J.; Sharp, Joshua S.

    2015-01-01

    Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes. PMID:25752613

  17. High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface.

    PubMed

    Li, Zixuan; Moniz, Heather; Wang, Shuo; Ramiah, Annapoorani; Zhang, Fuming; Moremen, Kelley W; Linhardt, Robert J; Sharp, Joshua S

    2015-04-24

    Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes.

  18. Supercharging by m-NBA Improves ETD-Based Quantification of Hydroxyl Radical Protein Footprinting

    PubMed Central

    Li, Xiaoyan; Li, Zixuan; Xie, Boer; Sharp, Joshua S.

    2015-01-01

    Hydroxyl radical protein footprinting (HRPF) is an MS-based technique for analyzing protein structure based on measuring the oxidation of amino acid side chains by hydroxyl radicals diffusing in solution. Spatial resolution of HRPF is limited by the smallest portion of the protein for which oxidation amounts can be accurately quantitated. Previous work has shown electron transfer dissociation (ETD) to be the most reliable method for quantifying the amount of oxidation of each amino acid side chain in a mixture of peptide oxidation isomers, but efficient ETD requires high peptide charge states which limits its applicability for HRPF. Supercharging reagents have been used to enhance peptide charge state for ETD analysis, but previous work have shown supercharging reagents to enhance charge state differently for different peptides sequences; it is currently unknown if different oxidation isomers will experience different charge enhancement effects. Here, we report the effect of m-nitrobenzyl alcohol (m-NBA) on the ETD-based quantification of peptide oxidation. The addition of m-NBA to both a defined mixture of synthetic isomeric oxidized peptides and Robo1 protein subjected to HRPF increased the abundance of higher charge state ions, improving our ability to perform efficient ETD of the mixture. No differences in the reported quantitation by ETD were noted in the presence or absence of m-NBA, indicating that all oxidation isomers were charge-enhanced to a similar extent. These results indicate the utility of m-NBA for residue-level quantification of peptide oxidation in HRPF and other applications. PMID:25916598

  19. Supercharging by m-NBA Improves ETD-Based Quantification of Hydroxyl Radical Protein Footprinting.

    PubMed

    Li, Xiaoyan; Li, Zixuan; Xie, Boer; Sharp, Joshua S

    2015-08-01

    Hydroxyl radical protein footprinting (HRPF) is an MS-based technique for analyzing protein structure based on measuring the oxidation of amino acid side chains by hydroxyl radicals diffusing in solution. Spatial resolution of HRPF is limited by the smallest portion of the protein for which oxidation amounts can be accurately quantitated. Previous work has shown electron transfer dissociation (ETD) to be the most reliable method for quantifying the amount of oxidation of each amino acid side chain in a mixture of peptide oxidation isomers, but efficient ETD requires high peptide charge states, which limits its applicability for HRPF. Supercharging reagents have been used to enhance peptide charge state for ETD analysis, but previous work has shown supercharging reagents to enhance charge state differently for different peptides sequences; it is currently unknown if different oxidation isomers will experience different charge enhancement effects. Here, we report the effect of m-nitrobenzyl alcohol (m-NBA) on the ETD-based quantification of peptide oxidation. The addition of m-NBA to both a defined mixture of synthetic isomeric oxidized peptides and Robo-1 protein subjected to HRPF increased the abundance of higher charge state ions, improving our ability to perform efficient ETD of the mixture. No differences in the reported quantitation by ETD were noted in the presence or absence of m-NBA, indicating that all oxidation isomers were charge-enhanced to a similar extent. These results indicate the utility of m-NBA for residue-level quantification of peptide oxidation in HRPF and other applications.

  20. Supercharging by m-NBA Improves ETD-Based Quantification of Hydroxyl Radical Protein Footprinting

    NASA Astrophysics Data System (ADS)

    Li, Xiaoyan; Li, Zixuan; Xie, Boer; Sharp, Joshua S.

    2015-08-01

    Hydroxyl radical protein footprinting (HRPF) is an MS-based technique for analyzing protein structure based on measuring the oxidation of amino acid side chains by hydroxyl radicals diffusing in solution. Spatial resolution of HRPF is limited by the smallest portion of the protein for which oxidation amounts can be accurately quantitated. Previous work has shown electron transfer dissociation (ETD) to be the most reliable method for quantifying the amount of oxidation of each amino acid side chain in a mixture of peptide oxidation isomers, but efficient ETD requires high peptide charge states, which limits its applicability for HRPF. Supercharging reagents have been used to enhance peptide charge state for ETD analysis, but previous work has shown supercharging reagents to enhance charge state differently for different peptides sequences; it is currently unknown if different oxidation isomers will experience different charge enhancement effects. Here, we report the effect of m-nitrobenzyl alcohol ( m-NBA) on the ETD-based quantification of peptide oxidation. The addition of m-NBA to both a defined mixture of synthetic isomeric oxidized peptides and Robo-1 protein subjected to HRPF increased the abundance of higher charge state ions, improving our ability to perform efficient ETD of the mixture. No differences in the reported quantitation by ETD were noted in the presence or absence of m-NBA, indicating that all oxidation isomers were charge-enhanced to a similar extent. These results indicate the utility of m-NBA for residue-level quantification of peptide oxidation in HRPF and other applications.

  1. [The inhibitor of free radical processes decrease of protein biosynthesis in gun short wound tissues and weaken development of the general adaptation syndrome].

    PubMed

    Todorov, I N; Bogdanov, G N; Mitrokhin, Iu I; Varfolomeev, V N; Sidorenko, L I; Mishchenko, D V

    2006-01-01

    The dynamics of total protein biosynthesis and procollagen biosynthesis in skeletal muscle of injury tissues with the antioxidant BHT (dibunol) treatment and with common healing were studied. The obtained date indicate that the AO treatment reduce the rate of biosynthesis both the total proteins and procollagen at the 3th day of healing. Dibunol also considerably reduce the protein biosynthesis in adrenals and brake of corticosteroids biogenesis as measured by ESR-signals intensity of reduced adrenodoxine. AO treatment also reduce the protein biosynthesis in thymus, spleen and bone marrow. The lowering of functional activity of endocrine and immune systems indicate that the AO significantly inhibit the systemic reactions of organism induced by acute wound affect. It was suggested that as "primary mediator" of stress-reaction may be considered lipoperoxide radicals and decay products of lipohydroperoide.

  2. Immuno-spin trapping of protein and DNA radicals: “tagging” free radicals to locate and understand the redox process

    PubMed Central

    Gomez-Mejiba, Sandra E.; Zhai, Zili; Akram, Hammad; Deterding, Leesa J.; Hensley, Kenneth; Smith, Nataliya; Towner, Rheal A.; Tomer, Kenneth B.; Mason, Ronald P.; Ramirez, Dario C.

    2009-01-01

    Biomolecule-centered radicals are intermediate species produced during both reversible (redox modulation) and irreversible (oxidative stress) oxidative modification of biomolecules. These oxidative processes must be studied in situ and in real time in order to understand the molecular mechanism of cell adaptation or death in response to changes in the extracellular environment. In this regard, we have developed and validated immuno-spin trapping to tag the redox process, tracing the oxidatively-generated modification of biomolecules, in situ and in real time, by detecting protein- and DNA-centered radicals. The purpose of this method article is to introduce and update the basic methods and applications of immuno-spin trapping for the study of redox biochemistry in oxidative stress and redox regulation. We describe in detail the production, detection and location of protein and DNA radicals in biochemical systems, cells, and tissues, and in the whole animal as well, by using immuno-spin trapping with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). PMID:19159679

  3. Survival and prognostic factors comparing stage IB 1 versus stage IB 2 cervical cancer treated with primary radical hysterectomy.

    PubMed

    Srisomboon, Jatupol; Kietpeerakool, Chumnan; Suprasert, Prapaporn; Manopanya, Manatsawee; Siriaree, Sitthicha; Charoenkwan, Kittipat; Cheewakriangkrai, Chalong; Sae-Teng, Charuwan

    2011-01-01

    This study was undertaken to compare the survival rates of stage IB 1 versus stage IB 2 cervical cancer patients and to evaluate the prognostic factors after treatment primarily with radical hysterectomy and pelvic lymphadenectomy (RHPL). Patients with stage IB cervical cancer undergoing primary RHPL at Chiang Mai University Hospital between January 2002 and December 2009 were evaluated for survival and recurrence. Clinicopathological variables were analyzed to identify the prognostic factors affecting the survival of the patients. During the study period, RHPL was performed on 570 stage IB 1 and 110 stage IB 2 cervical cancer patients. With a median follow-up of 48 months, the 5-year disease-free survivals were 98.1% and 82.8% respectively (p<0.001). Multivariate analysis identified four significant prognostic factors affecting survival including sub-staging, non-squamous cell carcinoma histology, lymph node metastasis and the presence of lymph-vascular space invasion. In conclusion, with a primary radical hysterectomy, stage IB 1 cervical cancer patients have a significantly better survival rate than those with stage IB 2. Significant prognostic factors for stage IB cervical cancer include tumor histology, nodal status, and the presence of lymph-vascular space invasion.

  4. Primary maxillary reconstruction after radical maxillectomy using a combined free flap and secondary dynamic suspension.

    PubMed

    Yoza, S; Gunji, H; Ono, I

    1997-01-01

    We report a case of maxillary reconstruction aimed at optimizing facial contour, oral function, and facial animation after radical maxillectomy. In the first stage, using combined latissimus dorsi serratus anterior flaps attached with ribs, we performed a three-dimensional restoration for midface defects. In the second stage, we performed dynamic reconstruction of the nasolabial fold and upper lip using a temporal muscle transposition. As a result, the upper lip was able to be elevated as much as 10 mm when the patient clenched his teeth, and the outcome was judged to be both aesthetically and functionally satisfactory.

  5. Changes in structural characteristics of antioxidative soy protein hydrolysates resulting from scavenging of hydroxyl radicals.

    PubMed

    Zhao, Jing; Xiong, Youling L; McNear, Dave H

    2013-02-01

    Antioxidant activity of soy protein (SP) and its hydrolyzed peptides has been widely reported. During scavenging of radicals, these antioxidative compounds would be oxidatively modified, but their fate is not understood. The objective of this study was to evaluate the structural characteristics of SP hydrolysates (SPHs), compared to intact SP, when used to neutralize hydroxyl radicals (•OH). SPHs with degree of hydrolysis (DH) 1 to 5 were prepared with Alcalase. Antioxidant activity of SPHs was confirmed by lipid oxidation inhibition measured with thiobarbituric acid-reactive substances, ability to scavenge 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radicals, and ferrous ion chelation capability. Oxidation of SPHs was initiated by reaction with •OH generated from 0.1 mM FeCl(3) , 20 mM H(2) O(2) , and 1.0 mM ascorbate. After oxidative stress, carbonyl content of SPHs increased by 2- to 3-fold and sulfhydryl groups decreased by up to 42% compared to nonoxidized samples (P < 0.05). Methionine, histidine, and lysine residues were significantly reduced as a result of inactivating •OH (P < 0.05). Attenuated total reflectance-Fourier transform infrared and circular dichroism spectroscopy suggested the conversion of helical structure to strands and turns. Oxidatively modified SPHs had a lower intrinsic fluorescence intensity but similar solubility when compared to nonoxidized samples. These structural changes due to •OH stress may impact the ingredient interaction and functionality of SPHs in food products. © 2013 Institute of Food Technologists®

  6. Oxygen radical induced fluorescence in proteins; identification of the fluorescent tryptophan metabolite, N-formyl kynurenine, as a biological index of radical damage.

    PubMed

    Griffiths, H R; Lunec, J; Blake, D R

    1992-06-01

    The effect of oxygen derived free radicals (OFR) on aromatic and sulphur containing amino acids has been investigated, both in their free form and within protein backbones. Aerated amino acids and proteins in solution were exposed to three discrete OFR generating systems; (1) gamma radiation in the presence or absence of formate (2) photolysis by UV light at 254 and 366 nm, and (3) site specific modification by H2O2 in the presence of CuII ions.A sensitive reverse-phase HPLC technique with dual detection systems (UV absorbance and fluorescence monitoring) was developed to analyse the products of amino acid oxidation. OFR denatured amino acids were chromatographed by this procedure, and all radical species generated, with the exception of the superoxide anion, resulted in the formation of identifiable fluorescent metabolites of tryptophan, kynurenines. The identity of peaks was confimed by spiking with authentic material and scanning absorption spectroscopy. After complete proteolytic hydrolysis, OFR treated proteins were also analysed by this technique; again the dose dependent production of kynurenines was detected in IgG,γ lens crystallins and albumin. Bityrosine was not detected in any of the proteins studied using this procedure, however, several novel unidentified fluorophores were detected in proteolytic hydrolysates, possibly the product of two different amino acid radicals.Immunoglobulin G isolated from the sera of normals and rheumatoid arthritis (RA) patients was examined for the presence of one specific tryptophan metabolite, N-formyl kynurenine. Significantly elevated levels of this metabolite were detected in rheumatoid sera, suggesting increased OFR activity in RA.These results have demonstrated firstly, that specific oxidised products of amino acids are retained in the protein backbone after exposure to OFR generating systems. Secondly, in aerated solution, oxidised tryptophan residues confer the major new visible fluorescence in non-haem proteins

  7. Genome Mining for Radical SAM Protein Determinants Reveals Multiple Sactibiotic-Like Gene Clusters

    PubMed Central

    Murphy, Kiera; O'Sullivan, Orla; Rea, Mary C.; Cotter, Paul D.; Ross, R. Paul; Hill, Colin

    2011-01-01

    Thuricin CD is a two-component bacteriocin produced by Bacillus thuringiensis that kills a wide range of clinically significant Clostridium difficile. This bacteriocin has recently been characterized and consists of two distinct peptides, Trnβ and Trnα, which both possess 3 intrapeptide sulphur to α-carbon bridges and act synergistically. Indeed, thuricin CD and subtilosin A are the only antimicrobials known to possess these unusual structures and are known as the sactibiotics (sulplur to alpha carbon-containing antibiotics). Analysis of the thuricin CD-associated gene cluster revealed the presence of genes encoding two highly unusual SAM proteins (TrnC and TrnD) which are proposed to be responsible for these unusual post-translational modifications. On the basis of the frequently high conservation among enzymes responsible for the post-translational modification of specific antimicrobials, we performed an in silico screen for novel thuricin CD–like gene clusters using the TrnC and TrnD radical SAM proteins as driver sequences to perform an initial homology search against the complete non-redundant database. Fifteen novel thuricin CD–like gene clusters were identified, based on the presence of TrnC and TrnD homologues in the context of neighbouring genes encoding potential bacteriocin structural peptides. Moreover, metagenomic analysis revealed that TrnC or TrnD homologs are present in a variety of metagenomic environments, suggesting a widespread distribution of thuricin-like operons in a variety of environments. In-silico analysis of radical SAM proteins is sufficient to identify novel putative sactibiotic clusters. PMID:21760885

  8. On-plate deposition of oxidized proteins to facilitate protein footprinting studies by radical probe mass spectrometry.

    PubMed

    Maleknia, Simin D; Downard, Kevin M

    2012-10-15

    The on-plate deposition of oxidized proteins is described to advance footprinting applications by radical probe mass spectrometry (RP-MS). An electrospray ionization (ESI) needle assembly mounted vertically over a 384-target matrix-assisted laser desorption/ionization (MALDI) plate enabled the limited oxidation of proteins as they were released in the charged droplets ahead of their deposition on the plate. This method combined with on-plate proteolytic digestion protocols expedites the analysis of proteins oxidized by RP-MS, and avoids the need to collect and reconstitute samples prior to analysis by MALDI mass spectrometry. Oxidation of peptides from solutions in water as well as an ammonium bicarbonate solution was investigated to test the optimal conditions required for on-plate oxidation of proteins. These comprised of peptides with a wide range of reactive amino acids including Phe, Tyr, Pro, His, Leu, Met and Lys that were previously shown to oxidize in both electrospray discharge and synchrotron radiolysis based footprinting experiments. The on-plate deposition of lysozyme oxidized at electrospray needle voltages of 6 and 9 kV were carried out to demonstrate conditions suitable for footprinting experiments as well as those that induce the onset of protein damage.

  9. Improved Identification and Relative Quantification of Sites of Peptide and Protein Oxidation for Hydroxyl Radical Footprinting

    NASA Astrophysics Data System (ADS)

    Li, Xiaoyan; Li, Zixuan; Xie, Boer; Sharp, Joshua S.

    2013-11-01

    Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein-ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric "oxidized" peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

  10. Involvement of protein kinase C, phospholipase C, and protein tyrosine kinase pathways in oxygen radical generation by asbestos-stimulated alveolar macrophage.

    PubMed

    Lim, Y; Kim, S H; Kim, K A; Oh, M W; Lee, K H

    1997-09-01

    Although asbestos stimulates oxygen radical generation in alveolar macrophages, the exact mechanism is still not clear. The purpose of this study was to compare the ability of three asbestos fibers (amosite, chrysotile, and crocidolite) to generate oxygen radicals in macrophages and examine the mechanism of this action. All asbestos fibers were able to induce chemiluminescence but chrysotile induced maximal chemiluminescence at higher concentrations than amosite and crocidolite. Protein kinase C (PKC) inhibitors (sphingosine and staurosporine) suppressed the ability of asbestos to induce oxygen radical generation. Phospholipase C (PLC) inhibitors (U73122 and neomycin) and protein tyrosine kinase (PTK) inhibitors (erbstatin and genistein) decreased oxygen radical generation of asbestos-stimulated alveolar macrophages. Oxygen radical generation was not suppressed by an adenylate cyclase activator (forskolin), a protein kinase A inhibitor (H-8), and a protein serine-threonine phosphatase inhibitor (okadaic acid). PLC and PTK inhibitors suppressed the increment of phosphoinositide turnover by amosite. These results suggest that asbestos fibers induce the generation of oxygen radicals through PTK, PLC, and PKC pathways in a dose-response pattern.

  11. Elucidating in Vivo Structural Dynamics in Integral Membrane Protein by Hydroxyl Radical Footprinting*

    PubMed Central

    Zhu, Yi; Guo, Tiannan; Park, Jung Eun; Li, Xin; Meng, Wei; Datta, Arnab; Bern, Marshall; Lim, Sai Kiang; Sze, Siu Kwan

    2009-01-01

    We describe here a novel footprinting technique to probe the in vivo structural dynamics of membrane protein. This method utilized in situ generation of hydroxyl radicals to oxidize and covalently modify biomolecules on living Escherichia coli cell surface. After enriching and purifying the membrane proteome, the modified amino acid residues of the protein were identified with tandem mass spectrometry to map the solvent-accessible surface of the protein that will form the footprint of in vivo structure of the protein. Of about 100 outer membrane proteins identified, we investigated the structure details of a typical β-barrel structure, the porin OmpF. We found that six modified tryptic peptides of OmpF were reproducibly detected with 19 amino acids modified under the physiological condition. The modified amino acid residues were widely distributed in the external loop area, β-strands, and periplasmic turning area, and all of them were validated as solvent-accessible according to the crystallography data. We further extended this method to study the dynamics of the voltage gating of OmpF in vivo using mimic changes of physiological circumstance either by pH or by ionic strength. Our data showed the voltage gating of porin OmpF in vivo for the first time and supported the proposed mechanism that the local electrostatic field changes in the eyelet region may alter the porin channels to switch. Thus, this novel method can be a potentially efficient method to study the structural dynamics of the membrane proteins of a living cell. PMID:19473960

  12. Combined radical radiation therapy and chemotherapy for primary squamous cell carcinoma of the anal canal

    SciTech Connect

    Cummings, B.J.; Rider, W.D.; Harwood, A.R.; Keane, T.J.; Thomas, G.M.; Erlichman, C.; Fine, S.

    1982-03-01

    Radical radiation therapy (5000 rads in 20 fractions in 4 weeks) combined with iv mitomycin (10 mg/m2) and 5-FU (1000 mg/m2/24 hours for 4 days) was used to treat 13 patients with locally advanced but operable squamous cell carcinoma of the anal canal. All patients achieved local control and retained anal continence, and none developed metastases. The patients were followed from 4 to 34 months (median, 12). Severe acute gastrointestinal toxic effects were seen in three patients; the same patients had significant thrombocytopenia or leukopenia. Treatment with this combined program may allow conservative management of squamous cell carcinoma of the anal canal and should be considered as an alternative to abdominoperineal resection.

  13. Effect of antifreeze proteins on frozen primary prostatic adenocarcinoma cells.

    PubMed

    Koushafar, H; Rubinsky, B

    1997-03-01

    Recent studies show that prostate adenocarcinoma cells can survive cryosurgery and that cell destruction depends on the specific thermal parameters used during freezing. The goal of this preliminary study is to determine whether certain chemical compounds, known as antifreeze proteins, can induce complete human primary prostatic adenocarcinoma cell destruction by freezing, regardless of the thermal parameters used. The study also examines the mechanism by which antifreeze proteins bring about cell destruction. Antifreeze proteins were added to solutions containing human primary prostatic adenocarcinoma cells. The cells were frozen with controlled thermal parameters using a directional solidification apparatus attached to a light microscope. Cell viability was determined after thawing as a function of antifreeze protein concentration and cooling rate during freezing. The dose response study shows that for all the cooling rates tested, 10-mg/mL solutions of antifreeze protein cause the complete destruction of human primary prostatic adenocarcinoma cells frozen to a temperature at which, without these proteins, the cells survive freezing. Light microscopy shows that the lethal effect of the antifreeze proteins is related to the formation of intracellular ice in the frozen cells. CONCLUSIONS; This preliminary study has demonstrated that antifreeze proteins have the ability to generate complete destruction of prostatic adenocarcinoma cells frozen to high subzero temperatures irrespective of the cooling rates used during freezing. This suggests that introducing antifreeze proteins into undesirable tissues prior to freezing may increase the efficacy and the control over tissue destruction by cryosurgery.

  14. Modulation of Protein Fragmentation Through Carbamylation of Primary Amines

    NASA Astrophysics Data System (ADS)

    Greer, Sylvester M.; Holden, Dustin D.; Fellers, Ryan; Kelleher, Neil L.; Brodbelt, Jennifer S.

    2017-08-01

    We evaluate the impact of carbamylation of the primary amines of the side-chains of Lys and the N-termini on the fragmentation of intact protein ions and the chromatographic properties of a mixture of E. coli ribosomal proteins. The fragmentation patterns of the six unmodified and carbamylated proteins obtained by higher energy collision dissociation (HCD) and ultraviolet photodissociation (UVPD) were compared. Carbamylation significantly reduced the total number of protons retained by the protein owing to the conversion of basic primary amines to non-basic carbamates. Carbamylation caused a significant negative impact on fragmentation of the protein by HCD (i.e., reduced sequence coverage and fewer diagnostic fragment ions) consistent with the mobile proton model, which correlates peptide fragmentation with charge distribution and the opportunity for charge-directed pathways. In addition, fragmentation was enhanced near the N- and C-termini upon HCD of carbamylated proteins. For LCMS/MS analysis of E. coli ribosomal proteins, the retention times increased by 16 min on average upon carbamylation, an outcome attributed to the increased hydrophobicity of the proteins after carbamylation. As noted for both the six model proteins and the ribosomal proteins, carbamylation had relatively little impact on the distribution or types of fragment ions product by UVPD, supporting the proposition that the mechanism of UVPD for intact proteins does not reflect the mobile proton model.

  15. Antioxidants, free radicals, storage proteins, puroindolines, and proteolytic activities in bread wheat (Triticum aestivum) seeds during accelerated aging.

    PubMed

    Calucci, Lucia; Capocchi, Antonella; Galleschi, Luciano; Ghiringhelli, Silvia; Pinzino, Calogero; Saviozzi, Franco; Zandomeneghi, Maurizio

    2004-06-30

    Seeds of bread wheat were incubated at 40 degrees C and 100% relative humidity for 0, 3, 4, 6, and 10 days. The effects of accelerated aging on seed germinability and some biochemical properties of flour (carotenoid, free radical, and protein contents and proteolytic activity) and gluten (free radical content and flexibility) were investigated. Seed germinability decreased during aging, resulting in seed death after 10 days. A progressive decrease of carotenoid content, in particular, lutein, was observed, prolonging the incubation, whereas the free radical content increased in both flour and gluten. A degradation of soluble and storage proteins was found, associated with a marked increase of proteolytic activity and a loss of viscoelastic properties of gluten. On the contrary, puroindolines were quite resistant to the treatment. The results are discussed in comparison with those previously obtained during accelerated aging of durum wheat seeds.

  16. INVESTIGATION OF THE RADICAL-MEDIATED PRODUCTION OF BENZENE OXIDE PROTEIN ADDUCTS IN VITRO AND IN VIVO

    EPA Science Inventory

    High background levels of benzene oxide (BO) adducts with hemoglobin and albumin (BO-Hb and BO-Alb) have been measured in unexposed humans and animals. To test the influence of radical-mediated pathways on production of these BO-protein adducts, we employed Fenton chemistry to...

  17. INVESTIGATION OF THE RADICAL-MEDIATED PRODUCTION OF BENZENE OXIDE PROTEIN ADDUCTS IN VITRO AND IN VIVO

    EPA Science Inventory

    High background levels of benzene oxide (BO) adducts with hemoglobin and albumin (BO-Hb and BO-Alb) have been measured in unexposed humans and animals. To test the influence of radical-mediated pathways on production of these BO-protein adducts, we employed Fenton chemistry to...

  18. Trends and Outcome from Radical Therapy for Primary Non-Metastatic Prostate Cancer in a UK Population

    PubMed Central

    Wright, Karen A.; Muir, Kenneth R.; Gnanapragasam, Vincent J.

    2015-01-01

    Background Increasing proportions of men diagnosed with prostate cancer in the UK are presenting with non-metastatic disease. We investigated how treatment trends in this demographic have changed. Patient and Methods Non-metastatic cancers diagnosed from 2000–2010 in the UK Anglian Cancer network stratified by age and risk group were analysed [n = 10,365]. Radiotherapy [RT] and prostatectomy [RP] cancer specific survival [CSS] were further compared [n = 4755]. Results Over the decade we observed a fall in uptake of primary androgen deprivation therapy but a rise in conservative management [CM] and radical therapy [p<0.0001]. CM in particular has become the primary management for low-risk disease by the decade end [p<0.0001]. In high-risk disease however both RP and RT uptake increased significantly but in an age dependent manner [p<0.0001]. Principally, increased RP in younger men and increased RT in men ≥ 70y. In multivariate analysis of radically treated men both high-risk disease [HR 8.0 [2.9–22.2], p<0.0001] and use of RT [HR 1.9 [1.0–3.3], p = 0.024] were significant predictors of a poorer CSM. In age-stratified analysis however, the trend to benefit of RP over RT was seen only in younger men [≤ 60 years] with high-risk disease [p = 0.07]. The numbers needed to treat by RP instead of RT to save one cancer death was 19 for this group but 67 for the overall cohort. Conclusion This study has identified significant shifts in non-metastatic prostate cancer management over the last decade. Low-risk disease is now primarily managed by CM while high-risk disease is increasingly treated radically. Treatment of high-risk younger men by RP is supported by evidence of better CSM but this benefit is not evident in older men. PMID:25742020

  19. Features of the immunohistochemical characteristics of primary tumors and recurrences of breast cancer after radical treatment.

    PubMed

    Prystash, Yurij Y

    Appearance of Recurrence (RC) of breast cancer (BC) is associated with a high risk of distant metastases, needs re-treatment and indicates the tumor aggressiveness. It has been remained unclear the molecular characteristics both of the RC and primary tumors in patients with invasive forms of breast cancer after mastectomy by Madden. To explore the changing of the receptor status of the primary tumor and local RC in patients with breast cancer. Immunohistochemical study were conducted on 262 patients with invasive breast cancer. Patients were divided into two groups: only local RC - 131 women and primary tumors of patients without local RC - also 131 persons. The difference of the receptor status of the tumors is presented. In the group of patients with recurrent "triplet negative" cancer and patients with positive reaction of epidermal growth factor (HER2neo) is more than 15.2%. In patients where RC (control group in the study) was not observed we have the mass greater proportion of tumors with positive hormone receptors in various combinations. Relapses are accompanied by lower levels of hormone receptors and increasing the frequency of "triplet negative" cancer as well as increasing of epidermal growth factor.

  20. Intensity-dependent direct solar radiation- and UVA-induced radical damage to human skin and DNA, lipids and proteins.

    PubMed

    Haywood, Rachel; Andrady, Carima; Kassouf, Nick; Sheppard, Nick

    2011-01-01

    Skin can be exposed to high-intensity UV-radiation in hot countries and during sunbed use; however, the free-radical damage at these intensities is unknown. We used electron spin resonance spectroscopy to measure free-radical generation in ex vivo human skin/substitutes +/- the spin-trap 5,5 dimethyl-1-pyrroline N-oxide (DMPO) exposed to solar-irradiation equivalent to Mediterranean sunlight. Skin-substitutes, model DNA-photosensitizer systems, lipids and proteins were also irradiated with low-intensity UVA/visible light. Without DMPO a broad singlet was detected (using both irradiations) in skin/substitutes, nail-keratin, tendon-collagen, phospholipid and DNA+melanin or riboflavin. In addition to lipid-derived (tentatively tert-alkoxyl/acyl-) and protein radicals detected with DMPO at lower intensities, isotropic carbon-, additional oxygen- and hydrogen-adducts were detected in solar-irradiated skin/substitutes at higher intensities. Carbon-adducts were detected in UVA-irradiated human skin cells, DNA+melanin or riboflavin and soybean-phospholipid. Anisotropic protein-adducts, comparable to adducts in solar-irradiated tendon-collagen, were absent in UVA-irradiated skin fibroblasts suggesting the trapping of extracellular collagen radicals. Absence of hydrogen-adducts in fibroblasts implies formation in the extracellular compartment. We conclude damage at high intensities is part cellular (carbon- and oxygen-radicals) and part extracellular (protein- and hydrogen/H(+)+e(-) ), and skin substitutes are suitable for sunscreen testing. While UVA absorption and lipid-oxidation is direct, DNA and protein-oxidation require photosensitisation.

  1. Patterns in protein primary sequences: classification, display and analysis.

    PubMed Central

    Saurugger, P. N.; Metfessel, B. A.

    1991-01-01

    The protein folding code, which is contained in the amino acid chain of a protein, has so far eluded elucidation. However, patterns of hydrophobic residues have previously been identified which show a specificity towards certain secondary structural elements. We are developing an analysis toolkit to find, visualize, and analyze patterns in primary sequences. Preliminary results show that there exist patterns in primary sequences which are useful for predicting the structural class of amino acid chains, performing especially well for the all-alpha helix and all-beta sheet classes. PMID:1807631

  2. The free-radical theory of ageing--older, wiser and still alive: modelling positional effects of the primary targets of ROS reveals new support.

    PubMed

    Kirkwood, Thomas B L; Kowald, Axel

    2012-08-01

    The continuing viability of the free-radical theory of ageing has been questioned following apparently incompatible recent results. We show by modelling positional effects of the generation and primary targets of reactive oxygen species that many of the apparently negative results are likely to be misleading. We conclude that there is instead a need to look more closely at the mechanisms by which free radicals contribute to age-related dysfunction in living systems. There also needs to be deeper understanding of the dynamics of accumulation and removal of the various kinds of molecular damage, in particular mtDNA mutations. Finally, the expectation that free-radical damage on its own might cause ageing needs to be relinquished in favour of the recognition that the free-radical theory is just one of the multiple mechanisms driving the ageing process.

  3. Nuclear envelope protein autoantibodies in primary biliary cirrhosis.

    PubMed

    Courvalin, J C; Worman, H J

    1997-02-01

    A subset of patients with primary biliary cirrhosis (PBC) have autoantibodies directed against nuclear envelope proteins. The major autoantigen is gp210, a 210 kilodalton (kD) transmembrane protein of the nuclear pore complex, that is recognized by antibodies in approximately 25% of patients. The predominant epitope in gp210 that is recognized by most of the autoantibodies is a 15 amino acid stretch in the cytoplasmic, carboxyl-terminal domain of the protein. Gp210 autoantibodies are specific for PBC, as are the less frequent autoantibodies directed against LBR, a transmembrane protein of the inner nuclear membrane. Although autoantibodies against nuclear lamins, abundant intermediate filament proteins associated with the inner nuclear membrane, may be found in PBC, they are not specific for this disease. Nuclear envelope protein autoantibodies are also present in some patients without detectable antimitochondrial antibodies and may be of particular utility in diagnosing individuals with atypical presentations of PBC.

  4. Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

    NASA Astrophysics Data System (ADS)

    Vahidi, Siavash; Konermann, Lars

    2016-07-01

    Hydroxyl radical (ṡOH) labeling with mass spectrometry detection reports on protein conformations and interactions. Fast photochemical oxidation of proteins (FPOP) involves ṡOH production via H2O2 photolysis by UV laser pulses inside a flow tube. The experiments are conducted in the presence of a scavenger (usually glutamine) that shortens the ṡOH lifetime. The literature claims that FPOP takes place within 1 μs. This ultrafast time scale implies that FPOP should be immune to labeling-induced artifacts that may be encountered with other techniques. Surprisingly, the FPOP time scale has never been validated in direct kinetic measurements. Here we employ flash photolysis for probing oxidation processes under typical FPOP conditions. Bleaching of the reporter dye cyanine-5 (Cy5) served as readout of the time-dependent radical milieu. Surprisingly, Cy5 oxidation extends over tens of milliseconds. This time range is four orders of magnitude longer than expected from the FPOP literature. We demonstrate that the glutamine scavenger generates metastable secondary radicals in the FPOP solution, and that these radicals lengthen the time frame of Cy5 oxidation. Cy5 and similar dyes are widely used for monitoring the radical dose experienced by proteins in solution. The measured Cy5 kinetics thus strongly suggest that protein oxidation in FPOP extends over a much longer time window than previously thought (i.e., many milliseconds instead of one microsecond). The optical approach developed here should be suitable for assessing the performance of future FPOP-like techniques with improved temporal labeling characteristics.

  5. [Free radical modification of proteins in brain structure of Sprague-Dawley rats and some behaviour indicators after prenatal stress].

    PubMed

    V'iushina, A V; Pritvorova, A V; Flerov, M A

    2012-08-01

    We studied the influence of late prenatal stress on free radical oxidation processes in Sprague-Dawley rats cortex, striatum, hippocampus, hypothalamus proteins. It was shown that after prenatal stress most changes were observed in hypothalamus and hippocampus. It was shown that in hypothalamus spontaneous oxidation level increased, but level of induced oxidation decreased, the opposite changes were found in hippocampus. Simultaneously minor changes of protein modification were observed in cortex and striatum. It was shown that prenatal stress changed both correlation of proteins free radical oxidation in studied structures and values of these data regarding to control. In test of "open field" motor activity in rats after prenatal stress decreased and time of freezing and grooming increased; opposite, in T-labyrinth motor activity and time of grooming in rats after prenatal stress increased, but time of freezing decreased.

  6. A comparative analysis of primary and secondary Gleason pattern predictive ability for positive surgical margins after radical prostatectomy.

    PubMed

    Sfoungaristos, S; Kavouras, A; Kanatas, P; Polimeros, N; Perimenis, P

    2011-01-01

    To compare the predictive ability of primary and secondary Gleason pattern for positive surgical margins in patients with clinically localized prostate cancer and a preoperative Gleason score ≤ 6. A retrospective analysis of the medical records of patients undergone a radical prostatectomy between January 2005 and October 2010 was conducted. Patients' age, prostate volume, preoperative PSA, biopsy Gleason score, the 1st and 2nd Gleason pattern were entered a univariate and multivariate analysis. The 1st and 2nd pattern were tested for their ability to predict positive surgical margins using receiver operating characteristic curves. Positive surgical margins were noticed in 56 cases (38.1%) out of 147 studied patients. The 2nd pattern was significantly greater in those with positive surgical margins while the 1st pattern was not significantly different between the 2 groups of patients. ROC analysis revealed that area under the curve was 0.53 (p=0.538) for the 1st pattern and 0.60 (p=0.048) for the 2nd pattern. Concerning the cases with PSA <10 ng/ml, it was also found that only the 2nd pattern had a predictive ability (p=0.050). When multiple logistic regression analysis was conducted it was found that the 2nd pattern was the only independent predictor. The second Gleason pattern was found to be of higher value than the 1st one for the prediction of positive surgical margins in patients with preoperative Gleason score ≤ 6 and this should be considered especially when a neurovascular bundle sparing radical prostatectomy is planned, in order not to harm the oncological outcome.

  7. Climbazole increases expression of cornified envelope proteins in primary keratinocytes.

    PubMed

    Pople, J E; Moore, A E; Talbot, D C S; Barrett, K E; Jones, D A; Lim, F L

    2014-10-01

    Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti-dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays. Microarray analysis was performed to profile gene expression changes in climbazole-treated primary human keratinocyte cells. Results were independently validated using qPCR and analysis of protein expression using ELISA and immunocytochemistry. Microarray analysis of climbazole-treated keratinocytes showed statistically significant expression changes in genes associated with the gene ontology groups encompassing epidermal differentiation, keratinization, cholesterol biosynthesis and immune response. Upregulated genes included a number encoding cornified envelope proteins such as group 3 late-cornified envelope proteins, LCE3 and group 2 small-proline-rich proteins, SPRR2. Protein analysis studies of climbazole-treated primary keratinocytes using ELISA and immunocytochemistry were able to demonstrate that the increase in gene transcripts translated into increased protein expression of these cornified envelope markers. Climbazole treatment of primary keratinocytes results in an upregulation in expression of a number of genes including those encoding proteins involved in cornified envelope formation with further studies demonstrating this did translate into increased protein expression. A climbazole-driven increase in cornified envelope proteins may improve the scalp skin barrier, which is known to be weaker

  8. A Metal and Base-Free Chemoselective Primary Amination of Boronic Acids Using Cyanamidyl/Arylcyanamidyl Radical as Aminating Species: Synthesis and Mechanistic Studies by Density Functional Theory.

    PubMed

    Chatterjee, Nachiketa; Arfeen, Minhajul; Bharatam, Prasad V; Goswami, Avijit

    2016-06-17

    An efficient, metal and base-free, chemoselective synthesis of aryl-, heteroaryl-, and alkyl primary amines from the corresponding boronic acids has been achieved at ambient temperature mediated by [bis(trifluoroacetoxy)iodo]benzene (PIFA) and N-bromosuccinimide (NBS) using cyanamidyl/arylcyanamidyl radicals as the aminating species. The primary amine compounds were initially obtained as their corresponding ammonium trifluoroacetate salts which, on treatment with aq NaOH, provide the free amines. Finally, the primary amines were isolated through column chromatography over silica-gel using hexane-EtOAc solvent system as the eluent. The reactions are sufficiently fast, completing within 1 h. Quantum chemical calculations in combination with experimental observations validate that the ipso amination of substituted boronic acids involves the formation of cyanamidyl/arylcyanamidyl radical, followed by regiospecific interaction of its nitrile-N center with boron atom of the boronic acids, leading to chemoselective primary amination.

  9. Teaching laparoscopic radical prostatectomy during the primary surgeon's early learning curve--analysis of our first 207 cases.

    PubMed

    Luke, Serge; Delprado, Warick; Louie-Johnsun, Mark

    2014-11-01

    To assess the feasibility of introducing laparoscopic radical prostatectomy (LRP) training during the primary surgeon's early learning curve in a regional Australian centre. From a prospective single surgeon database perioperative, oncological and functional outcome data was collected from the first 207 consecutive patients who underwent LRP immediately after a 12-month LRP Fellowship in a high-volume centre by the primary surgeon (M.L.J.). A training case was defined as the successful completion of at least two of 10 steps by a training Fellow. Perioperative and oncological outcomes were compared in training and non-training cohorts and overall learning curve was assessed by comparing consecutive 50-patient cohorts. In all, 31% of cases were training cases with a median (range) of 7 (2-10) steps of 10 steps performed by the training Fellow. Operative times were significantly longer in training cases (mean 269 vs 209 min; P < 0.001). There was no statistically significant difference in perioperative outcomes of length of stay (2.7 vs 2.6 days), transfusion rates (3.1% vs 2.1%), major complication (Clavien >3a) rates (1.6% vs 2.1%) or positive surgical margins (PSMs: pT2 2.8% vs 15.3% and pT3 52.0% vs 45.1%) between training and non-training groups, respectively. Overall, there were two open conversions (1.0%). Despite the challenging learning curve, LRP training can be commenced safely with a stepwise modular approach, even when the primary surgeon is in their early learning curve. Perioperative outcomes including PSMs and major complications were unaffected by trainee involvement. © 2014 The Authors. BJU International © 2014 BJU International.

  10. Secondary organic aerosol formation from primary aliphatic amines with NO3 radical

    NASA Astrophysics Data System (ADS)

    Malloy, Q. G. J.; Qi, Li; Warren, B.; Cocker, D. R., III; Erupe, M. E.; Silva, P. J.

    2008-07-01

    Primary aliphatic amines are an important class of nitrogen containing compounds found to be emitted from automobiles, waste treatment facilities and agricultural animal operations. A series of experiments conducted at the UC-Riverside/CE-CERT Environmental Chamber is presented in which oxidation of methylamine, ethylamine, propylamine, and butylamine with NO3 has been investigated. Very little aerosol formation is observed in the presence of O3 only. However, after addition of NO, and by extension NO3, large yields of aerosol mass loadings (~44% for butylamine) are seen. Aerosol generated was determined to be organic in nature due to the small fraction of NO and NO2 in the total signal (<17% for all amines tested) as detected by an aerosol mass spectrometer (AMS). We propose a reaction mechanism between carbonyl containing species and the parent amine leading to formation of particulate imine products. These findings can have significant impacts on rural communities and lead to elevated nighttime PM loadings, when significant levels on NO3 exist.

  11. Secondary organic aerosol formation from primary aliphatic amines with NO3 radical

    NASA Astrophysics Data System (ADS)

    Malloy, Q. G. J.; Qi, Li; Warren, B.; Cocker, D. R., III; Erupe, M. E.; Silva, P. J.

    2009-03-01

    Primary aliphatic amines are an important class of nitrogen containing compounds emitted from automobiles, waste treatment facilities and agricultural animal operations. A series of experiments conducted at the UC-Riverside/CE-CERT Environmental Chamber is presented in which oxidation of methylamine, ethylamine, propylamine, and butylamine with O3 and NO3 have been investigated. Very little aerosol formation is observed in the presence of O3 only. However, after addition of NO, and by extension NO3, large aerosol mass yields (~44% for butylamine) are seen. Aerosol generated was determined to be organic in nature due to the small fraction of NO and NO2 in the total signal (<1% for all amines tested) as detected by an aerosol mass spectrometer (AMS). We propose a reaction mechanism between carbonyl containing species and the parent amine leading to formation of particulate imine products. These findings can have significant impacts on rural communities with elevated nighttime PM loadings, when significant levels of NO3 exist.

  12. Role of heat shock proteins in oxygen radical-induced gastric apoptosis.

    PubMed

    Leung, Anna M; Redlak, Maria J; Miller, Thomas A

    2015-01-01

    The generation of reactive oxygen species (ROS) and their resultant oxidative damage is a common pathway for gastric mucosal injury. Developing strategies to protect the gastric epithelium against oxygen free radical damage is of profound pathophysiological interest. We have previously shown caspase-mediated apoptosis as a major cause of ROS-induced cell death in gastric mucosa. Because heat shock proteins (Hsps) confer protection against many cytotoxic agents, this study was undertaken to determine whether modulation of Hsps was protective against oxidative damage. AGS cells (human gastric mucosal cell line) received either no pretreatment, heat shock pretreatment (1 h at 42 ± 1°C), or pretreatment with an Hsp modulating drug (geldanamycin or quercetin). Cells were then exposed to hydrogen peroxide (H2O2), a representative ROS (1 mM, a physiologically relevant concentration), for 24 h. Caspase-3 activation and Poly ADP Ribose Polymerase (PARP) inactivation, as well as DNA-histone complex formation were used as measures of apoptosis. Inducible Hsps (Hsp70 and Hsp90) were detected using Western blot analysis. Results showed heat shock pretreatment induced increased expression of Hsp70 without change in Hsp90. In response to H2O2 exposure alone, there was significant increase in DNA-histone complex formation as well as caspase-3 activation and PARP cleavage in gastric epithelium. Heat shock pretreatment resulted in statistically significant prevention in these measures of apoptosis. Geldanamycin increased Hsp70, but elicited cleavage of Hsp90 and subsequently resulted in an increase in H2O2-induced apoptosis. Quercetin decreased Hsp70 and resulted again in increased H2O2-induced apoptosis. These findings indicate that heat shock pretreatment protects gastric mucosal cells against H2O2-induced apoptosis and that Hsp70 and Hsp90 may play key roles in this process. These results further suggest that perturbations in Hsp metabolism may induce mucosal injury in

  13. Long-lived radicals produced by γ-irradiation or vital activity in plants, animals, cells, and protein solution: their observation and inhomogeneous decay dynamics

    NASA Astrophysics Data System (ADS)

    Miyazaki, Tetsuo; Morikawa, Akiyuki; Kumagai, Jun; Ikehata, Masateru; Koana, Takao; Kikuchi, Shoshi

    2002-09-01

    Long-lived radicals produced by γ-irradiation or vital activity in plants, animals, cells, and protein (albumin) solution were studied by electron spin resonance spectroscopy. Long-lived radicals produced by vital activity exist in biological systems, such as plants, animals, and cells, in the range of 0.1-20 nmol g -1. Since vital organs keep the radicals at a constant concentration, the radicals are probably related to life conservation. Long-lived radicals are also produced by γ-irradiation of cells or protein solution. The radicals decay after death of living things or after γ-irradiation. We found that the decay dynamics in all biological systems can be expressed by the same kinetic equation of an inhomogeneous reaction.

  14. Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins

    NASA Astrophysics Data System (ADS)

    Tesch, Deanna M.; Nevzorov, Alexander A.

    2014-02-01

    Elucidating structure and topology of membrane proteins (MPs) is essential for unveiling functionality of these important biological constituents. Oriented-sample solid-state NMR (OS-NMR) is capable of providing such information on MPs under nearly physiological conditions. However, two dimensional OS-NMR experiments can take several days to complete due to long longitudinal relaxation times combined with the large number of scans to achieve sufficient signal sensitivity in biological samples. Here, free radicals 5-DOXYL stearic acid, TEMPOL, and CAT-1 were added to uniformly 15N-labeled Pf1 coat protein reconstituted in DMPC/DHPC bicelles, and their effect on the longitudinal relaxation times (T1Z) was investigated. The dramatically shortened T1Z's allowed for the signal gain per unit time to be used for either: (i) up to a threefold reduction of the total experimental time at 99% magnetization recovery or (ii) obtaining up to 74% signal enhancement between the control and radical samples during constant experimental time at “optimal” relaxation delays. In addition, through OS-NMR and high-field EPR studies, free radicals were able to provide positional constraints in the bicelle system, which provide a description of the location of each residue in Pf1 coat protein within the bicellar membranes. This information can be useful in the determination of oligomerization states and immersion depths of larger membrane proteins.

  15. Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins.

    PubMed

    Tesch, Deanna M; Nevzorov, Alexander A

    2014-02-01

    Elucidating structure and topology of membrane proteins (MPs) is essential for unveiling functionality of these important biological constituents. Oriented-sample solid-state NMR (OS-NMR) is capable of providing such information on MPs under nearly physiological conditions. However, two dimensional OS-NMR experiments can take several days to complete due to long longitudinal relaxation times combined with the large number of scans to achieve sufficient signal sensitivity in biological samples. Here, free radicals 5-DOXYL stearic acid, TEMPOL, and CAT-1 were added to uniformly (15)N-labeled Pf1 coat protein reconstituted in DMPC/DHPC bicelles, and their effect on the longitudinal relaxation times (T1Z) was investigated. The dramatically shortened T1Z's allowed for the signal gain per unit time to be used for either: (i) up to a threefold reduction of the total experimental time at 99% magnetization recovery or (ii) obtaining up to 74% signal enhancement between the control and radical samples during constant experimental time at "optimal" relaxation delays. In addition, through OS-NMR and high-field EPR studies, free radicals were able to provide positional constraints in the bicelle system, which provide a description of the location of each residue in Pf1 coat protein within the bicellar membranes. This information can be useful in the determination of oligomerization states and immersion depths of larger membrane proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Expression of DNA damage response proteins in cervical cancer patients treated with radical chemoradiotherapy.

    PubMed

    Ho, C K; Kornaga, E N; Klimowicz, A C; Enwere, E K; Dean, M; Bebb, G D; Phan, T; Ghatage, P; Magliocco, A M; Lees-Miller, S P; Doll, C M

    2017-04-01

    The management of locally advanced cervical cancer has improved significantly with the advent of cisplatin-based chemoradiotherapy (CRT) as the primary treatment regimen. Nevertheless, a significant proportion of patients fail to respond or relapse on this treatment and have a very poor prognosis. Our goal was to determine the prognostic value of a panel of proteins involved in detection and repair of DNA damage. We performed fluorescence immunohistochemistry, and used software analysis to assess expression of DNA damage response proteins ATM, DNA-PKcs, PARP-1, Ku70 and Ku86 in 117 pre-treatment specimens from patients with locally advanced cervical cancer. We compared expression to clinicopathologic correlates to determine prognostic significance. Five-year progression-free survival was significantly lower in the low expressors than in high expressors of ATM (35% vs. 58%, p=0.044) and PARP-1 (24% vs. 61%, p=0.003), and showed a trend to significance for DNA-PKcs (30% vs. 60%, p=0.050). Low expression of the same proteins also correlated significantly with lower overall survival. In multivariable analysis, adjusted for FIGO stage and tumor size, low ATM and PARP-1 expression was significantly associated with both poorer progression-free and overall survival. Pairwise analyses indicated that expression levels of these proteins were correlated. Expression of DNA damage response proteins in cervical cancer is associated with outcome in patients treated with CRT. Immunohistochemical analysis of these proteins may be useful in guiding treatment decisions in such patients. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Utility of RhoC and ZAG protein expression as biomarkers for prediction of PSA failure following radical prostatectomy for high grade prostate cancer.

    PubMed

    Mills, John; Oliver, Alice; Sherwin, Justin C; Frydenberg, Mark; Peters, Justin S; Costello, Anthony; Harewood, Laurence; Love, Christopher; Redgrave, Nicholas; van Golen, Kenneth L; Bailey, Michael; Pedersen, John

    2012-10-01

    To assess the prognostic utility of semi-quantiative expression of RhoC protein in whole prostates from patients who had radical prostatectomies for high grade prostate cancer (PCa). Subjects who had surgery >55 months previously with primary Gleason pattern 4 PCa were identified from practice records, archival tissues were retrieved for review and RhoC immunohistochemistry, and ZAG expression was also assessed as a control. Eighty-nine subjects were included in the study; 57 had a rising prostate specific antigen (PSA) post-operatively ('cases') and 32 did not ('controls'). By univariate analysis, expression of both RhoC and ZAG proteins was greater in controls than cases, but this was significant only for ZAG. By multivariate analysis, Gleason variables (patterns and score), extraprostatic extension and decreased RhoC staining all contributed to predicting PSA failure (p < 0.05). ZAG expression was inversely correlated with Gleason pattern and hence was not independently predictive in our multivariate model. Increased RhoC expression predicted a good outcome after radical prostatectomy. ZAG staining also correlated with a favourable outcome but was not independently predictive due to its relationship with Gleason pattern.

  18. Characterization of a Cross-Linked Protein-Nucleic Acid Substrate Radical in the Reaction Catalyzed by RlmN

    SciTech Connect

    Silakov, Alexey; Grove, Tyler L.; Radle, Matthew I.; Bauerle, Matthew R.; Green, Michael T.; Rosenzweig, Amy C.; Boal, Amie K.; Booker, Squire J.

    2014-08-14

    RlmN and Cfr are methyltransferases/methylsynthases that belong to the radical S-adenosylmethionine superfamily of enzymes. RlmN catalyzes C2 methylation of adenosine 2503 (A2503) of 23S rRNA, while Cfr catalyzes C8 methylation of the exact same nucleotide, and will subsequently catalyze C2 methylation if the site is unmethylated. A key feature of the unusual mechanisms of catalysis proposed for these enzymes is the attack of a methylene radical, derived from a methylcysteine residue, onto the carbon center undergoing methylation to generate a paramagnetic protein–nucleic acid cross-linked species. This species has been thoroughly characterized during Cfr-dependent C8 methylation, but does not accumulate to detectible levels in RlmN-dependent C2 methylation. Herein, we show that inactive C118S/A variants of RlmN accumulate a substrate-derived paramagnetic species. Characterization of this species by electron paramagnetic resonance spectroscopy in concert with strategic isotopic labeling shows that the radical is delocalized throughout the adenine ring of A2503, although predominant spin density is on N1 and N3. Moreover, 13C hyperfine interactions between the radical and the methylene carbon of the formerly [methyl-13C]Cys355 residue show that the radical species exists in a covalent cross-link between the protein and the nucleic acid substrate. X-ray structures of RlmN C118A show that, in the presence of SAM, the substitution does not alter the active site structure compared to that of the wild-type enzyme. Together, these findings have new mechanistic implications for the role(s) of C118 and its counterpart in Cfr (C105) in catalysis, and suggest involvement of the residue in resolution of the cross-linked species via a radical mediated process

  19. Thermodynamics and Kinetics for the Free Radical Oxygen Protein Oxidation Pathway in a Model for β-Structured Peptides.

    PubMed

    Green, Mandy C; Dubnicka, Laura J; Davis, Alex C; Rypkema, Heather A; Francisco, Joseph S; Slipchenko, Lyudmila V

    2016-04-28

    Oxidative stress plays a role in many biological phenomena, but involved mechanisms and individual reactions are not well understood. Correlated electronic structure calculations with the MP2, MP4, and CCSD(T) methods detail thermodynamic and kinetic information for the free radical oxygen protein oxidation pathway studied in a trialanine model system. The pathway includes aerobic, anaerobic and termination reactions. The course of the oxidation process depends on local conditions and availability of specific reactive oxygen species (ROS). A chemical mechanism is proposed for how oxidative stress promotes β-structure formation in the amyloid diseases. The work can be used to aid experimentalists as they explore individual reactions and mechanisms involving oxygen free radicals and oxidative stress in β-structured proteins.

  20. Evaluation of 2D-ESEEM data of 15N-labeled radical cations of the primary donor P 700 in photosystem I and chlorophyll a

    NASA Astrophysics Data System (ADS)

    Käβ, H.; Lubitz, W.

    1996-03-01

    Hyperfine couplings (hfc's) of the nitrogen nuclei in the 15N-labeled radical cations of chlorophyll a and the primary donor P 700 in photosystem I of spinach were investigated in frozen solution by two-dimensional stimulated echo ESEEM. 15N hfc tensors were evaluated by comparison of the experimental data with simulations of the time domain and frequency domain ESEEM signals. The results are discussed in the framework of a chlorophyll a dimer model for the radical cation of P 700.

  1. Aluminum potentiates glutamate-induced calcium accumulation and iron-induced oxygen free radical formation in primary neuronal cultures.

    PubMed

    Mundy, W R; Freudenrich, T M; Kodavanti, P R

    1997-01-01

    Aluminum is a neurotoxic metal that may be involved in the progression of neurodegenerative diseases, including Alzheimer disease and amyotrophic lateral sclerosis (ALS). Although the mechanism of action is not known, aluminum has been shown to alter Ca2+ flux and homeostasis, and facilitate peroxidation of membrane lipids. Since abnormal increases of intracellular Ca2+ and oxygen free radicals have both been implicated in pathways leading to neurodegeneration, we examined the effect of aluminum on these parameters in vitro using primary cultures of cerebellar granule cells. Exposure to glutamate (1-300 microM) caused a concentration-dependent uptake of 45Ca in granule cells to a maximum of 280% of basal. Pretreatment with AlCl3 (1-1000 microM) had no effect on 45Ca accumulation, but increased the uptake induced by glutamate. Similarly, AlCl3 had no effect on intracellular free Ca2+ levels measured using fluorescent probe fura-2, but potentiated the increase induced by glutamate. The production of reactive oxygen species (ROS) was examined using the fluorescent probe dichlorofluorescin. By itself, AlCl3 had little effect on ROS production. However, AlCl3 pretreatment potentiated the ROS production induced by 50 microM Fe2+. These results suggest that aluminum may facilitate increases in intracellular Ca2+ and ROS, and potentially contribute to neurotoxicity induced by other neurotoxicants.

  2. Reaction of protein chloramines with DNA and nucleosides: evidence for the formation of radicals, protein-DNA cross-links and DNA fragmentation.

    PubMed Central

    Hawkins, Clare L; Pattison, David I; Davies, Michael J

    2002-01-01

    Stimulated phagocyte cells produce the oxidant HOCl, via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is important in bacterial cell killing, but excessive or misplaced generation can damage the host tissue and may lead to the development of certain diseases such as cancer. The role of HOCl in the oxidation of isolated proteins, DNA and their components has been investigated extensively, but little work has been performed on the protein-DNA (nucleosome) complexes present in eukaryotic cell nuclei. Neither the selectivity of damage in such complexes nor the possibility of transfer of damage from the protein to DNA or vice versa, has been studied. In the present study, kinetic modelling has been employed to predict that reaction occurs predominantly with the protein and not with the DNA in the nucleosome, using molar HOCl excesses of up to 200-fold. With 50-200-fold excesses, 50-80% of the HOCl is predicted to react with histone lysine and histidine residues to yield chloramines. The yield and stability of such chloramines predicted by these modelling studies agrees well with experimental data. Decomposition of these species gives protein-derived, nitrogen-centred radicals, probably on the lysine side chains, as characterized by the EPR and spin-trapping experiments. It is shown that isolated lysine, histidine, peptide and protein chloramines can react with plasmid DNA to cause strand breaks. The protection against such damage afforded by the radical scavengers Trolox (a water-soluble alpha-tocopherol derivative) and 5,5-dimethyl-1-pyrroline-N-oxide suggests a radical-mediated process. The EPR experiments and product analyses have also provided evidence for the rapid addition of protein radicals, formed on chloramine decomposition, to pyrimidine nucleosides to give nucleobase radicals. Further evidence for the formation of such covalent cross-links has been obtained from experiments performed using (3)H-lysine and (14)C-histidine chloramines

  3. Reaction of protein chloramines with DNA and nucleosides: evidence for the formation of radicals, protein-DNA cross-links and DNA fragmentation.

    PubMed

    Hawkins, Clare L; Pattison, David I; Davies, Michael J

    2002-08-01

    Stimulated phagocyte cells produce the oxidant HOCl, via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is important in bacterial cell killing, but excessive or misplaced generation can damage the host tissue and may lead to the development of certain diseases such as cancer. The role of HOCl in the oxidation of isolated proteins, DNA and their components has been investigated extensively, but little work has been performed on the protein-DNA (nucleosome) complexes present in eukaryotic cell nuclei. Neither the selectivity of damage in such complexes nor the possibility of transfer of damage from the protein to DNA or vice versa, has been studied. In the present study, kinetic modelling has been employed to predict that reaction occurs predominantly with the protein and not with the DNA in the nucleosome, using molar HOCl excesses of up to 200-fold. With 50-200-fold excesses, 50-80% of the HOCl is predicted to react with histone lysine and histidine residues to yield chloramines. The yield and stability of such chloramines predicted by these modelling studies agrees well with experimental data. Decomposition of these species gives protein-derived, nitrogen-centred radicals, probably on the lysine side chains, as characterized by the EPR and spin-trapping experiments. It is shown that isolated lysine, histidine, peptide and protein chloramines can react with plasmid DNA to cause strand breaks. The protection against such damage afforded by the radical scavengers Trolox (a water-soluble alpha-tocopherol derivative) and 5,5-dimethyl-1-pyrroline-N-oxide suggests a radical-mediated process. The EPR experiments and product analyses have also provided evidence for the rapid addition of protein radicals, formed on chloramine decomposition, to pyrimidine nucleosides to give nucleobase radicals. Further evidence for the formation of such covalent cross-links has been obtained from experiments performed using (3)H-lysine and (14)C-histidine chloramines

  4. Inhibition of protein radical reactions of ferrylmyoglobin by the water-soluble analog of vitamin E, Trolox C.

    PubMed

    Giulivi, C; Cadenas, E

    1993-05-15

    The reactivity of Trolox C, a water-soluble analog of vitamin E, toward protein radicals formed during the oxidation of metmyoglobin and the therefrom derived molecular products was examined in terms of the ability of the phenolic antioxidant to prevent specific oxidative reactions involving tyrosyl radicals and to reduce the molecular products to functional hemoproteins. (i) Trolox prevented the oxidative reactions initiated upon oxidation of the hemoprotein by H2O2 and involving tyrosyl radicals: on one hand, it inhibited the covalent binding of protein to the heme group and, on the other hand, it inhibited dimerization of sperm whale myoglobin, the latter process entailing the intermolecular covalent binding of tyrosines. The inhibition of both processes required concentrations of Trolox 20- to 50-fold higher than those needed to reduce the hypervalent heme iron to its ferric form. Likewise, Trolox inhibited dimerization and polymerization of sperm whale myoglobin upon its treatment with chloroiridate, a process associated with formation of tyrosyl radicals and negligible oxidation of the heme iron. However, these results did not provide unambiguous evidence for the reactivity of the phenolic antioxidant toward amino acid radicals in myoglobin, for Trolox displayed a high reactivity toward the oxidant, chloroiridate. (ii) The stable oxidation product originating from sperm whale myoglobin oxidation, i.e., the dimeric hemoprotein, was redox active, inasmuch as it retained its capacity to form an oxy complex upon reduction and an oxoferryl complex upon oxidation by H2O2. The latter complex was reduced by Trolox to a compound which exhibited a 10- to 12-nm blue shift of the 632-nm absorption typical of the native metmyoglobin. Although this absorption shift was likely to express oxidative modifications of the porphyrin ring, the modified hemoprotein retained its capacity to react with peroxides and displayed a peroxidative activity.

  5. Changes in the radical scavenging activity of bacterial-type douchi, a traditional fermented soybean product, during the primary fermentation process.

    PubMed

    Fan, Junfeng; Zhang, Yanyan; Chang, Xiaojie; Saito, Masayoshi; Li, Zaigui

    2009-12-01

    We studied the effects of isoflavones and peptides on antioxidant activities of bacterial-type douchi during fermentation. Radical scavenging activities increased with increasing fermentation time. Isoflavone conversion was not obvious, while soy protein hydrolyzed dramatically during fermentation. These results suggest that soybean peptides rather than isoflavones result in variations in antioxidant activity in bacterial-type douchi.

  6. Kinetic evidence that a radical transfer pathway in protein R2 of mouse ribonucleotide reductase is involved in generation of the tyrosyl free radical.

    PubMed

    Schmidt, P P; Rova, U; Katterle, B; Thelander, L; Gräslund, A

    1998-08-21

    Class I ribonucleotide reductases consist of two subunits, R1 and R2. The active site is located in R1; active R2 contains a diferric center and a tyrosyl free radical (Tyr.), both essential for enzymatic activity. The proposed mechanism for the enzymatic reaction includes the transport of a reducing equivalent, i.e. electron or hydrogen radical, across a 35-A distance between Tyr. in R2 and the active site in R1, which are connected by a hydrogen-bonded chain of conserved, catalytically essential amino acid residues. Asp266 and Trp103 in mouse R2 are part of this radical transfer pathway. The diferric/Tyr. site in R2 is reconstituted spontaneously by mixing iron-free apoR2 with Fe(II) and O2. The reconstitution reaction requires the delivery of an external reducing equivalent to form the diferric/Tyr. site. Reconstitution kinetics were investigated in mouse apo-wild type R2 and the three mutants D266A, W103Y, and W103F by rapid freeze-quench electron paramagnetic resonance with >/=4 Fe(II)/R2 at various reaction temperatures. The kinetics of Tyr. formation in D266A and W103Y is on average 20 times slower than in wild type R2. More strikingly, Tyr. formation is completely suppressed in W103F. No change in the reconstitution kinetics was found starting from Fe(II)-preloaded proteins, which shows that the mutations do not affect the rate of iron binding. Our results are consistent with a reaction mechanism using Asp266 and Trp103 for delivery of the external reducing equivalent. Further, the results with W103F suggest that an intact hydrogen-bonded chain is crucial for the reaction, indicating that the external reducing equivalent is a H. Finally, the formation of Tyr. is not the slowest step of the reaction as it is in Escherichia coli R2, consistent with a stronger interaction between Tyr. and the iron center in mouse R2. A new electron paramagnetic resonance visible intermediate named mouse X, strikingly similar to species X found in E. coli R2, was detected only

  7. 15N electron nuclear double resonance of the primary donor cation radical P+.865 in reaction centers of Rhodopseudomonas sphaeroides: additional evidence for the dimer model.

    PubMed Central

    Lubitz, W; Isaacson, R A; Abresch, E C; Feher, G

    1984-01-01

    Four 15N hyperfine coupling constants, including signs, have been measured by electron nuclear double resonance (ENDOR) and electron nuclear nuclear triple resonance (TRIPLE) for the bacteriochlorophyll a radical cation, BChla+., in vitro and for the light-induced primary donor radical cation, P+.865, in reaction centers of Rhodopseudomonas sphaeroides R-26. A comparison of the data shows that the hyperfine coupling constants have the same sign in both radicals and are, on the average, smaller by a factor of 2 in P+.865. These results provide additional evidence that P+.865 is a bacteriochlorophyll dimer and are in contradiction with the monomer structure of P+.865 recently proposed by O'Malley and Babcock. The reduction factors of the individual 15N couplings, together with the evidence from proton ENDOR data and molecular orbital calculations, indicate a dimer structure in which only two rings (either I and I or III and III) of the bacteriochlorophyll macrocycles overlap. PMID:6096857

  8. ENDOR and ESEEM of the 15N labelled radical cations of chlorophyll a and the primary donor P 700 in photosystem I

    NASA Astrophysics Data System (ADS)

    Käβ, H.; Bittersmann-Weidlich, E.; Andréasson, L.-E.; Bönigk, B.; Lubitz, W.

    1995-05-01

    The hyperfine couplings of the nitrogen nuclei in the radical cations of both 15N-labelled chlorophyll a and the primary donor P 700 in Photosystem I of Synechococcus elongatus and spinach ( Spinacea oleracea) in frozen solutions were investigated by ENDOR and, for confirmation, by two-dimensional ESEEM techniques. In addition, 1H ENDOR experiments were performed on these compounds. The experimental 15N hyperfine couplings of the chlorophyll a radical cation are compared with theoretical ones obtained by RHF-INDO/SP calculations and with the respective hyperfine couplings in the closely related 15N-bacteriochlorophyll a radical cation. Based on the observed 15N and 1H hyperfine couplings two possible models are discussed for P 700+: (a) the special pair model with a strongly asymmetric spin density distribution over the dimer halves; (b) the model of a strongly perturbed chlorophyll a monomer.

  9. [Influence of antitumor preparations on the concentration of free radicals in cells of Fusarium bulbigenum var. blasticola fungus during primary and tumour-like secondary growth].

    PubMed

    Riabikin, Iu A; Nikitina, E T; Balgimbatva, A S; Zashkvara, O V; Shakiev, S Sh

    2007-01-01

    The fungus Fusarium bulbigenum var. blasticola in which secondary tumor-like formations appear under certain conditions in aging was used as a new test system to examine the action of antitumor preparations. Free radicals in the primary mycelium and tumor-like formations without introduction of preparations (control samples) and after the introduction of preparation into the cultivation medium of the fungus have been studied by EPR spectroscopy. The EPR spectra of the fungus represent single, somewhat asymmetrical lines with a width of deltaH = 0.4 divided by 0.6 mT and g = 2.0036 +/- 0.006, which enabled one to assign the paramagnetic centers observed to melanine radicals. It was found that the concentration of free radicals in tumor-like formations is always higher than in the primary mycelium, which may be related to intensive metabolism in tumor-like formations. It has been established that several antitumor preparations (fluorouracil, hydrea, methotrexat, and vepezide) completely inhibit the growth of tumor-like formations. Another group of preparations (cyclophosphanum, dacarbazin, adriablastin, and vinblastin), on the contrary, stimulate their growth, which is accompanied by an increase in the concentration of free radicals in cells of the primary mycelium and tumor-like formations. The preparations of the third group (mercaptopurine, lanvis, and farmorubicin), despite the increased level of free radicals in cells, have a weak inhibitory effect. It has been shown that, in the concentration range studied, vitamins B2, B12, C, and PP stimulate the growth of tumor-like formations, and, when used in combination with antitumor preparations, enhance or reduce the inhibitory properties of these preparations.

  10. Identification of a unique Fe-S cluster binding site in a glycyl-radical type microcompartment shell protein.

    PubMed

    Thompson, Michael C; Wheatley, Nicole M; Jorda, Julien; Sawaya, Michael R; Gidaniyan, Soheil D; Ahmed, Hoda; Yang, Zhongyu; McCarty, Krystal N; Whitelegge, Julian P; Yeates, Todd O

    2014-09-23

    Recently, progress has been made toward understanding the functional diversity of bacterial microcompartment (MCP) systems, which serve as protein-based metabolic organelles in diverse microbes. New types of MCPs have been identified, including the glycyl-radical propanediol (Grp) MCP. Within these elaborate protein complexes, BMC-domain shell proteins [bacterial microcompartment (in reference to the shell protein domain)] assemble to form a polyhedral barrier that encapsulates the enzymatic contents of the MCP. Interestingly, the Grp MCP contains a number of shell proteins with unusual sequence features. GrpU is one such shell protein whose amino acid sequence is particularly divergent from other members of the BMC-domain superfamily of proteins that effectively defines all MCPs. Expression, purification, and subsequent characterization of the protein showed, unexpectedly, that it binds an iron-sulfur cluster. We determined X-ray crystal structures of two GrpU orthologs, providing the first structural insight into the homohexameric BMC-domain shell proteins of the Grp system. The X-ray structures of GrpU, both obtained in the apo form, combined with spectroscopic analyses and computational modeling, show that the metal cluster resides in the central pore of the BMC shell protein at a position of broken 6-fold symmetry. The result is a structurally polymorphic iron-sulfur cluster binding site that appears to be unique among metalloproteins studied to date.

  11. Evidence for dissolved organic matter as the primary source and sink of photochemically produced hydroxyl radical in arctic surface waters.

    PubMed

    Page, Sarah E; Logan, J Robert; Cory, Rose M; McNeill, Kristopher

    2014-04-01

    Hydroxyl radical (˙OH) is an indiscriminate oxidant that reacts at near-diffusion-controlled rates with organic carbon. Thus, while ˙OH is expected to be an important oxidant of dissolved organic matter (DOM) and other recalcitrant compounds, the role of ˙OH in the oxidation of these compounds in aquatic ecosystems is not well known due to the poorly constrained sources and sinks of ˙OH, especially in pristine (unpolluted) natural waters. We measured the rates of ˙OH formation and quenching across a range of surface waters in the Arctic varying in concentrations of expected sources and sinks of ˙OH. Photochemical formation of ˙OH was observed in all waters tested, with rates of formation ranging from 2.6 ± 0.6 to 900 ± 100 × 10(-12) M s(-1). Steady-state concentrations ranged from 2 ± 1 to 290 ± 60 × 10(-17) M, and overlapped with previously reported values in surface waters. While iron-mediated photo-Fenton reactions likely contributed to the observed ˙OH production, several lines of evidence suggest that DOM was the primary source and sink of photochemically produced ˙OH in pristine arctic surface waters. DOM from first-order or headwater streams was more efficient in producing ˙OH than what has previously been reported for DOM, and ˙OH formation decreased with increasing residence time of DOM in sunlit surface waters. Despite the ubiquitous formation of ˙OH in arctic surface waters observed in this study, photochemical ˙OH formation was estimated to contribute ≤4% to the observed photo-oxidation of DOM; however, key uncertainties in this estimate must be addressed before ruling out the role of ˙OH in the oxidation of DOM in these waters.

  12. Identification of a Unique Fe-S Cluster Binding Site in a Glycyl-Radical Type Microcompartment Shell Protein

    PubMed Central

    Thompson, Michael C.; Wheatley, Nicole M.; Jorda, Julien; Sawaya, Michael R.; Gidaniyan, Soheil D.; Ahmed, Hoda; Yang, Zhongyu; McCarty, Krystal N.; Whitelegge, Julian P.; Yeates, Todd O.

    2014-01-01

    Recently, progress has been made toward understanding the functional diversity of bacterial microcompartment (MCP) systems, which serve as protein-based metabolic organelles in diverse microbes. New types of MCPs have been identified, including the glycyl-radical propanediol (Grp) MCP. Within these elaborate protein complexes, BMC-domain shell proteins assemble to form a polyhedral barrier that encapsulates the enzymatic contents of the MCP. Interestingly, the Grp MCP contains a number of shell proteins with unusual sequence features. GrpU is one such shell protein, whose amino acid sequence is particularly divergent from other members of the BMC-domain superfamily of proteins that effectively defines all MCPs. Expression, purification, and subsequent characterization of the protein showed, unexpectedly, that it binds an iron-sulfur cluster. We determined X-ray crystal structures of two GrpU orthologs, providing the first structural insight into the homohexameric BMC-domain shell proteins of the Grp system. The X-ray structures of GrpU, both obtained in the apo form, combined with spectroscopic analyses and computational modeling, show that the metal cluster resides in the central pore of the BMC shell protein at a position of broken 6-fold symmetry. The result is a structurally polymorphic iron-sulfur cluster binding site that appears to be unique among metalloproteins studied to date. PMID:25102080

  13. Radical scavenging capacity and antimutagenic properties of purified proteins from Solanum betaceum fruits and Solanum tuberosum tubers.

    PubMed

    Ordóñez, Roxana Mabel; Zampini, Iris Catiana; Rodríguez, Fabiola; Cattaneo, Florencia; Sayago, Jorge Esteban; Isla, María Inés

    2011-08-24

    In this study, antioxidant activities in free-radical-mediated oxidative systems and the genotoxic/antigenotoxic effects of two proteins with molecular mass around 17 kDa, purified from Solanum betaceum fruits (cyphomine) and Solanum tuberosum tubers (solamarine), were investigated. Both proteins inhibited uric acid formation with IC(50) values between 55 and 60 μg/mL, and both proteins were able to reduce oxidative damage by scavenging hydroxyl radicals and superoxide anion in a dose-dependent manner. Furthermore, the DPPH• reduction assay showed SC(50) values of 55-73 μg/mL. Cyphomine and solamarine were able to retain their antioxidant activity after heat treatment at 80 °C for 15 min. Allium cepa and Salmonella /microsome assays showed no genotoxic and mutagenic effects. Solamarine showed an antimutagenic effect against a direct mutagen (4-nitro-o-phenylenediamine). Consequently, the present study showed that the investigated proteins are promising ingredients for the development of functional foods with a beneficial impact on human health and an important source for the production of bioactive peptides.

  14. Protein radical formation on thyroid peroxidase during turnover as detected by immuno-spin trapping.

    PubMed

    Ehrenshaft, Marilyn; Mason, Ronald P

    2006-08-01

    Thyroid peroxidase (TPO) is a 933 amino acid residue, heme-containing, integral membrane glycoprotein that catalyzes two steps in the maturation of the thyroid hormone precursor. As with other peroxidases, these reactions require hydrogen peroxide and initial enzyme oxidation. Previous researchers studied the oxidative state of the TPO heme moiety using spectrophotometric and catalytic analyses. We use a novel antiserum to 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to detect radical-derived DMPO spin-trapped TPO. Our work reveals that TPO generates radical adducts in the presence of H2O2, but that the generation of these adducts can be suppressed by the addition of substrates and inhibitors. Chemical alteration of the tyrosine residues of TPO greatly reduces the generation of TPO-DMPO adducts. Iodide strongly suppresses the H2O2-generated production of TPO radical adducts and protects the enzyme from loss of enzyme activity. Because the normal catalytic mechanism of TPO involves the production of radical species, TPO is potentially more susceptible to oxidative damage than most enzymes which do not require H2O2 as a substrate. We hypothesize that oxidatively damaged TPO may trigger the production of anti-TPO autoantibodies, resulting in the development of autoimmune thyroid disorders. Evidence that correlates iodine deficiencies with development of thyroid autoimmune disorders supports this conjecture.

  15. Modulation of primary radical pair kinetics and energetics in photosystem II by the redox state of the quinone electron acceptor Q(A).

    PubMed Central

    Gibasiewicz, K; Dobek, A; Breton, J; Leibl, W

    2001-01-01

    Time-resolved photovoltage measurements on destacked photosystem II membranes from spinach with the primary quinone electron acceptor Q(A) either singly or doubly reduced have been performed to monitor the time evolution of the primary radical pair P680(+)Pheo(-). The maximum transient concentration of the primary radical pair is about five times larger and its decay is about seven times slower with doubly reduced compared with singly reduced Q(A). The possible biological significance of these differences is discussed. On the basis of a simple reversible reaction scheme, the measured apparent rate constants and relative amplitudes allow determination of sets of molecular rate constants and energetic parameters for primary reactions in the reaction centers with doubly reduced Q(A) as well as with oxidized or singly reduced Q(A). The standard free energy difference DeltaG degrees between the charge-separated state P680(+)Pheo(-) and the equilibrated excited state (Chl(N)P680)* was found to be similar when Q(A) was oxidized or doubly reduced before the flash (approximately -50 meV). In contrast, single reduction of Q(A) led to a large change in DeltaG degrees (approximately +40 meV), demonstrating the importance of electrostatic interaction between the charge on Q(A) and the primary radical pair, and providing direct evidence that the doubly reduced Q(A) is an electrically neutral species, i.e., is doubly protonated. A comparison of the molecular rate constants shows that the rate of charge recombination is much more sensitive to the change in DeltaG degrees than the rate of primary charge separation. PMID:11259277

  16. [Cow milk protein allergies: primary prevention. Nutritional aspects].

    PubMed

    Dalmau Serra, J; Martorell Aragonés, A

    2008-03-01

    In 2001 the Nutrition Committee of the Spanish Association of Pediatrics, published a position paper on "Recommendations on the use of formulae for the treatment and prevention of adverse reactions to cow milk proteins" (An Esp Pediatr. 2001;54:372-9). Since most of the recommendations given in this report still remain effective, the Nutrition Committee of the Spanish Association of Pediatrics reaffirms its support of the data expressed in it. In the present publication we only approach the aspects that have changed due to new knowledge and the accumulated clinical experience, especially on the primary prevention of allergy to cow milk proteins, and on the basis of it, the present recommendations for this prevention are set out. In addition, the importance of the nutritional aspects in all infants and small children receiving special nutritional formulae and exclusion diets are reviewed.

  17. G-protein-coupled receptors, Hedgehog signaling and primary cilia.

    PubMed

    Mukhopadhyay, Saikat; Rohatgi, Rajat

    2014-09-01

    The Hedgehog (Hh) pathway has become an important model to study the cell biology of primary cilia, and reciprocally, the study of ciliary processes provides an opportunity to solve longstanding mysteries in the mechanism of vertebrate Hh signal transduction. The cilium is emerging as an unique compartment for G-protein-coupled receptor (GPCR) signaling in many systems. Two members of the GPCR family, Smoothened and Gpr161, play important roles in the Hh pathway. We review the current understanding of how these proteins may function to regulate Hh signaling and also highlight some of the critical unanswered questions being tackled by the field. Uncovering GPCR-regulated mechanisms important in Hh signaling may provide therapeutic strategies against the Hh pathway that plays important roles in development, regeneration and cancer.

  18. Protein-resistant polyurethane via surface-initiated atom transfer radical polymerization of oligo(ethylene glycol) methacrylate.

    PubMed

    Jin, Zhilin; Feng, Wei; Zhu, Shiping; Sheardown, Heather; Brash, John L

    2009-12-15

    Protein-resistant polyurethane (PU) surfaces were prepared by surface-initiated simultaneous normal and reverse atom transfer radical polymerization (s-ATRP) of poly(oligo(ethylene glycol) methacrylate) (poly (OEGMA)). Oxygen plasma treatment was employed for initial activation of the PU surface. The grafted polymer chain length was adjusted by varying the molar ratio of monomer to sacrificial initiator in solution from 5:1 to 200:1. The modified PU surfaces were characterized by water contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Protein adsorption experiments from tris-buffered saline (TBS) and plasma were carried out to evaluate the protein-resistance of the surfaces. Adsorption from single and binary protein solutions as well as from plasma was significantly reduced after modification. Adsorption decreased with increasing poly(OEGMA) chain length. Fibrinogen (Fg) adsorption on the 200:1 monomer/initiator surface was in the range of 3-33 ng/cm(2) representing 96-99% reduction compared with the unmodified PU. Fg adsorption from 0.01-10% plasma was as low as 1-5 ng/cm(2). Moreover, binary protein adsorption experiments using Fg and lysozyme (Lys) showed that protein size is a factor in the protein resistance of these surfaces.

  19. Free Radical Reactions in Food.

    ERIC Educational Resources Information Center

    Taub, Irwin A.

    1984-01-01

    Discusses reactions of free radicals that determine the chemistry of many fresh, processed, and stored foods. Focuses on reactions involving ascorbic acid, myoglobin, and palmitate radicals as representative radicals derived from a vitamin, metallo-protein, and saturated lipid. Basic concepts related to free radical structure, formation, and…

  20. Free Radical Reactions in Food.

    ERIC Educational Resources Information Center

    Taub, Irwin A.

    1984-01-01

    Discusses reactions of free radicals that determine the chemistry of many fresh, processed, and stored foods. Focuses on reactions involving ascorbic acid, myoglobin, and palmitate radicals as representative radicals derived from a vitamin, metallo-protein, and saturated lipid. Basic concepts related to free radical structure, formation, and…

  1. Primary photodissociation pathways of epichlorohydrin and analysis of the C-C bond fission channels from an O(3P)+allyl radical intermediate

    NASA Astrophysics Data System (ADS)

    FitzPatrick, Benjamin L.; Alligood, Bridget W.; Butler, Laurie J.; Lee, Shih-Huang; Lin, Jim-Min, Jr.

    2010-09-01

    This study initially characterizes the primary photodissociation processes of epichlorohydrin, c-(H2COCH)CH2Cl. The three dominant photoproduct channels analyzed are c-(H2COCH)CH2+Cl, c-(H2COCH)+CH2Cl, and C3H4O+HCl. In the second channel, the c-(H2COCH) photofission product is a higher energy intermediate on C2H3O global potential energy surface and has a small isomerization barrier to vinoxy. The resulting highly vibrationally excited vinoxy radicals likely dissociate to give the observed signal at the mass corresponding to ketene, H2CCO. The final primary photodissociation pathway HCl+C3H4O evidences a recoil kinetic energy distribution similar to that of four-center HCl elimination in chlorinated alkenes, so is assigned to production of c-(H2COC)=CH2; the epoxide product is formed with enough vibrational energy to isomerize to acrolein and dissociate. The paper then analyzes the dynamics of the C3H5O radical produced from C-Cl bond photofission. When the epoxide radical photoproduct undergoes facile ring opening, it is the radical intermediate formed in the O(P3)+allyl bimolecular reaction when the O atom adds to an end C atom. We focus on the HCO+C2H4 and H2CO+C2H3 product channels from this radical intermediate in this report. Analysis of the velocity distribution of the momentum-matched signals from the HCO+C2H4 products at m/e=29 and 28 shows that the dissociation of the radical intermediate imparts a high relative kinetic energy, peaking near 20 kcal/mol, between the products. Similarly, the energy imparted to relative kinetic energy in the H2CO+C2H3 product channel of the O(P3)+allyl radical intermediate also peaks at high-recoil kinetic energies, near 18 kcal/mol. The strongly forward-backward peaked angular distributions and the high kinetic energy release result from tangential recoil during the dissociation of highly rotationally excited nascent radicals formed photolytically in this experiment. The data also reveal substantial branching to an HCCH+H3

  2. Reactions of amino acids, peptides, and proteins with oxidized metabolites of tris(p-carboxyltetrathiaaryl)methyl radical EPR probes.

    PubMed

    Decroos, Christophe; Boucher, Jean-Luc; Mansuy, Daniel; Xu-Li, Yun

    2014-04-21

    Oxidation of the tris(p-carboxyltetrathiaaryl)methyl (TAM) EPR radical probe, TAMa(•), by rat liver microsomes (RLM) + NADPH, or horseradish peroxidase (HRP) + H2O2, or K2IrCl6, led to an intermediate cation, TAMa(+), which was treated with glutathione (GSH), with formation of an adduct, TAMa-SG(•), resulting from the substitution of a TAMa(•) carboxylate group with the SG group. L-α-Amino acids containing a strong nucleophilic residue (NuH), such as L-cysteine or L-histidine, also reacted with TAMa(+), with formation of radical adducts TAMa-Nu(•) in which a carboxylate group of TAMa(•) was replaced with Nu. Other less nucleophilic L-α-amino acids, such as L-arginine, L-serine, L-threonine, L-tyrosine, or L-aspartate, as well as the tetrapeptide H-(Gly)4-OH, reacted with TAMa(+) via their α-NH2 group, with formation of an iminoquinone methide, IQMa, deriving from an oxidative decarboxylation and amination of TAMa(•). Upon reaction of TAMa(+) with L-proline and L-lysine, N-substituted iminoquinone methide adducts, IQMa-Pro and IQMa-Lys, were formed. Finally, preliminary results showed that oxidation of TAMa(•) in the presence of bovine serum albumin (BSA), led to the covalent binding of TAMa-derived metabolites to BSA. Oxidation of another frequently used TAM probe, TAMb(•) (Oxo63), in the presence of GSH, N-acetyl-cysteine methyl ester, or histidine also led to TAMb-Nu(•) adducts equivalent to the corresponding TAMa-Nu(•) adducts, suggesting that the oxidative metabolism of such TAM(•) probes could lead to protein covalent binding. Moreover, the above data describe an easy access to new TAM radical EPR probes coupled to amino acids, peptides or proteins that could be useful for addressing various biological targets.

  3. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    NASA Astrophysics Data System (ADS)

    Yuan, Huihui; Qian, Bin; Zhang, Wei; Lan, Minbo

    2016-02-01

    An anti-fouling surface of polyurethane (PU) film grafted with Poly(N-vinylpyrrolidone) (PVP) was prepared through surface-initiated atom transfer radical polymerization (SI-ATRP). And the polymerization time was investigated to obtain PU films with PVP brushes of different lengths. The surface properties and protein adsorption of modified PU films were evaluated. The results showed that the hydrophilicity of PU-PVP films were improved with the increase of polymerization time, which was not positive correlation with the surface roughness due to the brush structure. Additionally, the protein resistance performance was promoted when prolonging the polymerization time. The best antifouling PU-PVP (6.0 h) film reduced the adsoption level of bovine serum albumin (BSA), lysozyme (LYS), and brovin serum fibrinogen (BFG) by 93.4%, 68.3%, 85.6%, respectively, compared to the unmodified PU film. The competitive adsorption of three proteins indicated that LYS preferentially adsorbed on the modified PU film, while BFG had the lowest adsorption selectivity. And the amount of BFG on PU-PVP (6.0 h) film reduced greatly to 0.08 μg/cm2, which was almost one-tenth of its adsorption from the single-protein system. Presented results suggested that both hydrophilicity and surface roughness might be the important factors in all cases of protein adsorption, and the competitive or selective adsorption might be related to the size of the proteins, especially on the non-charged films.

  4. Endogenous 3,4-dihydroxyphenylalanine and dopaquinone modifications on protein tyrosine: links to mitochondrially derived oxidative stress via hydroxyl radical.

    PubMed

    Zhang, Xu; Monroe, Matthew E; Chen, Baowei; Chin, Mark H; Heibeck, Tyler H; Schepmoes, Athena A; Yang, Feng; Petritis, Brianne O; Camp, David G; Pounds, Joel G; Jacobs, Jon M; Smith, Desmond J; Bigelow, Diana J; Smith, Richard D; Qian, Wei-Jun

    2010-06-01

    Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3,4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first proteome survey of endogenous site-specific modifications, i.e. DOPA and its further oxidation product dopaquinone in mouse brain and heart tissues. Results from LC-MS/MS analyses included 50 and 14 DOPA-modified tyrosine sites identified from brain and heart, respectively, whereas only a few nitrotyrosine-containing peptides, a more commonly studied marker of oxidative stress, were detectable, suggesting the much higher abundance for DOPA modification as compared with tyrosine nitration. Moreover, 20 and 12 dopaquinone-modified peptides were observed from brain and heart, respectively; nearly one-fourth of these peptides were also observed with DOPA modification on the same sites. For both tissues, these modifications are preferentially found in mitochondrial proteins with metal binding properties, consistent with metal-catalyzed hydroxyl radical formation from mitochondrial superoxide and hydrogen peroxide. These modifications also link to a number of mitochondrially associated and other signaling pathways. Furthermore, many of the modification sites were common sites of previously reported tyrosine phosphorylation, suggesting potential disruption of signaling pathways. Collectively, the results suggest that these modifications are linked with mitochondrially derived oxidative stress and may serve as sensitive markers for disease pathologies.

  5. The use of the methods of radiolysis to explore the mechanisms of free radical modifications in proteins.

    PubMed

    Houée-Levin, Chantal; Bobrowski, Krzysztof

    2013-10-30

    The method of radiolysis is based upon the interaction of ionising radiation with the solvent (water). One can form the same free radicals as in conditions of oxidative stress ((•)OH, O2(•)(-), NO2(•)…). Moreover, the quantity of reactive oxygen (ROS) or nitrogen (RNS) species formed in the irradiated medium can be calculated knowing the dose and the radiation chemical yield, G, thus this method is quantitative. The use of the method of radiolysis has provided a wealth of data, especially about the kinetics of the oxidation by various free radicals and their mechanisms, the identification of transients formed, their lifetimes and the possibility to repair them by the so-called antioxidants. In this review we have collected the most recent data about protein oxidation that might be useful to a proteomic approach. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Beneficial effect of internal hydrogen bonding interactions on the beta-fragmentation of primary alkoxyl radicals. Two-step conversion of D-xylo- and D-ribofuranoses into L-threose and D-erythrose, respectively.

    PubMed

    Hernandez-García, Luís; Quintero, Leticia; Sánchez, Mario; Sartillo-Piscil, Fernando

    2007-10-26

    Primary alkoxyl free radicals were generated from their readily synthesized N-phthalimido derivatives under reductive conditions. Primary alkoxyl radicals derived from their corresponding xylo- and ribofuranose derivatives underwent, exclusively, an unusual beta-fragmentation affording L-threose and D-erythrose derivatives, respectively. This occurs because the alkoxyl radical is capable of achieving an internal hydrogen-bonding interaction leading to a stable six-membered ring intramolecular hydrogen-bonded structure. When the hydroxyl group is protected, the beta-fragmentation pathway is prevented and the hydrogen atom transfer (HAT) pathway occurs. Computational studies provided strong support for the experimental observations.

  7. EPR studies on a stable sulfinyl radical observed in the iron-oxygen-reconstituted Y177F/I263C protein R2 double mutant of ribonucleotide reductase from mouse.

    PubMed

    Adrait, Annie; Ohrström, Maria; Barra, Anne-Laure; Thelander, Lars; Gräslund, Astrid

    2002-05-21

    Ribonucleotide reductase (RNR) catalyzes the biosynthesis of deoxyribonucleotides. The active enzyme contains a diiron center and a tyrosyl free radical required for enzyme activity. The radical is located at Y177 in the R2 protein of mouse RNR. The radical is formed concomitantly with the mu-oxo-bridged diferric center in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. EPR at 9.6 and 285 GHz was used to investigate the reconstitution reaction in the double-mutant Y177F/I263C of mouse protein R2. The aim was to produce a protein-linked radical derived from the Cys residue in the mutant protein to investigate its formation and characteristics. The mutation Y177F hinders normal radical formation at Y177, and the I263C mutation places a Cys residue at the same distance from the iron center as Y177 in the native protein. In the reconstitution reaction, we observed small amounts of a transient radical with a probable assignment to a peroxy radical, followed by a stable sulfinyl radical, most likely located on C263. The unusual radical stability may be explained by the hydrophobic surroundings of C263, which resemble the hydrophobic pocket surrounding Y177 in native protein R2. The observation of a sulfinyl radical in RNR strengthens the relationship between RNR and another free radical enzyme, pyruvate formate-lyase, where a similar relatively stable sulfinyl radical has been observed in a mutant. Sulfinyl radicals may possibly be considered as stabilized forms of very short-lived thiyl radicals, proposed to be important intermediates in the radical chemistry of RNR.

  8. Mast Cells Density Positive to Tryptase Correlate with Microvascular Density in both Primary Gastric Cancer Tissue and Loco-Regional Lymph Node Metastases from Patients That Have Undergone Radical Surgery

    PubMed Central

    Ammendola, Michele; Sacco, Rosario; Zuccalà, Valeria; Luposella, Maria; Patruno, Rosa; Gadaleta, Pietro; Zizzo, Nicola; Gadaleta, Cosmo Damiano; De Sarro, Giovambattista; Sammarco, Giuseppe; Oltean, Mihai; Ranieri, Girolamo

    2016-01-01

    Mast Cells (MCs) play a role in immune responses and more recently MCs have been involved in tumoral angiogenesis. In particular MCs can release tryptase, a potent in vivo and in vitro pro-angiogenic factor via proteinase-activated receptor-2 (PAR-2) activation and mitogen-activated protein kinase (MAPK) phosphorylation. MCs can release tryptase following c-Kit receptor activation. Nevertheless, no data are available concerning the relationship among MCs Density Positive to Tryptase (MCDPT) and Microvascular Density (MVD) in both primary gastric cancer tissue and loco-regional lymph node metastases. A series of 75 GC patients with stage T2–3N2–3M0 (by AJCC for Gastric Cancer Seventh Edition) undergone to radical surgery were selected for the study. MCDPT and MVD were evaluated by immunohistochemistry and by image analysis system and results were correlated each to other in primary tumor tissue and in metastatic lymph nodes harvested. Furthermore, tissue parameters were correlated with important clinico-pathological features. A significant correlation between MCDPT and MVD was found in primary gastric cancer tissue and lymph node metastases. Pearson t-test analysis (r ranged from 0.74 to 0.79; p-value ranged from 0.001 to 0.003). These preliminary data suggest that MCDPT play a role in angiogenesis in both primary tumor and in lymph node metastases from GC. We suggest that MCs and tryptase could be further evaluated as novel targets for anti-angiogenic therapies. PMID:27854307

  9. Mast Cells Density Positive to Tryptase Correlate with Microvascular Density in both Primary Gastric Cancer Tissue and Loco-Regional Lymph Node Metastases from Patients That Have Undergone Radical Surgery.

    PubMed

    Ammendola, Michele; Sacco, Rosario; Zuccalà, Valeria; Luposella, Maria; Patruno, Rosa; Gadaleta, Pietro; Zizzo, Nicola; Gadaleta, Cosmo Damiano; De Sarro, Giovambattista; Sammarco, Giuseppe; Oltean, Mihai; Ranieri, Girolamo

    2016-11-15

    Mast Cells (MCs) play a role in immune responses and more recently MCs have been involved in tumoral angiogenesis. In particular MCs can release tryptase, a potent in vivo and in vitro pro-angiogenic factor via proteinase-activated receptor-2 (PAR-2) activation and mitogen-activated protein kinase (MAPK) phosphorylation. MCs can release tryptase following c-Kit receptor activation. Nevertheless, no data are available concerning the relationship among MCs Density Positive to Tryptase (MCDPT) and Microvascular Density (MVD) in both primary gastric cancer tissue and loco-regional lymph node metastases. A series of 75 GC patients with stage T2-3N2-3M₀ (by AJCC for Gastric Cancer Seventh Edition) undergone to radical surgery were selected for the study. MCDPT and MVD were evaluated by immunohistochemistry and by image analysis system and results were correlated each to other in primary tumor tissue and in metastatic lymph nodes harvested. Furthermore, tissue parameters were correlated with important clinico-pathological features. A significant correlation between MCDPT and MVD was found in primary gastric cancer tissue and lymph node metastases. Pearson t-test analysis (r ranged from 0.74 to 0.79; p-value ranged from 0.001 to 0.003). These preliminary data suggest that MCDPT play a role in angiogenesis in both primary tumor and in lymph node metastases from GC. We suggest that MCs and tryptase could be further evaluated as novel targets for anti-angiogenic therapies.

  10. Variations in C-reactive protein, plasma free radicals and fibrinogen values in patients with osteoarthritis treated with Pycnogenol.

    PubMed

    Belcaro, G; Cesarone, M R; Errichi, S; Zulli, C; Errichi, B M; Vinciguerra, G; Ledda, A; Di Renzo, A; Stuard, S; Dugall, M; Pellegrini, L; Gizzi, G; Ippolito, E; Ricci, A; Cacchio, M; Cipollone, G; Ruffini, I; Fano, F; Hosoi, M; Rohdewald, P

    2008-01-01

    In a previous, double-blind, placebo-controlled study we evaluated the efficacy of a 3-month treatment with Pycnogenol for 156 patients with osteoarthritis of the knee. Pycnogenol significantly decreased joint pain and improved joint function as evaluated using the WOMAC score and walking performance of patients on a treadmill. In this study, we further investigated the anti-inflammatory and antioxidant activity of Pycnogenol in a subset of the osteoarthritis patients presenting with elevated C-reactive protein (CRP) and plasma-free radicals. Elevated CRP levels have been suggested to be associated with disease progression in osteoarthritis. In our study, 29 subjects of the Pycnogenol group and 26 patients in the placebo group showed CRP levels higher than 3 mg/l at baseline. Comparison of blood specimens drawn at baseline and after 3-month treatment showed that Pycnogenol significantly decreased plasma free radicals to 70.1% of baseline values. Plasma CRP levels decreased from baseline 3.9 mg/l to 1.1 mg/l in the Pycnogenol group whereas the control group had initial values of 3.9 mg/l which decreased to 3.6 mg/l. The CRP decrease in the Pycnogenol was statistical significant as compared to the control group (P < 0.05). Fibrinogen levels were found to be lowered to 62.8% of initial values (P < 0.05) in response to Pycnogenol. No significant changes for plasma free radicals, CRP and fibrinogen were found in the placebo-treated group. The decrease of systemic inflammatory markers suggests that Pycnogenol may exert anti-inflammatory activity in osteoarthritic joints and patients did not present with other ailments or infections. The nature of the anti-inflammatory effects of Pycnogenol with regard to CRP warrants further investigation.

  11. Endogenous 3, 4- Dihydroxyphenylalanine and Dopaquinone Modifications on Protein Tyrosine: links to mitochondrially derived oxidative stress via hydroxyl radical

    SciTech Connect

    Zhang, Xu; Monroe, Matthew E.; Chen, Baowei; Chin, Mark H.; Heibeck, Tyler H.; Schepmoes, Athena A.; Yang, Feng; Petritis, Brianne O.; Camp, David G.; Pounds, Joel G.; Jacobs, Jon M.; Smith, Desmond J.; Bigelow, Diana J.; Smith, Richard D.; Qian, Weijun

    2010-06-02

    Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3, 4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first global proteome survey of endogenous site-specific modifications, i.e, DOPA and its further oxidation product dopaquinone (DQ) in mouse brain and heart tissues. Results from LC-MS/MS analyses included 203 and 71 DOPA-modified tyrosine sites identified from brain and heart, respectively, with a false discovery rate of ~1%; while only a few nitrotyrosine containing peptides, a more commonly studied marker of oxidative stress, were detectable, suggesting the much higher abundance for DOPA modification as compared with tyrosine nitration. Moreover, 57 and 29 DQ modified peptides were observed from brain and heart, respectively; nearly half of these peptides were also observed with DOPA modification on the same sites. For both tissues, these modifications are preferentially found in mitochondrial proteins with metal-binding properties, consistent with metal catalyzed hydroxyl radical formation from mitochondrial superoxide and hydrogen peroxide. These modifications also link to a number of mitochondria-associated and other signaling pathways. Furthermore, many of the modification sites were common sites of previously reported tyrosine phosphorylation suggesting potential disruption of signaling pathways. Structural aspects of DOPA-modified tyrosine sequences are distinct from those of nitrotyrosines suggesting that each type of modifications provides a marker for different in vivo reactive chemistries and can be used to predict sensitive protein targets. Collectively, the results suggest that these modifications are linked with mitochondrially-derived oxidative stress, and may serve as sensitive markers for disease pathologies.

  12. Primary radical radiotherapy for squamous cell carcinoma of the middle ear and external auditory cana--an historical series.

    PubMed

    Pemberton, L S; Swindell, R; Sykes, A J

    2006-06-01

    To evaluate patients treated with radical radiotherapy alone for squamous cell carcinoma of the middle ear (MEC) and external auditory canal (EAC) in terms of freedom from local recurrence, cancer-specific survival and morbidity. Between 1965 and 1988, 123 patients were treated, 70 with MEC and 53 with EAC. The median age was 64 years (range 21-86) and 78% presented as late stage. The median dose was 55 Gy (range 39-55) in 16 once daily fractions (range 13-21). At 5 and 10 years, respectively, freedom from local recurrence was 56 and 56%, disease-free survival was 45 and 43%, cancer-specific survival was 53 and 51%, and overall survival was 40 and 21%. Cancer-specific survival was significantly worse with late stage as opposed to early stage (P = 0.0026), as was local recurrence (P = 0.0088). No differences in survival and local control were seen according to site. Radionecrosis developed in 6% of patients. Combined treatment using radiotherapy and radical surgery is often favoured. This large series shows that radical radiotherapy achieves comparable results in terms of local control and cancer-specific survival. Our radiotherapy regimen is now 55 Gy in 20 daily fractions to reduce late morbidity. Radiotherapy alone remains a viable option, especially as morbidity can be minimised and target volume delineation optimised using computer planning in the future.

  13. Selective radical addition with a designed heterobifunctional halide: a primary study toward sequence-controlled polymerization upon template effect.

    PubMed

    Ida, Shohei; Terashima, Takaya; Ouchi, Makoto; Sawamoto, Mitsuo

    2009-08-12

    A ruthenium(II)-catalyzed, highly selective, quantitative radical addition of an alkene, methacrylic acid (MAA), has been achieved by using a template halide (2) containing a built-in amine group as a recognition site for the carboxyl group of the substrate. The specific ionic binding of MAA by the amine template (1:1 molar ratio) led to preferential formation of the 1:1 MAA-2 adduct, whereas a similar halide without a template induced MAA oligomerization even in the presence of an externally added amine. A competitive radical addition of MAA versus its ester form [methyl methacrylate (MMA)] on the halide further demonstrated that the substrate selectivity [k'(MAA)/k'(MMA)] for 2 is enhanced more than 10 times by the intramolecular introduction of the template relative to the result for the nontemplate halide. These specificities are most likely triggered by the specific interaction (recognition) of the carboxyl group in MAA via the acid-selective template amine, which is implanted in the close vicinity of the radical addition site in 2. These results intimate possibility of control over the repeat-unit sequence in precision polymerization.

  14. Efficacy of DL-alpha-lipoic acid on methanol induced free radical changes, protein oxidative damages and hsp70 expression in folate deficient rat nervous tissue.

    PubMed

    Rajamani, Rathinam; Muthuvel, Arumugam; Manikandan, Sundaramahalingam; Srikumar, Ramasundaram; Sheeladevi, Rathinasamy

    2007-05-01

    DL-alpha-Lipoic acid (LPA) was reported to be effective in reducing free radicals generated by oxidative stress. The protective of effect of LPA on methanol (MeOH) induced free radical changes and oxidative damages in discrete regions of rat brain have been reported in this study. Folate deficient rat (FDD) model was used. The five animal groups (saline control, FDD control, FDD+MeOH, FDD+LPA+MeOH, LPA control) were used. The FDD+MeOH and FDD+LPA+MeOH animals were injected intraperitoneally with methanol (3gm/kg). After 24h, the level of free radical scavengers such as, superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione was estimated in six discrete regions of brain, retina and optic nerve. Level of protein thiol, protein carbonyl and lipid peroxidation was also estimated. Expression of heat shock protein 70 mRNA (hsp70) was studied in the cerebellum and hippocampus by reverse transcriptase PCR. All the samples showed elevation in the level of free radical scavenging enzymes and reduced level of glutathione in the FDD+MeOH group in relation to the other groups. hsp70 expression was more in FDD+MeOH group when compared to FDD+LPA+MeOH group. In conclusion, MeOH exposure leads to increased free radical generation and protein oxidative damages in the rat nervous tissue. Treatment with LPA prevents oxidative damage induced by MeOH exposure.

  15. Principles of hydrogen radical mediated peptide/protein fragmentation during matrix-assisted laser desorption/ionization mass spectrometry.

    PubMed

    Asakawa, Daiki

    2016-07-01

    Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) is a very easy way to obtain large sequence tags and, thereby, reliable identification of peptides and proteins. Recently discovered new matrices have enhanced the MALDI-ISD yield and opened new research avenues. The use of reducing and oxidizing matrices for MALDI-ISD of peptides and proteins favors the production of fragmentation pathways involving "hydrogen-abundant" and "hydrogen-deficient" radical precursors, respectively. Since an oxidizing matrix provides information on peptide/protein sequences complementary to that obtained with a reducing matrix, MALDI-ISD employing both reducing and oxidizing matrices is a potentially useful strategy for de novo peptide sequencing. Moreover, a pseudo-MS(3) method provides sequence information about N- and C-terminus extremities in proteins and allows N- and C-terminal side fragments to be discriminated within the complex MALDI-ISD mass spectrum. The combination of high mass resolution of a Fourier transform-ion cyclotron resonance (FTICR) analyzer and the software suitable for MALDI-ISD facilitates the interpretation of MALDI-ISD mass spectra. A deeper understanding of the MALDI-ISD process is necessary to fully exploit this method. Thus, this review focuses first on the mechanisms underlying MALDI-ISD processes, followed by a discussion of MALDI-ISD applications in the field of proteomics. © 2014 Wiley Periodicals, Inc., Mass Spec Rev 35:535-556, 2016. © 2014 Wiley Periodicals, Inc.

  16. Visualization of a radical B12 enzyme with its G-protein chaperone

    SciTech Connect

    Jost, Marco; Cracan, Valentin; Hubbard, Paul A.; Banerjee, Ruma; Drennan, Catherine L.

    2015-02-09

    G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. In this paper, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms of IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Finally and notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.

  17. Visualization of a radical B12 enzyme with its G-protein chaperone

    DOE PAGES

    Jost, Marco; Cracan, Valentin; Hubbard, Paul A.; ...

    2015-02-09

    G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. In this paper, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms ofmore » IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Finally and notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.« less

  18. Reduction of the tyrosyl radical and the iron center in protein R2 of ribonucleotide reductase from mouse, herpes simplex virus and E. coli by p-alkoxyphenols.

    PubMed

    Pötsch, S; Sahlin, M; Langelier, Y; Gräslund, A; Lassmann, G

    1995-10-23

    The rate of reduction of the tyrosyl radical in the small subunit of ribonucleotide reductase (protein R2) from E. coli, mouse, and herpes simplex virus (HSV-2) by a series of p-alkoxyphenols with different alkyl chains, have been studied by stopped-flow UV-vis and stopped-flow EPR spectroscopy. The reduction and release of iron in R2 by the inhibitors was followed using bathophenanthroline as chelator of Fe2+. p-Alkoxyphenols reduce the mouse R2 tyrosyl radical 1-2 orders of magnitude faster than the HSV-2 and E. coli radical. In contrast to E. coli, the iron center in R2 from mouse and HSV-2 is reduced by the inhibitors. For mouse R2, the rate of reduction of the tyrosyl radical increases in parallel with increasing alkyl chain length of the inhibitor, an observation which may be important for the design of new antiproliferative drugs.

  19. Interdisciplinary model of care (RADICALS) for early detection and management of chronic obstructive pulmonary disease (COPD) in Australian primary care: study protocol for a cluster randomised controlled trial.

    PubMed

    Liang, Jenifer; Abramson, Michael J; Zwar, Nicholas; Russell, Grant; Holland, Anne E; Bonevski, Billie; Mahal, Ajay; Hecke, Benjamin van; Phillips, Kirsten; Eustace, Paula; Paul, Eldho; Petrie, Kate; Wilson, Sally; George, Johnson

    2017-09-18

    Up to half of all smokers develop clinically significant chronic obstructive pulmonary disease (COPD). Gaps exist in the implementation and uptake of evidence-based guidelines for managing COPD in primary care. We describe the methodology of a cluster randomised controlled trial (cRCT) evaluating the efficacy and cost-effectiveness of an interdisciplinary model of care aimed at reducing the burden of smoking and COPD in Australian primary care settings. A cRCT is being undertaken to evaluate an interdisciplinary model of care (RADICALS - Review of Airway Dysfunction and Interdisciplinary Community-based care of Adult Long-term Smokers). General practice clinics across Melbourne, Australia, are identified and randomised to the intervention group (RADICALS) or usual care. Patients who are current or ex-smokers, of at least 10 pack years, including those with an existing diagnosis of COPD, are being recruited to identify 280 participants with a spirometry-confirmed diagnosis of COPD. Handheld lung function devices are being used to facilitate case-finding. RADICALS includes individualised smoking cessation support, home-based pulmonary rehabilitation and home medicines review. Patients at control group sites receive usual care and Quitline referral, as appropriate. Follow-ups occur at 6 and 12 months from baseline to assess changes in quality of life, abstinence rates, health resource utilisation, symptom severity and lung function. The primary outcome is change in St George's Respiratory Questionnaire score of patients with COPD at 6 months from baseline. This project has been approved by the Monash University Human Research Ethics Committee and La Trobe University Human Ethics Committee (CF14/1018 - 2014000433). Results of the study will be disseminated in peer-reviewed journals and research conferences. If the intervention is successful, the RADICALS programme could potentially be integrated into general practices across Australia and sustained over time. ACTRN

  20. Extended radical mastectomy versus simple mastectomy followed by radiotherapy in primary breast cancer. A fifty-year follow-up to the Copenhagen Breast Cancer randomised study.

    PubMed

    Johansen, Helge; Kaae, Sigvard; Jensen, Maj-Britt; Mouridsen, Henning T

    2008-01-01

    From November 1951 to December 1957, 666 consecutive patients with untreated primary breast cancer admitted to the Radium Center in Copenhagen were randomised before their operability was evaluated into two groups, simple mastectomy with postoperative radiotherapy or extended radical mastectomy. Following physical examination 241 of the patients were excluded, primarily due to tumours deemed inoperable due to clinical criteria (n =107) and due to poor general condition (n =69). Twenty-five years results of disease-free free survival and fifty years results of survival are presented, showing no difference between the two groups. Patients with clinical stage I did significantly better than patients with stage II-III tumours. Patients with grade I tumours had a better survival than patients with grade II-III. The breast cancer associated mortality was lower in premenopausal patients compared to postmenopausal patients. An excess mortality due to breast cancer was evident up to 20-25 years following the primary diagnosis.

  1. Binding of Gallic Acid and Epigallocatechin Gallate to Heat-Unfolded Whey Proteins at Neutral pH Alters Radical Scavenging Activity of in Vitro Protein Digests.

    PubMed

    Cao, Yanyun; Xiong, Youling L

    2017-09-27

    Preheated (80 °C for 9 min) whey protein isolate (HWPI) was reacted with 20, 120, and 240 μmol/g (protein basis) gallic acid (GA) or epigallocatechin gallate (EGCG) at neutral pH and 25 °C. Isothermal titration calorimetry and fluorometry showed a similar trend that GA binding to HWPI was moderate but weaker than EGCG binding. However, the shift of maximal fluorescence emission wavelength in opposite directions in response to GA (blue) and EGCG (red) suggests discrepant binding patterns. Electrophoresis results showed that EGCG induced formation of HWPI complexes while GA only had a marginal effect. Both free and phenolic-bound HWPI exhibited mild antiradical activity. However, when subjected to in vitro digestion, synergistic radical-scavenging activity was produced between the phenolics and peptides with the highest synergism being observed on 120 μmol/g phenolics.

  2. Oil, protein, antioxidants and free radical scavenging activity of stone from wild olive trees (Olea europaea L.).

    PubMed

    Hannachi, Hédia; Elfalleh, Walid; Marzouk, Sizaiem

    2013-05-01

    The wild olive trees or oleaster (var. sylvestris) and the cultivated olive trees (var. europaea) constitute the two botanical varieties of Olea europaea L. from Mediterranean. In this study, a partial chemical profile was conducted including the total lipids, the fatty acid profiles, soluble proteins, polyphenols, flavanoids contents and antioxidants activities of stone from six oleaster trees. The comparison was made by two olive cultivars cultivated in the same region. The oleaster and cultivar stones were richer in oil content having an average of 8.99 and 7.38 % dry weight basis (DW), respectively. Qualitatively, all studied oils have the same fatty acids profile with the oleic acid C18:1n-9 as the major fatty acid. The oleaster stone oils were richer in monounsaturated fatty acids having an average of 64.87%. They, also, richer in protein content with an average of 198.86 mg/g DW.The globulin is the major fraction, followed by the albumin, the prolamin and the glutemin fractions. The oleaster stone extracts contain polyphenols, flavonoids with an average of 151.14 and 11.91 mg gallic acid equivalent/100g of DW, respectively. The studied extracts showed antioxidant activity using the free radical scavenging activity determined by DPPH and ABTS. The unexploited oleaster stone seems to be a source of oil with good fatty acids balance, in protein and antioxidants metabolites and would be useful for the formulation of supplements and/or pharmaceutical ingredients.

  3. The sensitivity of a radical pair compass magnetoreceptor can be significantly amplified by radical scavengers.

    PubMed

    Kattnig, Daniel R; Hore, P J

    2017-09-14

    Birds have a remarkable ability to obtain navigational information from the Earth's magnetic field. The primary detection mechanism of this compass sense is uncertain but appears to involve the quantum spin dynamics of radical pairs formed transiently in cryptochrome proteins. We propose here a new version of the current model in which spin-selective recombination of the radical pair is not essential. One of the two radicals is imagined to react with a paramagnetic scavenger via spin-selective electron transfer. By means of simulations of the spin dynamics of cryptochrome-inspired radical pairs, we show that the new scheme offers two clear and important benefits. The sensitivity to a 50 μT magnetic field is greatly enhanced and, unlike the current model, the radicals can be more than 2 nm apart in the magnetoreceptor protein. The latter means that animal cryptochromes that have a tetrad (rather than a triad) of tryptophan electron donors can still be expected to be viable as magnetic compass sensors. Lifting the restriction on the rate of the spin-selective recombination reaction also means that the detrimental effects of inter-radical exchange and dipolar interactions can be minimised by placing the radicals much further apart than in the current model.

  4. Chemical modification of lysozyme, glucose 6-phosphate dehydrogenase, and bovine eye lens proteins induced by peroxyl radicals: role of oxidizable amino acid residues.

    PubMed

    Arenas, Andrea; López-Alarcón, Camilo; Kogan, Marcelo; Lissi, Eduardo; Davies, Michael J; Silva, Eduardo

    2013-01-18

    Chemical and structural alterations to lysozyme (LYSO), glucose 6-phosphate dehydrogenase (G6PD), and bovine eye lens proteins (BLP) promoted by peroxyl radicals generated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions were investigated. SDS-PAGE analysis of the AAPH-treated proteins revealed the occurrence of protein aggregation, cross-linking, and fragmentation; BLP, which are naturally organized in globular assemblies, were the most affected proteins. Transmission electron microscopy (TEM) analysis of BLP shows the formation of complex protein aggregates after treatment with AAPH. These structural modifications were accompanied by the formation of protein carbonyl groups and protein hydroperoxides. The yield of carbonyls was lower than that for protein hydroperoxide generation and was unrelated to protein fragmentation. The oxidized proteins were also characterized by significant oxidation of Met, Trp, and Tyr (but not other) residues, and low levels of dityrosine. As the dityrosine yield is too low to account for the observed cross-linking, we propose that aggregation is associated with tryptophan oxidation and Trp-derived cross-links. It is also proposed that Trp oxidation products play a fundamental role in nonrandom fragmentation and carbonyl group formation particularly for LYSO and G6PD. These data point to a complex mechanism of peroxyl-radical mediated modification of proteins with monomeric (LYSO), dimeric (G6PD), and multimeric (BLP) structural organization, which not only results in oxidation of protein side chains but also gives rise to radical-mediated protein cross-links and fragmentation, with Trp species being critical intermediates.

  5. Cigarette smoke-induced reduction in binding of the salivary translocator protein is not mediated by free radicals.

    PubMed

    Nagler, R; Savulescu, D; Gavish, M

    2016-02-01

    Oral cancer is the most common malignancy of the head and neck and its main inducer is exposure to cigarette smoke (CS) in the presence of saliva. It is commonly accepted that CS contributes to the pathogenesis of oral cancer via reactive free radicals and volatile aldehydes. The 18 kDa translocator protein (TSPO) is an intracellular receptor involved in proliferation and apoptosis, and has been linked to various types of cancer. The presence of TSPO in human saliva has been linked to oral cancer, and its binding affinity to its ligand is reduced following exposure to CS. In the present study we wished to further investigate the mechanism behind the CS-induced reduction of TSPO binding by exploring the possible mediatory role of reactive oxygen species (ROS) and volatile aldehydes in this process. We first analyzed TSPO binding in control saliva and in saliva exposed to CS in the presence and absence of various antioxidants. These experiments found that TSPO binding ability was not reversed by any of the antioxidants added, suggesting that CS exerts its effect on TSPO via mechanisms that do not involve volatile aldehydes and free radicals tested. Next, we analyzed TSPO binding in saliva following addition of exogenous ROS in the form of H2O2. These experiments found that TSPO binding was enhanced due to the treatment, once again showing that the CS-induced TSPO binding reduction is not mediated by this common form of ROS. However, the previously reported CS-induced reduction in salivary TSPO binding together with the role of TSPO in cells and its link to cancer strongly suggest that TSPO has a critical role in the pathogenesis of CS-induced oral cancer. The importance of further elucidating the mechanisms behind it should be emphasized. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  6. Superparamagnetic lysozyme surface-imprinted polymer prepared by atom transfer radical polymerization and its application for protein separation.

    PubMed

    Gai, Qing-Qing; Qu, Feng; Liu, Zong-Jian; Dai, Rong-Ji; Zhang, Yu-Kui

    2010-07-30

    Molecular imprinting as a promising and facile separation technique has received much attention because of their high selectivity for target molecules. In this study, the superparamagnetic lysozyme surface-imprinted polymer was prepared by a novel fabricating protocol, the grafting of the imprinted polymer on magnetic particles in aqueous media was done by atom transfer radical polymerization (ATRP), and the properties of the imprinted polymer were characterized in detail. Its high selective adsorption and recognition to lysozyme demonstrated the separation ability of the magnetic imprinted material to template molecule, and it has been used for quick and direct separation of lysozyme from the mixture of standard proteins and real egg white samples under an external magnetic field. Furthermore, the elution of lysozyme from the imprinted material was achieved by PEG/sulphate aqueous two-phase system, which caused lysozyme not only desorption from the imprinted materials but also redistribution in the top and bottom phase of aqueous two-phase system. The aqueous two-phase system exhibited some of the extraction and enrichment effect to desorbed lysozyme. Our results showed that ATRP is a promising method for the protein molecularly imprinted polymer preparation.

  7. An iron-binding protein, Dpr, from Streptococcus mutans prevents iron-dependent hydroxyl radical formation in vitro.

    PubMed

    Yamamoto, Yuji; Poole, Leslie B; Hantgan, Roy R; Kamio, Yoshiyuki

    2002-06-01

    The dpr gene is an antioxidant gene which was isolated from the Streptococcus mutans chromosome by its ability to complement an alkyl hydroperoxide reductase-deficient mutant of Escherichia coli, and it was proven to play an indispensable role in oxygen tolerance in S. mutans. Here, we purified the 20-kDa dpr gene product, Dpr, from a crude extract of S. mutans as an iron-binding protein and found that Dpr formed a spherical oligomer about 9 nm in diameter. Molecular weight determinations of Dpr in solution by analytical ultracentrifugation and light-scattering analyses gave values of 223,000 to 292,000, consistent with a subunit composition of 11.5 to 15 subunits per molecule. The purified Dpr contained iron and zinc atoms and had an ability to incorporate up to 480 iron and 11.2 zinc atoms per molecule. Unlike E. coli Dps and two other members of the Dps family, Dpr was unable to bind DNA. One hundred nanomolar Dpr prevented by more than 90% the formation of hydroxyl radical generated by 10 microM iron(II) salt in vitro. The data shown in this study indicate that Dpr may act as a ferritin-like iron-binding protein in S. mutans and may allow this catalase- and heme-peroxidase-deficient bacterium to grow under air by limiting the iron-catalyzed Fenton reaction.

  8. Oxidative and nitrosative stress induced in myofibrillar proteins by a hydroxyl-radical-generating system: impact of nitrite and ascorbate.

    PubMed

    Villaverde, Adriana; Parra, Vita; Estévez, Mario

    2014-03-12

    Understanding the chemistry behind the redox properties of nitrite and ascorbate is essential to identify the impact of curing agents on food quality and optimize the formulation of cured meat products. This study was designed to gain insight into the interactions between curing agents and myofibrillar proteins (MPs) during in vitro oxidation by a hydroxyl-radical-generating system. MPs (4 mg/mL) were oxidized for 4 days at 37 °C under constant stirring with 25 μM iron(III) and 2.5 mM hydrogen peroxide. Dependent upon the addition of nitrite (0, 75, and 150 mg/L) and ascorbate (0, 250, and 500 mg/L), nine different reaction units were prepared in triplicate (n = 3) according to a total factorial design. Upon completion of the oxidation assay, samples were analyzed for the concentration of tryptophan (TRP), α-aminoadipic semialdehyde (AAS), Schiff bases (SBs), and 3-nitrotyrosine (3NT). Ascorbate at 250 mg/L significantly inhibited the depletion of TRP (∼20% inhibition) and the formation of AAS and SBs (>90% inhibition) in MP suspensions. Nitrite, alone, had a negligible effect on protein oxidation but induced the formation of a specific marker of nitrosative stress, namely, 3NT. Ascorbate was also efficient at inhibiting the formation of 3NT by a dose-dependent anti-nitrosative effect and enabled the antioxidant action of nitrite.

  9. Chelating and radical scavenging activities of soy protein hydrolysates prepared from microbial proteases and their effect on meat lipid peroxidation.

    PubMed

    Zhang, Lei; Li, Jianrong; Zhou, Kequan

    2010-04-01

    Three commercial microbial proteases, neutral protease from Bacillus subtilis (NP), validase from Aspergillus oryze (Val), and alkaline protease from Bacillus licheniformis (AP), were investigated for producing antioxidant hydrolysates from soy protein. The resulting hydrolysates were fractioned by sequential ultrafiltration and their antioxidant properties were examined. All the 12 hydrolysate fractions showed noticeable oxygen radical absorbance capacity (ORAC) but significantly varied from 23.8 to 83.8 micromol Trolox equivalents (TE)/g. The hydrolysates also possessed significantly different 1,1-diphenyl-2-picrylhydrazyl (DPPH()) scavenging activities and transition metal chelating activities. Three fractions with strong antioxidant activities, NP-F1, Val-F1, and AP-F3, were incorporated into ground beef to determine their dose-response on lipid peroxidation during 15-day storage. AP-F3 and NP-F1 but not Val-F1 significantly reduced meat lipid peroxidation by 20.1% and 12.9%, respectively. Our results suggested that the commercial microbial proteases such as B. subtilis and B. licheniformis could be used to produce effective antioxidant hydrolysates from food proteins. Copyright 2009 Elsevier Ltd. All rights reserved.

  10. Primary photodissociation pathways of epichlorohydrin and analysis of the C-C bond fission channels from an O({sup 3}P)+allyl radical intermediate

    SciTech Connect

    FitzPatrick, Benjamin L.; Alligood, Bridget W.; Butler, Laurie J.; Lee, Shih-Huang; Lin, Jim Jr-Min

    2010-09-07

    This study initially characterizes the primary photodissociation processes of epichlorohydrin, c-(H{sub 2}COCH)CH{sub 2}Cl. The three dominant photoproduct channels analyzed are c-(H{sub 2}COCH)CH{sub 2}+Cl, c-(H{sub 2}COCH)+CH{sub 2}Cl, and C{sub 3}H{sub 4}O+HCl. In the second channel, the c-(H{sub 2}COCH) photofission product is a higher energy intermediate on C{sub 2}H{sub 3}O global potential energy surface and has a small isomerization barrier to vinoxy. The resulting highly vibrationally excited vinoxy radicals likely dissociate to give the observed signal at the mass corresponding to ketene, H{sub 2}CCO. The final primary photodissociation pathway HCl+C{sub 3}H{sub 4}O evidences a recoil kinetic energy distribution similar to that of four-center HCl elimination in chlorinated alkenes, so is assigned to production of c-(H{sub 2}COC)=CH{sub 2}; the epoxide product is formed with enough vibrational energy to isomerize to acrolein and dissociate. The paper then analyzes the dynamics of the C{sub 3}H{sub 5}O radical produced from C-Cl bond photofission. When the epoxide radical photoproduct undergoes facile ring opening, it is the radical intermediate formed in the O({sup 3}P)+allyl bimolecular reaction when the O atom adds to an end C atom. We focus on the HCO+C{sub 2}H{sub 4} and H{sub 2}CO+C{sub 2}H{sub 3} product channels from this radical intermediate in this report. Analysis of the velocity distribution of the momentum-matched signals from the HCO+C{sub 2}H{sub 4} products at m/e=29 and 28 shows that the dissociation of the radical intermediate imparts a high relative kinetic energy, peaking near 20 kcal/mol, between the products. Similarly, the energy imparted to relative kinetic energy in the H{sub 2}CO+C{sub 2}H{sub 3} product channel of the O({sup 3}P)+allyl radical intermediate also peaks at high-recoil kinetic energies, near 18 kcal/mol. The strongly forward-backward peaked angular distributions and the high kinetic energy release result from

  11. c-erbB-2 protein overexpression and p53 immunoreaction in primary and recurrent breast cancer tissues.

    PubMed

    Shimizu, C; Fukutomi, T; Tsuda, H; Akashi-Tanaka, S; Watanabe, T; Nanasawa, T; Sugihara, K

    2000-01-01

    We investigated whether expression levels of c-erbB-2 and p53 proteins in breast cancer tissues differ in primary and metastatic lesions. Immunohistochemical staining or sandwich enzyme immunoassay was used to determine expression levels of c-erbB-2 and p53 proteins in 42 breast cancer samples from 21 patients. Estrogen (ER) and progesterone receptors (PgR) were also measured by enzyme immunoassay in each case. All patients had undergone radical surgery for primary tumors and surgical resection of asynchronous metastatic lesions. Thirteen patients (62%) were premenopausal and 14 (67%) received postoperative adjuvant therapies. Median disease-free survival time was 26 months (range, 5-104). The resected metastatic lesions included 1 in the liver, 3 in the lung, and 3 in the supraclavicular lymph nodes. The remaining 14 were local skin lesions. There was no difference in the positivity rate of c-erbB-2 (38%: 8/21) and p53 (39%: 7/18) expression between the primary tumors and the recurrent lesions. In addition, no discordant c-erbB-2 or p53 expression was observed between the primary tumors and their respective metastatic lesions. Positivity rates for ER and PgR were 50% (10/20) and 60% (12/20) for the primary tumors, but only 25% (5/20) and 30% (6/20) for the recurrent lesions, respectively (P = 0. 19 for ER and P = 0.11 for PgR). c-erbB-2 and p53 expression levels in breast cancer cells were almost unchanged as the disease progressed and/or in response to adjuvant therapies, regardless of the hormone receptor status. Copyright 2000 Wiley-Liss, Inc.

  12. AdoMet radical proteins—from structure to evolution—alignment of divergent protein sequences reveals strong secondary structure element conservation

    PubMed Central

    Nicolet, Yvain; Drennan, Catherine L.

    2004-01-01

    Eighteen subclasses of S-adenosyl-l-methionine (AdoMet) radical proteins have been aligned in the first bioinformatics study of the AdoMet radical superfamily to utilize crystallographic information. The recently resolved X-ray structure of biotin synthase (BioB) was used to guide the multiple sequence alignment, and the recently resolved X-ray structure of coproporphyrinogen III oxidase (HemN) was used as the control. Despite the low 9% sequence identity between BioB and HemN, the multiple sequence alignment correctly predicted all but one of the core helices in HemN, and correctly predicted the residues in the enzyme active site. This alignment further suggests that the AdoMet radical proteins may have evolved from half-barrel structures (αβ)4 to three-quarter-barrel structures (αβ)6 to full-barrel structures (αβ)8. It predicts that anaerobic ribonucleotide reductase (RNR) activase, an ancient enzyme that, it has been suggested, serves as a link between the RNA and DNA worlds, will have a half-barrel structure, whereas the three-quarter barrel, exemplified by HemN, will be the most common architecture for AdoMet radical enzymes, and fewer members of the superfamily will join BioB in using a complete (αβ)8 TIM-barrel fold to perform radical chemistry. These differences in barrel architecture also explain how AdoMet radical enzymes can act on substrates that range in size from 10 atoms to 608 residue proteins. PMID:15289575

  13. Copper brain protein protection against free radical-induced neuronal death: Survival ratio in SH-SY5Y neuroblastoma cell cultures.

    PubMed

    Deloncle, Roger; Fauconneau, Bernard; Guillard, Olivier; Delaval, José; Lesage, Gérard; Pineau, Alain

    2017-01-01

    In Creutzfeldt Jakob, Alzheimer and Parkinson diseases, copper metalloproteins such as prion, amyloid protein precursor and α-synuclein are able to protect against free radicals by reduction from cupric Cu(+2) to cupreous Cu(+). In these pathologies, a regional copper (Cu) brain decrease correlated with an iron, zinc or manganese (Mn) increase has previously been observed, leading to local neuronal death and abnormal deposition of these metalloproteins in β-sheet structures. In this study we demonstrate the protective effect of Cu metalloproteins against deleterious free-radical effects. With neuroblastoma SH-SY5Y cell cultures, we show that bovine brain prion protein in Cu but not Mn form prevents free radical-induced neuronal death. The survival ratio of SH-SY5Y cells has been measured after UV irradiation (free radical production), when the incubating medium is supplemented with bovine brain homogenate in native, Cu or Mn forms. This ratio, about 28% without any addition or with bovine brain protein added in Mn form, increases by as much as 54.73% with addition to the culture medium of native bovine brain protein and by as much as 95.95% if the addition is carried out in cupric form. This protective effect of brain copper protein against free radical-induced neuronal death has been confirmed with Inductively Coupled Plasma Mass Spectrometry Mn and Cu measurement in bovine brain homogenates: respectively lower than detection limit and 9.01μg/g dry weight for native form; lower than detection limit and 825.85μg/g dry weight for Cu-supplemented form and 1.75 and 68.1μg/g dry weight in Mn-supplemented brain homogenate. Copyright © 2016 Elsevier GmbH. All rights reserved.

  14. Comparison of two adjuvant hormone therapy regimens in patients with high-risk localized prostate cancer after radical prostatectomy: primary results of study CU1005.

    PubMed

    Chang, Kun; Qin, Xiao-Jian; Zhang, Hai-Liang; Dai, Bo; Zhu, Yao; Shi, Guo-Hai; Shen, Yi-Jun; Zhu, Yi-Ying; Ye, Ding-Wei

    2016-01-01

    The role of adjuvant hormonal therapy and optimized regimens for high-risk localized prostate cancer after radical prostatectomy remains controversial. Herein, the clinical trial CU1005 prospectively evaluated two regimens of maximum androgen blockage  or bicalutamide 150 mg daily as immediate adjuvant therapy for high-risk localized prostate cancer. Overall, 209 consecutive patients were recruited in this study, 107 of whom received 9 months of adjuvant maximum androgen blockage, whereas 102 received 9 months of adjuvant bicalutamide 150 mg. The median postoperative follow-up time was 27.0 months. The primary endpoint was biochemical recurrence. Of the 209 patients, 59 patients developed biochemical recurrence. There was no difference between the two groups with respect to clinical characteristics, including age, pretreatment prostate-specific antigen, Gleason score, surgical margin status, or pathological stages. The maximum androgen blockage group experienced longer biochemical recurrence-free survival (P = 0.004) compared with the bicalutamide 150 mg group. Side-effects in the two groups were similar and could be moderately tolerated in all patients. In conclusion, immediate, 9-month maximum androgen blockage should be considered as an alternative to bicalutamide 150 mg as adjuvant treatment for high-risk localized prostate cancer patients after radical prostatectomy.

  15. Comparison of two adjuvant hormone therapy regimens in patients with high-risk localized prostate cancer after radical prostatectomy: primary results of study CU1005

    PubMed Central

    Chang, Kun; Qin, Xiao-Jian; Zhang, Hai-Liang; Dai, Bo; Zhu, Yao; Shi, Guo-Hai; Shen, Yi-Jun; Zhu, Yi-Ying; Ye, Ding-Wei

    2016-01-01

    The role of adjuvant hormonal therapy and optimized regimens for high-risk localized prostate cancer after radical prostatectomy remains controversial. Herein, the clinical trial CU1005 prospectively evaluated two regimens of maximum androgen blockage or bicalutamide 150 mg daily as immediate adjuvant therapy for high-risk localized prostate cancer. Overall, 209 consecutive patients were recruited in this study, 107 of whom received 9 months of adjuvant maximum androgen blockage, whereas 102 received 9 months of adjuvant bicalutamide 150 mg. The median postoperative follow-up time was 27.0 months. The primary endpoint was biochemical recurrence. Of the 209 patients, 59 patients developed biochemical recurrence. There was no difference between the two groups with respect to clinical characteristics, including age, pretreatment prostate-specific antigen, Gleason score, surgical margin status, or pathological stages. The maximum androgen blockage group experienced longer biochemical recurrence-free survival (P = 0.004) compared with the bicalutamide 150 mg group. Side-effects in the two groups were similar and could be moderately tolerated in all patients. In conclusion, immediate, 9-month maximum androgen blockage should be considered as an alternative to bicalutamide 150 mg as adjuvant treatment for high-risk localized prostate cancer patients after radical prostatectomy. PMID:26323560

  16. The fabrication of superlow protein absorption zwitterionic coating by electrochemically mediated atom transfer radical polymerization and its application.

    PubMed

    Hu, Yichuan; Yang, Guang; Liang, Bo; Fang, Lu; Ma, Guanglong; Zhu, Qin; Chen, Shengfu; Ye, Xuesong

    2015-02-01

    A well-controllable electrochemically mediated surface-initiated atom transfer radical polymerization (e-siATRP) method for the fabrication of superlow protein absorption zwitterionic hydrogel coatings based on poly(sulbetaine methacrylate) (pSBMA) was developed in this work. The effects of the electric condition on polymerization as well as its antifouling performances both in vitro and in vivo were also investigated. Different potentials (-0.08 V, -0.15 V and -0.22 V) and polymerization times (from 8 to 48 h) were chosen to study the polymerization procedure. X-ray photoelectron spectroscopy, atomic force microscopy and ellipsometry measurements were used to characterize the properties of the polymer layers. Ellipsometry measurements showed that a higher potential provided faster polymerization and thicker polymer layers; however, the protein absorption experiments showed that the best polymerization condition was under a constant potential of -0.15 V and 32 h, under which the protein absorption was 0.8% in an enzyme-linked immunosorbent assay (compared to a bare gold electrode). The electrodes with a pSBMA coating effectively deduced the current sensitivity decay both in undiluted serum and in vivo. The usage of the commercially available polymerization monomer of SBMA, the simple convenient synthesis process regardless of the presence of oxygen and the excellent controllability of e-siATRP make it a very promising and universal technique in the preparation of zwitterionic polymer coatings, especially in the development of biocompatible material for implantable devices such as neural and biosensor electrodes.

  17. Structural Analysis of the Glycosylated Intact HIV-1 gp120-b12 Antibody Complex Using Hydroxyl Radical Protein Footprinting.

    PubMed

    Li, Xiaoyan; Grant, Oliver C; Ito, Keigo; Wallace, Aaron; Wang, Shixia; Zhao, Peng; Wells, Lance; Lu, Shan; Woods, Robert J; Sharp, Joshua S

    2017-02-21

    Glycoprotein gp120 is a surface antigen and virulence factor of human immunodeficiency virus 1. Broadly neutralizing antibodies (bNAbs) that react to gp120 from a variety of HIV isolates offer hope for the development of broadly effective immunogens for vaccination purposes, if the interactions between gp120 and bNAbs can be understood. From a structural perspective, gp120 is a particularly difficult system because of its size, the presence of multiple flexible regions, and the large amount of glycosylation, all of which are important in gp120-bNAb interactions. Here, the interaction of full-length, glycosylated gp120 with bNAb b12 is probed using high-resolution hydroxyl radical protein footprinting (HR-HRPF) by fast photochemical oxidation of proteins. HR-HRPF allows for the measurement of changes in the average solvent accessible surface area of multiple amino acids without the need for measures that might alter the protein conformation, such as mutagenesis. HR-HRPF of the gp120-b12 complex coupled with computational modeling shows a novel extensive interaction of the V1/V2 domain, probably with the light chain of b12. Our data also reveal HR-HRPF protection in the C3 domain caused by interaction of the N330 glycan with the b12 light chain. In addition to providing information about the interactions of full-length, glycosylated gp120 with b12, this work serves as a template for the structural interrogation of full-length glycosylated gp120 with other bNAbs to better characterize the interactions that drive the broad specificity of the bNAb.

  18. The radical induced cell death protein 1 (RCD1) supports transcriptional activation of genes for chloroplast antioxidant enzymes

    PubMed Central

    Hiltscher, Heiko; Rudnik, Radoslaw; Shaikhali, Jehad; Heiber, Isabelle; Mellenthin, Marina; Meirelles Duarte, Iuri; Schuster, Günter; Kahmann, Uwe; Baier, Margarete

    2014-01-01

    The rimb1 (redox imbalanced 1) mutation was mapped to the RCD1 locus (radical-induced cell death 1; At1g32230) demonstrating that a major factor involved in redox-regulation genes for chloroplast antioxidant enzymes and protection against photooxidative stress, RIMB1, is identical to the regulator of disease response reactions and cell death, RCD1. Discovering this link let to our investigation of its regulatory mechanism. We show in yeast that RCD1 can physically interact with the transcription factor Rap2.4a which provides redox-sensitivity to nuclear expression of genes for chloroplast antioxidant enzymes. In the rimb1 (rcd1-6) mutant, a single nucleotide exchange results in a truncated RCD1 protein lacking the transcription factor binding site. Protein-protein interaction between full-length RCD1 and Rap2.4a is supported by H2O2, but not sensitive to the antioxidants dithiotreitol and ascorbate. In combination with transcript abundance analysis in Arabidopsis, it is concluded that RCD1 stabilizes the Rap2.4-dependent redox-regulation of the genes encoding chloroplast antioxidant enzymes in a widely redox-independent manner. Over the years, rcd1-mutant alleles have been described to develop symptoms like chlorosis, lesions along the leaf rims and in the mesophyll and (secondary) induction of extra- and intra-plastidic antioxidant defense mechanisms. All these rcd1 mutant characteristics were observed in rcd1-6 to succeed low activation of the chloroplast antioxidant system and glutathione biosynthesis. We conclude that RCD1 protects plant cells from running into reactive oxygen species (ROS)-triggered programs, such as cell death and activation of pathogen-responsive genes (PR genes) and extra-plastidic antioxidant enzymes, by supporting the induction of the chloroplast antioxidant system. PMID:25295044

  19. Changes in ascorbic acid content in primary cultured rat hepatocytes exposed to 2,2'-azobis (2-amidinopropane) dihydrochloride, a radical initiator.

    PubMed

    Sasakii, K; Kimura, K; Kitaguchi, Y; Onoue, T; Ogura, H; Aoyagi, Y

    2001-08-01

    Changes in ascorbate content in primary cultured rat hepatocytes exposed to oxidative stress derived from water soluble radical initiator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) were examined. Cells were exposed to 0.05 and 5 mg/ml of AAPH as 'mild' and severe' oxidative stresses, respectively. Lipid peroxidation in hepatocytes was induced by 'severe' oxidative stress, but not by 'mild' oxidative stress. Ascorbate decreased at 6 hr after administration of both mild' and severe' oxidative stresses, and recovered to the control level after a further 6 hr. In cells treated with 'severe oxidative stress, however, total ascorbate (reduced form plus oxidized form) had increased 24 hr after administration. These results indicated that consumption alone did not account for the increase of ascorbate in hepatocytes under oxidative stress.

  20. [Probabilistic evaluation of homology of primary protein structures].

    PubMed

    Ismailov, I I; Sharov, M V; Sharov, M V; Bekmukhametova, Z U

    1994-11-01

    Criteria have been developed for resemblance of segments of certain length of different proteins to be considered as an evidence of those proteins relationship. A table is formed of minimal numbers of identical amino acids for which the probability of the causal coincidence in a segment of certain length is less then 1%. The criteria were corroborated by comparison of natural proteins and model amino acid sequences concocted by means of the random numbers generator.

  1. Protein Primary Structure of the Vaccinia Virion at Increased Resolution

    PubMed

    Ngo, Tuan; Mirzakhanyan, Yeva; Moussatche, Nissin; Gershon, Paul David

    2016-11-01

    Here we examine the protein covalent structure of the vaccinia virus virion. Within two virion preparations, >88% of the theoretical vaccinia virus-encoded proteome was detected with high confidence, including the first detection of products from 27 open reading frames (ORFs) previously designated "predicted," "uncharacterized," "inferred," or "hypothetical" polypeptides containing as few as 39 amino acids (aa) and six proteins whose detection required nontryptic proteolysis. We also detected the expression of four short ORFs, each of which was located within an ORF ("ORF-within-ORF"), including one not previously recognized or known to be expressed. Using quantitative mass spectrometry (MS), between 58 and 74 proteins were determined to be packaged. A total of 63 host proteins were also identified as candidates for packaging. Evidence is provided that some portion of virion proteins are "nicked" via a combination of endoproteolysis and concerted exoproteolysis in a manner, and at sites, independent of virus origin or laboratory procedures. The size of the characterized virion phosphoproteome was doubled from 189 (J. Matson, W. Chou, T. Ngo, and P. D. Gershon, Virology 452-453:310-323, 2014, doi:http://dx.doi.org/10.1016/j.virol.2014.01.012) to 396 confident, unique phosphorylation sites, 268 of which were within the packaged proteome. This included the unambiguous identification of phosphorylation "hot spots" within virion proteins. Using isotopically enriched ATP, 23 sites of intravirion kinase phosphorylation were detected within nine virion proteins, all at sites already partially occupied within the virion preparations. The clear phosphorylation of proteins RAP94 and RP19 was consistent with the roles of these proteins in intravirion early gene transcription. In a blind search for protein modifications, cysteine glutathionylation and O-linked glycosylation featured prominently. We provide evidence for the phosphoglycosylation of vaccinia virus proteins.

  2. The primary structure of a plant storage protein: zein.

    PubMed Central

    Geraghty, D; Peifer, M A; Rubenstein, I; Messing, J

    1981-01-01

    The protein sequence of a representative of the zeins, the major storage proteins of maize, has been derived from the nucleotide sequence of a zein cDNA clone. This cDNA was sequence both by the Maxam and Gilbert and the M13-dideoxy techniques. The nucleotide sequence encompasses the non-translated 3' terminus of the mRNA, the entire coding sequence specifying both the mature zein protein and a small signal peptide, and a portion of the non-translated 5' region. The deduced amino acid composition and the amino-terminal amino acid sequence closely resemble those derived from chemical analysis of the zein protein fraction. The data presented represent the first complete amino acid sequence of a plant storage protein. PMID:6895552

  3. Care pathways to improve care co-ordination and quality between primary and hospital care for patients with radical prostatectomy: a quality improvement project.

    PubMed

    Van Houdt, Sabine; Heyrman, Jan; Vanhaecht, Kris; Sermeus, Walter; De Lepeleire, Jan

    2013-01-01

    Care pathways are widely used in hospitals to improve quality. There is a growing interest in extending care pathways into primary care. There is little evidence on the relationship between care pathways across the primary-hospital care continuum and improvement in quality of care. Members of primary and hospital care services in the region of Bruges (Belgium) developed a care pathway for radical prostatectomy patients. An evaluation of this care pathway encountered some problems. To assess if a revision of the care pathway would improve quality of care enhancing patient outcomes. An exploratory trial was performed to test the feasibility of quality measurement, the possible intervention effect and recruitment. A pre-post-intervention postal survey was used. Quality of care was translated into process and outcome indicators. These indicators were measured in two groups receiving a postal questionnaire: one group before (pre-intervention) and another group after implementation (post-intervention). A Fisher's exact test was used to compare differences for dichotomous variables, and a Mann-Whitney U-test to compare ordinal and continuous variables. Observed improvements in process and outcome indicators were not statistically significant after correcting for multiple testing: 95.1% of patients received the information pack during the pre-operative consultation (versus 81.0% in the pre-intervention), 86.0% of the patients consulted a physiotherapist who specialised in pelvic floor muscle exercise treatment (versus 56.0% in the pre-intervention) and no patients experienced pain (versus 13.6% in the pre-intervention). No changes were observed for communication and co-ordination between caregivers. Given the background of scarce evidence on the quality improvement effect of care pathways between primary and hospital care, this exploratory trial provides information about the quality measurement, the possible intervention effect and recruitment. The quality improvement

  4. Locomotor proteins in tissues of primary tumors and metastases of ovarian and breast cancer

    NASA Astrophysics Data System (ADS)

    Kondakova, I. V.; Yunusova, N. V.; Spirina, L. V.; Shashova, E. E.; Kolegova, E. S.; Kolomiets, L. A.; Slonimskaya, E. M.; Villert, A. B.

    2016-08-01

    The paper discusses the capability for active movement in an extracellular matrix, wherein remodeling of the cytoskeleton by actin binding proteins plays a significant role in metastases formation. We studied the expression of actin binding proteins and β-catenin in tissues of primary tumors and metastases of ovarian and breast cancer. Contents of p45 Ser β-catenin and the actin severing protein gelsolin were decreased in metastases of ovarian cancer relative to primary tumors. The level of the cofilin, functionally similar to gelsolin, was significantly higher in metastases compared to primary ovarian and breast tumor tissue. In breast cancer, significant increase in the number of an actin monomer binder protein thymosin-β4 was observed in metastases as compared to primary tumors. The data obtained suggest the involvement of locomotor proteins in metastases formation in ovarian and breast cancer.

  5. Fast computational methods for predicting protein structure from primary amino acid sequence

    DOEpatents

    Agarwal, Pratul Kumar

    2011-07-19

    The present invention provides a method utilizing primary amino acid sequence of a protein, energy minimization, molecular dynamics and protein vibrational modes to predict three-dimensional structure of a protein. The present invention also determines possible intermediates in the protein folding pathway. The present invention has important applications to the design of novel drugs as well as protein engineering. The present invention predicts the three-dimensional structure of a protein independent of size of the protein, overcoming a significant limitation in the prior art.

  6. Inhibition of lipid oxidation in cooked beef patties by hydrolyzed potato protein is related to its reducing and radical scavenging ability.

    PubMed

    Wang, Lynn L; Xiong, Youling L

    2005-11-16

    Protein hydrolysates were prepared by limited alcalase hydrolysis (0.5, 1, and 6 h, corresponding to degrees of hydrolysis of 0.72, 1.9, and 2.3, respectively) of heat-coagulated potato protein. The hydrolysates were characterized for peptide composition, ferric reducing/antioxidant power (FRAP), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activity, and Fe2+- and Cu2+-chelation capacity. Hydrolyzed and intact proteins were formulated (4%, w/w) into beef patties to determine in situ antioxidant efficacy. Thiobarbituric acid-reactive substances (TBARS) and peroxide value (PV) formed in cooked and PVC-packaged patties during storage (4 degrees C, 0-7 days) were analyzed. Hydrolysis increased the protein solubility by 14-19-fold and produced numerous short peptides (< 6 kDa). The FRAP values of the protein sample (23 micromol/g) increased markedly after hydrolysis but were similar between the three hydrolysates (597-643 micromol/g). Similarly, the ABTS radical-scavenging activity also was increased by hydrolysis and was the greatest for the 1-h hydrolysate. Hydrolysis increased the Cu2+-chelation activity but decreased the Fe2+-chelation ability of the protein. The production of PV in patties after 7 days of storage was lowered 44.9% and 74.5% (P < 0.05), and that of TBARS was reduced 40.9% and 50.3% (P < 0.05), by intact and hydrolyzed proteins, respectively.

  7. Protein Oxidation Implicated as the Primary Determinant of Bacterial Radioresistance

    SciTech Connect

    Daly, Michael J.; Gaidamakova, E.; Matrosova, V.; Vasilenko, A.; Zhai, M.; leapman, Richard D.; Lai, Barry; Ravel, Bruce; Li, Shu-Mei W.; Kemner, Kenneth M.; Fredrickson, Jim K.

    2007-04-02

    In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of manganese-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications which introduce carbonyl groups. Conditions which inhibited Mn-accumulation or Mn redox-cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence (XRF) microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR resistant and sensitive bacteria, our findings support that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.

  8. A novel index for preoperative, non-invasive prediction of macro-radical primary surgery in patients with stage IIIC-IV ovarian cancer-a part of the Danish prospective pelvic mass study.

    PubMed

    Karlsen, Mona Aarenstrup; Fagö-Olsen, Carsten; Høgdall, Estrid; Schnack, Tine Henrichsen; Christensen, Ib Jarle; Nedergaard, Lotte; Lundvall, Lene; Lydolph, Magnus Christian; Engelholm, Svend Aage; Høgdall, Claus

    2016-09-01

    The purpose of this study was to develop a novel index for preoperative, non-invasive prediction of complete primary cytoreduction in patients with FIGO stage IIIC-IV epithelial ovarian cancer. Prospectively collected clinical data was registered in the Danish Gynecologic Cancer Database. Blood samples were collected within 14 days of surgery and stored by the Danish CancerBiobank. Serum human epididymis protein 4 (HE4), serum cancer antigen 125 (CA125), age, performance status, and presence/absence of ascites at ultrasonography were evaluated individually and combined to predict complete tumor removal. One hundred fifty patients with advanced epithelial ovarian cancer were treated with primary debulking surgery (PDS). Complete PDS was achieved in 41 cases (27 %). The receiver operating characteristic curves demonstrated an area under the curve of 0.785 for HE4, 0.678 for CA125, and 0.688 for age. The multivariate model (Cancer Ovarii Non-invasive Assessment of Treatment Strategy (CONATS) index), consisting of HE4, age, and performance status, demonstrated an AUC of 0.853. According to the Danish indicator level, macro-radical PDS should be achieved in 60 % of patients admitted to primary surgery (positive predictive value of 60 %), resulting in a negative predictive value of 87.5 %, sensitivity of 68.3 %, specificity of 83.5 %, and cutoff of 0.63 for the CONATS index. Non-invasive prediction of complete PDS is possible with the CONATS index. The CONATS index is meant as a supplement to the standard preoperative evaluation of each patient. Evaluation of the CONATS index combined with radiological and/or laparoscopic findings may improve the assessment of the optimal treatment strategy in patients with advanced epithelial ovarian cancer.

  9. Post-translational Modification of Ribosomal Proteins: Structural and Functional Characterization of RimO from Thermotoga maritima, a Radical S-adenosylmethionine methylthiotransferase

    SciTech Connect

    Arragain, S.; Latour, J; Forouhar, F; Neely, H; Montelione, G; Hunt, J; Mulliez, E; Fontecave, M; Atta, M; et al.

    2010-01-01

    Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826-1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 {angstrom} crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.

  10. Combination of electrografting and atom-transfer radical polymerization for making the stainless steel surface antibacterial and protein antiadhesive.

    PubMed

    Ignatova, Milena; Voccia, Samuel; Gilbert, Bernard; Markova, Nadya; Cossement, Damien; Gouttebaron, Rachel; Jérôme, Robert; Jérôme, Christine

    2006-01-03

    A two-step "grafting from" method has been successfully carried out, which is based on the electrografting of polyacrylate chains containing an initiator for the atom transfer radical polymerization (ATRP) of 2-(tert-butylamino)ethyl methacrylate (TBAEMA) or copolymerization of TBAEMA with either monomethyl ether of poly(ethylene oxide) methacrylate (PEOMA) or acrylic acid (AA) or styrene. The chemisorption of this type of polymer brushes onto stainless steel surfaces has potential in orthopaedic surgery. These films have been characterized by ATR-FTIR, Raman spectroscopy, atomic force microscopy (AFM), and measurement of contact angles of water. The polymer formed in solution by ATRP and that one detached on purpose from the surface have been analyzed by size exclusion chromathography (SEC) and (1)H NMR spectroscopy. The strong adherence of the films onto stainless steel has been assessed by peeling tests. AFM analysis has shown that addition of hydrophilic comonomers to the grafted chains decreases the surface roughness. According to dynamic quartz crystal microbalance experiments, proteins (e.g., fibrinogen) are more effectively repelled whenever copolymer brushes contain neutral hydrophilic (PEOMA) co-units rather than negatively charged groups (PAA salt). Moreover, a 2- to 3-fold decrease in the fibrinogen adsorption is observed when TBAEMA is copolymerized with either PEOMA or AA rather than homopolymerized or copolymerized with styrene. Compared to the bare stainless steel surface, brushes of polyTBAEMA, poly(TBAEMA-co-PEOMA) and poly(TBAEMA-co-AA) decrease the bacteria adhesion by 3 to 4 orders of magnitude as revealed by Gram-positive bacteria S. aureus adhesion tests.

  11. CFTR protein expression in primary and cultured epithelia.

    PubMed Central

    Zeitlin, P L; Crawford, I; Lu, L; Woel, S; Cohen, M E; Donowitz, M; Montrose, M H; Hamosh, A; Cutting, G R; Gruenert, D

    1992-01-01

    The gene responsible for the lethal disorder cystic fibrosis encodes a 1480-amino acid glycoprotein, CFTR. Using polyclonal antibodies directed against separate phosphorylation sites in the pre-nucleotide-binding fold (exon 9) and the R domain (exon 13), we have identified a 165-kDa protein in Xenopus laevis oocytes injected with recombinant CFTR cRNA transcribed from the full-length CFTR plasmid pBQ4.7. A protein of the same mobility was also detected with Western blotting techniques in whole cell extracts of cells that express CFTR mRNA (T84, FHTE, HT-29), including biopsied human nasal and bronchial tissue. Immunodetectable 165-kDa protein was concentrated in the apical membrane fraction of ileal villus tissue. We also report that the 165-kDa protein levels can be modulated pharmacologically, and these levels are appropriately correlated with second-messenger-regulated Cl- efflux. Thus, native or recombinant CFTR can be recognized by these anti-CFTR peptide polyclonal antibodies. Images PMID:1370353

  12. Definition of antigen specificity for antimitochondrial proteins detected by Western blotting using native mitochondrial proteins in primary biliary cirrhosis.

    PubMed

    Miyakawa, H; Kawaguchi, N; Kikuchi, K; Fujikawa, H; Kitazawa, E; Matsushita, M

    2001-10-01

    The major autoantigens to anti-mitochondrial antibody (AMA) in primary biliary cirrhosis (PBC) have previously been identified to be PDC-E2, BCOADC-E2, and OGDC-E2. However, analysis of these autoantigens to AMA cannot be examined using the two routine assays; immmunofluorescence and ELISA. Moreover, there are some problems in specificity and sensitivity in these routine assays. So, analysis with Western blotting using native mitochondrial protein as the antigen is required; it allows the identification of the molecular weights for the proteins which react with AMA in patients' sera. However, since the antigen-proteins used are not unified, molecular weights of AMA corresponding proteins vary among laboratories. In the present study, as the first step to help address this issue, we investigated the antigen specificity of protein bands detected by Western blotting using our in-house bovine and porcine heart mitochondrial proteins. Three major recombinant mitochondrial proteins were prepared. The antigen specificity was examined by the absorption tests preincubated with the three recombinant mitochondrial proteins. The molecular weights of developing our bovine and porcine heart mitochondrial proteins using SDS-PAGE were multiple protein bands including 74, 52, 50, and 43 kDa protein bands. Of them, the 74, 50, and 43 kDa protein bands were absorbed with preincubations of recombinant PDC-E2, BCOADC-E2, and OGDC-E2 protein, respectively. AMA specificity of these three major proteins with our Western blotting was confirmed.

  13. Molecular basis of intramolecular electron transfer in proteins during radical-mediated oxidations: Computer simulation studies in model tyrosine-cysteine peptides in solution

    PubMed Central

    Petruk, Ariel A.; Bartesaghi, Silvina; Trujillo, Madia; Estrin, Darío A.; Murgida, Daniel; Kalyanaraman, Balaraman; Marti, Marcelo A.; Radi, Rafael

    2012-01-01

    Experimental studies in hemeproteins and model Tyr/Cys-containing peptides exposed to oxidizing and nitrating species suggest that intramolecular electron transfer (IET) between tyrosyl radicals (Tyr-O●) and Cys residues controls oxidative modification yields. The molecular basis of this IET process is not sufficiently understood with structural atomic detail. Herein, we analyzed using molecular dynamics and quantum mechanics-based computational calculations, mechanistic possibilities for the radical transfer reaction in Tyr/Cys-containing peptides in solution and correlated them with existing experimental data. Our results support that Tyr-O● to Cys radical transfer is mediated by an acid/base equilibrium that involves deprotonation of Cys to form the thiolate, followed by a likely rate-limiting transfer process to yield cysteinyl radical and a Tyr phenolate; proton uptake by Tyr completes the reaction. Both, the pKa values of the Tyr phenol and Cys thiol groups and the energetic and kinetics of the reversible IET are revealed as key physico-chemical factors. The proposed mechanism constitutes a case of sequential, acid/base equilibrium-dependent and solvent-mediated, proton-coupled electron transfer and explains the dependency of oxidative yields in Tyr/Cys peptides as a function of the number of alanine spacers. These findings contribute to explain oxidative modifications in proteins that contain sequence and/or spatially close Tyr-Cys residues. PMID:22640642

  14. Primary structure of the Aequorea victoria green-fluorescent protein.

    PubMed

    Prasher, D C; Eckenrode, V K; Ward, W W; Prendergast, F G; Cormier, M J

    1992-02-15

    Many cnidarians utilize green-fluorescent proteins (GFPs) as energy-transfer acceptors in bioluminescence. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca(2+)-activated phosphoprotein. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid (aa) residues within the polypeptide. This report describes the cloning and sequencing of both cDNA and genomic clones of GFP from the cnidarian, Aequorea victoria. The gfp10 cDNA encodes a 238-aa-residue polypeptide with a calculated Mr of 26,888. Comparison of A. victoria GFP genomic clones shows three different restriction enzyme patterns which suggests that at least three different genes are present in the A. victoria population at Friday Harbor, Washington. The gfp gene encoded by the lambda GFP2 genomic clone is comprised of at least three exons spread over 2.6 kb. The nucleotide sequences of the cDNA and the gene will aid in the elucidation of structure-function relationships in this unique class of proteins.

  15. 2D ESEEM of the [sup 15]N-labeled radical cations of bacteriochlorophyll a and of the primary donor in reaction centers of Rhodobacter sphaeroides

    SciTech Connect

    Kaess, H.; Rautter, J.; Boenigk, B.; Lubitz, W. ); Hoefer, P. )

    1995-01-05

    The radical cations of [[sup 15]N]bacteriochlorophyll a and of the primary donor P[sub 865] in [sup 15]N-labeled reaction centers of Rhodobacter spaeroides R-26.1 were investigated in frozen solutions using two-dimensional ESEEM methods. Both three-pulse (stimulated echo) and four-pulse (HYSCORE) sequences were employed to avoid ambiguities in data analysis. Computer simulations of the experimental powder spectra were performed, yielding a complete set of nitrogen hyperfine coupling tensors for both systems. The obtained tensor values are compared with those from ENDOR measurements and from semiempirical INDO-type MO calculations. The results obtained from 2D stimulated echo ESEEM and HYSCORE for P[sub 865][sup [center dot]+] are interpreted in terms of an asymmetric spin density distribution over the halves of the bacteriochlorophyll dimer ([open quote]special pair[close quote]) in the reaction center with a ratio of approximately 5:1. This asymmetry is considerably larger at low temperatures in the frozen state than at room temperature. It is postulated that two different conformational states of the dimer exist at these temperatures with different spin density distributions. 60 refs., 9 figs., 4 tabs.

  16. Quantitative Protein Topography Analysis and High-Resolution Structure Prediction Using Hydroxyl Radical Labeling and Tandem-Ion Mass Spectrometry (MS)*

    PubMed Central

    Kaur, Parminder; Kiselar, Janna; Yang, Sichun; Chance, Mark R.

    2015-01-01

    Hydroxyl radical footprinting based MS for protein structure assessment has the goal of understanding ligand induced conformational changes and macromolecular interactions, for example, protein tertiary and quaternary structure, but the structural resolution provided by typical peptide-level quantification is limiting. In this work, we present experimental strategies using tandem-MS fragmentation to increase the spatial resolution of the technique to the single residue level to provide a high precision tool for molecular biophysics research. Overall, in this study we demonstrated an eightfold increase in structural resolution compared with peptide level assessments. In addition, to provide a quantitative analysis of residue based solvent accessibility and protein topography as a basis for high-resolution structure prediction; we illustrate strategies of data transformation using the relative reactivity of side chains as a normalization strategy and predict side-chain surface area from the footprinting data. We tested the methods by examination of Ca+2-calmodulin showing highly significant correlations between surface area and side-chain contact predictions for individual side chains and the crystal structure. Tandem ion based hydroxyl radical footprinting-MS provides quantitative high-resolution protein topology information in solution that can fill existing gaps in structure determination for large proteins and macromolecular complexes. PMID:25687570

  17. Sensitivity enhancement for membrane proteins reconstituted in parallel and perpendicular oriented bicelles obtained by using repetitive cross-polarization and membrane-incorporated free radicals.

    PubMed

    Koroloff, Sophie N; Tesch, Deanna M; Awosanya, Emmanuel O; Nevzorov, Alexander A

    2017-02-01

    Multidimensional separated local-field and spin-exchange experiments employed by oriented-sample solid-state NMR are essential for structure determination and spectroscopic assignment of membrane proteins reconstituted in macroscopically aligned lipid bilayers. However, these experiments typically require a large number of scans in order to establish interspin correlations. Here we have shown that a combination of optimized repetitive cross polarization (REP-CP) and membrane-embedded free radicals allows one to enhance the signal-to-noise ratio by factors 2.4-3.0 in the case of Pf1 coat protein reconstituted in magnetically aligned bicelles with their normals being either parallel or perpendicular to the main magnetic field. Notably, spectral resolution is not affected at the 2:1 radical-to-protein ratio. Spectroscopic assignment of Pf1 coat protein in the parallel bicelles has been established as an illustration of the method. The proposed methodology will advance applications of oriented-sample NMR technique when applied to samples containing smaller quantities of proteins and three-dimensional experiments.

  18. Quantitative protein topography analysis and high-resolution structure prediction using hydroxyl radical labeling and tandem-ion mass spectrometry (MS).

    PubMed

    Kaur, Parminder; Kiselar, Janna; Yang, Sichun; Chance, Mark R

    2015-04-01

    Hydroxyl radical footprinting based MS for protein structure assessment has the goal of understanding ligand induced conformational changes and macromolecular interactions, for example, protein tertiary and quaternary structure, but the structural resolution provided by typical peptide-level quantification is limiting. In this work, we present experimental strategies using tandem-MS fragmentation to increase the spatial resolution of the technique to the single residue level to provide a high precision tool for molecular biophysics research. Overall, in this study we demonstrated an eightfold increase in structural resolution compared with peptide level assessments. In addition, to provide a quantitative analysis of residue based solvent accessibility and protein topography as a basis for high-resolution structure prediction; we illustrate strategies of data transformation using the relative reactivity of side chains as a normalization strategy and predict side-chain surface area from the footprinting data. We tested the methods by examination of Ca(+2)-calmodulin showing highly significant correlations between surface area and side-chain contact predictions for individual side chains and the crystal structure. Tandem ion based hydroxyl radical footprinting-MS provides quantitative high-resolution protein topology information in solution that can fill existing gaps in structure determination for large proteins and macromolecular complexes.

  19. The effectiveness of clove extracts in the inhibition of hydroxyl radical oxidation-induced structural and rheological changes in porcine myofibrillar protein.

    PubMed

    Chen, Hongsheng; Diao, Jingjing; Li, Yuanyuan; Chen, Qian; Kong, Baohua

    2016-01-01

    Oxidation is a major cause of protein quality deterioration during the storage and processing of food. This study investigated the effects of clove extract (CE) on structural and rheological changes in porcine longissimus myofibrillar proteins (MP) and the effects of oxidizing radicals produced by a Fenton reaction system (FRS). Increased oxidation time was accompanied by increased carbonyl content, reduced Ca-ATPase activity, decreased enthalpy of denaturation, decreased thermal transition temperatures (P<0.05), and increased protein susceptibility to thermal aggregation. The addition of CE significantly inhibited carbonyl formation (P<0.05), enhanced solubility and thermal stability, and improved the gel formation ability (storage modulus, loss modulus) of MP. The protective effect of CE on protein denaturation was demonstrated by its efficacy in maintaining Ca-ATPase activity and decreasing the degree of protein aggregation. Overall, the hydroxyl radical-induced loss of the structural and functional properties of MP was significantly reduced by the presence of CE. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Successful treatment of primary extramedullary leukemia (EML) of the uterus with radical surgery, chemotherapy, autologous bone marrow transplantation (BMT) and prophylactic local irradiation.

    PubMed

    Huter, O; Brezinka, C; Nachbaur, D; Schwaighofer, H; Lang, A; Niederwieser, D

    1996-09-01

    Extramedullary myeloid cell tumors are rare manifestations of acute nonlymphocytic leukemia (ANLL). While many advances in diagnosis have been made, dilemmas remain concerning the treatment of this disease. In primary extramedullary leukemia (EML) most reports agree upon a local therapy followed by systemic chemotherapy such as is used for ANLL. However, further prophylactic local or systemic therapy with stem cell support remains controversial. A 20-year-old patient was diagnosed as having granulocytic sarcoma (GS) of the uterus without evidence of ANLL in 1991. After resection of the tumor at the uterine cervix and chemotherapy with daunorubicin 50 mg/m2 (days 1-3) and cytosine-arabinoside 200 mg/m2 (days 1-7) in September 1991, complete remission was achieved. In October 1991 cytosine-arabinoside 1000 mg/m2 every 12 h from day 1 to day 6 and amsacrine 200 mg from day 5 to day 7 were given as consolidation. Two years later relapse occurred in the adnexae. After radical hysterectomy, the same induction and consolidation chemotherapy was administrated. Subsequently, cytoxane 60 mg/m2 and fractionated total body irradiation (6 x 200 cGy) were given as conditioning and the previously cryopreserved bone marrow was reinfused. Finally, after hematopoietic engraftment, prophylactic local irradiation (4500 cGy) to the pelvis was given resulting in a disease-free long-term survival of more than 36 months after relapse. Although this experience is confined to one patient, it may contribute to the design of prospective therapeutic studies in patients with primary EML.

  1. Cephaloridine induces translocation of protein kinase C delta into mitochondria and enhances mitochondrial generation of free radicals in the kidney cortex of rats causing renal dysfunction.

    PubMed

    Kohda, Yuka; Gemba, Munekazu

    2005-05-01

    We have previously reported that the enhancement of free radical generation in mitochondria isolated from the kidney cortex of rats exposed to cephaloridine (CER) is probably mediated by the activation of protein kinase C (PKC). We examined which isoenzymes of PKC might be involved in the development of nephrotoxicity induced by CER in rats. The CER-induced renal dysfunction observed 24 h after its injection was prevented by a potent antioxidant DPPD and well-known PKC inhibitors like H-7 and rottlerin. At 1.5 and 3.5 h after the CER injection, the free radical generation was increased markedly and this was associated with translocation of PKCdelta into the mitochondria of renal cortex tissue. Pretreatment of rats with H-7, a PKC inhibitor, significantly inhibited the CER-derived increase in mitochondrial generation of free radicals, suggesting that H-7 probably gets into the mitochondria and inhibits the activity of translocated PKC within the mitochondria. It was also shown that pretreatment of rats with rottlerin, a specific inhibitor of PKCdelta, suppressed the early translocation of PKCdelta into mitochondria and inhibited the CER-derived development of renal dysfunction. These results suggest that the CER-derived early translocation of PKCdelta into mitochondria probably leads to the enhanced production of free radicals through the mitochondrial respiratory chain during the development of the nephrotoxicity caused by CER. Understanding the role of PKCdelta in mitochondria may provide an important clue to the molecular mechanisms of mitochondrial production of reactive oxygen species and the free radical-induced renal failure in rats treated with CER.

  2. Primary structure elements of spider dragline silks and their contribution to protein solubility.

    PubMed

    Huemmerich, Daniel; Helsen, Christopher W; Quedzuweit, Susanne; Oschmann, Jan; Rudolph, Rainer; Scheibel, Thomas

    2004-10-26

    Spider silk proteins have mainly been investigated with regard to their contribution to mechanical properties of the silk thread. However, little is known about the molecular mechanisms of silk assembly. As a first step toward characterizing this process, we aimed to identify primary structure elements of the garden spider's (Araneus diadematus) major dragline silk proteins ADF-3 and ADF-4 that determine protein solubility. In addition, we investigated the influence of conditions involved in mediating natural thread assembly on protein aggregation. Genes encoding spider silk-like proteins were generated using a cloning strategy, which is based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences. Comparing secondary structure, solubility, and aggregation properties of the synthesized proteins revealed that single primary structure elements have diverse influences on protein characteristics. Repetitive regions representing the largest part of dragline silk proteins determined the solubility of the synthetic proteins, which differed greatly between constructs derived from ADF-3 and ADF-4. Factors, such as acidification and increases in phosphate concentration, which promote silk assembly in vivo generally decreased silk protein solubility in vitro. Strikingly, this effect was pronounced in engineered proteins comprising the carboxyl-terminal nonrepetitive regions of ADF-3 or ADF-4, indicating that these regions might play an important role in initiating assembly of spider silk proteins.

  3. Expression of Yes-associated protein modulates Survivin expression in primary liver malignancies.

    PubMed

    Bai, Haibo; Gayyed, Mariana F; Lam-Himlin, Dora M; Klein, Alison P; Nayar, Suresh K; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A

    2012-09-01

    Hepatocellular carcinoma and intrahepatic cholangiocarcinoma account for 95% of primary liver cancer. For each of these malignancies, the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice after genetic manipulation of Yes-associated protein, a transcription coactivator. Here, we comprehensively documented Yes-associated protein expression in the human liver and primary liver cancers. We showed that nuclear Yes-associated protein expression is significantly increased in human intrahepatic cholangiocarcinoma and hepatocellular carcinoma. We found that increased Yes-associated protein levels in hepatocellular carcinoma are due to multiple mechanisms including gene amplification and transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein family, has been reported as an independent prognostic factor for poor survival in both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. We found that nuclear Yes-associated protein expression correlates significantly with nuclear Survivin expression for both intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Furthermore, using mice engineered to conditionally overexpress Yes-associated protein in the liver, we found that Survivin messenger RNA expression depends upon Yes-associated protein levels. Our findings suggested that Yes-associated protein contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Ramipril-induced delayed myocardial protection against free radical injury involves bradykinin B2 receptor-NO pathway and protein synthesis

    PubMed Central

    Jin, Zhu-Qiu; Chen, Xiu

    1998-01-01

    The aim of the present study was to examine whether ramipril induces delayed myocardial protection against free radical injuries ex vivo and to determine the possible role of the bradykinin B2–nitric oxide (NO) pathway, prostaglandins(PGs) and protein synthesis in this delayed adaptive response.Rats were pretreated with ramipril (10 or 50 μg kg−1, i.v.) and hearts were isolated after 24, 48 and 72 h. Langendorff hearts were subjected to 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical-induced injury.Left ventricular developed pressure (LVDP) and its maximal increase velocity (+dP/dtmax), coronary flow (CF), heart rate (HR), lactate dehydrogenase (LDH) in coronary effluent and thiobarbituric acid reactive substances (TBARS) in the myocardium were measured.The results showed that in the DPPH control group, 20 min after free radical-induced injury, LVDP, +dP/dtmax, CF, HR declined, whereas TBARS and LDH increased significantly. The above cardiac function parameters were significantly improved in RAM-pretreated rats after 24 and 48 h.Pretreatment with HOE 140, the selective bradykinin B2 receptor antagonist, NG-nitro-L-arginine, the NO synthase inhibitor, and actinomycin D, the RNA transcription inhibitor, prior to ramipril injection abolished the beneficial effects of ramipril at 24 h while indomethacin, a cyclooxygenase inhibitor, pretreatment had no effect on ramipril-induced delayed protection.In conclusion, ramipril induces delayed myocardial protection against free radical injury in the rat heart. This delayed protection was sustained for 48 h, is associated with the bradykinin B2 receptor–NO pathway and depends on protein but not prostaglandin synthesis. PMID:9806340

  5. Differential extraction and protein sequencing reveals major differences in patterns of primary cell wall proteins from plants.

    PubMed

    Robertson, D; Mitchell, G P; Gilroy, J S; Gerrish, C; Bolwell, G P; Slabas, A R

    1997-06-20

    The proteins of the primary cell walls of suspension cultured cells of five plant species, Arabidopsis, carrot, French bean, tomato, and tobacco, have been compared. The approach that has been adopted is differential extraction followed by SDS-polyacrylamide gel electrophoresis (PAGE), rather than two-dimensional gel analysis, to facilitate protein sequencing. Whole cells were washed sequentially with the following aqueous solutions, CaCl2, CDTA (cyclohexane diaminotetraacetic acid, DTT (dithiothreitol), NaCl, and borate. SDS-PAGE analysis showed consistent differences between species. From the 233 proteins that were selected for sequencing, 63% gave N-terminal data. This analysis shows that (i) patterns of proteins revealed by SDS-PAGE are strikingly different for all five species, (ii) a large number of these proteins cannot be identified by data base searches indicating that a significant proportion of wall proteins have not been previously described, (iii) the major proteins that can be identified belong to very different classes of proteins, (iv) the majority of proteins found in the extracellular growth media are absent from their respective cell wall extracts, and (v) the results of the extraction process are indicative of higher order structure. It appears that aspects of speciation reside in the complement of extracellular wall proteins. The data represent a protein resource for cell wall studies complementary to EST (expressed sequence tag) and DNA sequencing strategies.

  6. The Exocyst Protein Sec10 Is Necessary for Primary Ciliogenesis and Cystogenesis In Vitro

    PubMed Central

    Zuo, Xiaofeng; Guo, Wei

    2009-01-01

    Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, in which they are felt to participate in flow sensing and have been linked to the pathogenesis of cystic renal disorders such as autosomal dominant polycystic kidney disease. We previously localized the exocyst, an eight-protein complex involved in membrane trafficking, to the primary cilium of Madin-Darby canine kidney cells and showed that it was involved in cystogenesis. Here, using short hairpin RNA (shRNA) to knockdown exocyst expression and stable transfection to induce exocyst overexpression, we show that the exocyst protein Sec10 regulates primary ciliogenesis. Using immunofluorescence, scanning, and transmission electron microscopy, primary cilia containing only basal bodies are seen in the Sec10 knockdown cells, and increased ciliogenesis is seen in Sec10-overexpressing cells. These phenotypes do not seem to be because of gross changes in cell polarity, as apical, basolateral, and tight junction proteins remain properly localized. Sec10 knockdown prevents normal cyst morphogenesis when the cells are grown in a collagen matrix, whereas Sec10 overexpression results in increased cystogenesis. Transfection with human Sec10 resistant to the canine shRNA rescues the phenotype, demonstrating specificity. Finally, Par3 was recently shown to regulate primary cilia biogenesis. Par3 and the exocyst colocalized by immunofluorescence and coimmunoprecipitation, consistent with a role for the exocyst in targeting and docking vesicles carrying proteins necessary for primary ciliogenesis. PMID:19297529

  7. The exocyst protein Sec10 is necessary for primary ciliogenesis and cystogenesis in vitro.

    PubMed

    Zuo, Xiaofeng; Guo, Wei; Lipschutz, Joshua H

    2009-05-01

    Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, in which they are felt to participate in flow sensing and have been linked to the pathogenesis of cystic renal disorders such as autosomal dominant polycystic kidney disease. We previously localized the exocyst, an eight-protein complex involved in membrane trafficking, to the primary cilium of Madin-Darby canine kidney cells and showed that it was involved in cystogenesis. Here, using short hairpin RNA (shRNA) to knockdown exocyst expression and stable transfection to induce exocyst overexpression, we show that the exocyst protein Sec10 regulates primary ciliogenesis. Using immunofluorescence, scanning, and transmission electron microscopy, primary cilia containing only basal bodies are seen in the Sec10 knockdown cells, and increased ciliogenesis is seen in Sec10-overexpressing cells. These phenotypes do not seem to be because of gross changes in cell polarity, as apical, basolateral, and tight junction proteins remain properly localized. Sec10 knockdown prevents normal cyst morphogenesis when the cells are grown in a collagen matrix, whereas Sec10 overexpression results in increased cystogenesis. Transfection with human Sec10 resistant to the canine shRNA rescues the phenotype, demonstrating specificity. Finally, Par3 was recently shown to regulate primary cilia biogenesis. Par3 and the exocyst colocalized by immunofluorescence and coimmunoprecipitation, consistent with a role for the exocyst in targeting and docking vesicles carrying proteins necessary for primary ciliogenesis.

  8. Localized surface plasmon resonance nanosensing of C-reactive protein with poly(2-methacryloyloxyethyl phosphorylcholine)-grafted gold nanoparticles prepared by surface-initiated atom transfer radical polymerization.

    PubMed

    Kitayama, Yukiya; Takeuchi, Toshifumi

    2014-06-03

    Highly sensitive and selective protein nanosensing based on localized surface plasmon resonance (LSPR) of gold nanoparticles (AuNPs) on which polymerized specific ligands were grafted as an artificial protein recognition layer for the target protein were demonstrated. As a model, optical nanosensing for C-reactive protein (CRP), a known biomarker for chronic inflammation that predicts the risk of arteriosclerosis or heart attacks, was achieved by measuring the shift of LSPR spectra derived from the change of permittivity of poly(2-methacryloyloxyethyl phosphorylcholine)-grafted AuNPs (PMPC-g-AuNPs) upon interacting with CRP, in which the PMPC-g-AuNPs layer were grafted on AuNPs by surface-initiated atom transfer radical polymerization (ATRP). This nanosensing system was effective even for detecting CRP concentrations in a human serum solution diluted to 1% (w/w), at which point a limit of detection was ~50 ng/mL and nonspecific adsorption of other proteins was negligible. The nanosensing system using specific ligand-grafted AuNPs has several strengths, such as low preparation cost, avoiding the need for expensive instruments, no necessary complex pretreatments, and high stability, because it does not contain biobased molecules. We believe this novel synthetic route for protein nanosensors, composed of AuNPs and a polymerized specific ligand utilizing surface-initiated controlled/living radical polymerization, will provide a foundation for the design and synthesis of nanosensors targeting various other biomarker proteins, paving the way for future advances in the field of biosensing.

  9. Protein import into the photosynthetic organelles of Paulinella chromatophora and its implications for primary plastid endosymbiosis.

    PubMed

    Mackiewicz, Paweł; Bodył, Andrzej; Gagat, Przemysław

    2012-12-01

    The rhizarian amoeba Paulinella chromatophora harbors two photosynthetically active organelles of cyanobacterial origin that have been acquired independently of classic primary plastids. Because their acquisition did take place relatively recently, they are expected to provide new insight into the ancient cyanobacterial primary endosymbiosis. During the process of Paulinella endosymbiont-to-organelle transformation, more than 30 genes have been transferred from the organelle to the host nuclear genome via endosymbiotic gene transfer (EGT). The article discusses step-by-step protein import of EGT-derived proteins into Paulinella photosynthetic organelles with the emphasis on the nature of their targeting signals and the final passage of proteins through the inner organelle membrane. The latter most probably involves a simplified Tic translocon composed of Tic21- and Tic32-like proteins as well as a Hsp70-based motor responsible for pulling of imported proteins into the organelle matrix. Our results indicate that although protein translocation across the inner membrane of Paulinella photosynthetic organelles seems to resemble the one in classic primary plastids, the transport through the outer membrane does not. The differences could result from distinct integration pathways of Paulinella photosynthetic organelles and primary plastids with their respective host cells.

  10. Osteoclast radicals.

    PubMed

    Silverton, S

    1994-11-01

    In biological research, new ideas arise and quickly spread to encompass the entire field. Thus, the evolution of molecular biology has significantly changed our methods of approaching our research. A similar far-reaching finding has been the advent of radical reactions into biology. Although radical chemistry has been utilized for many technological advances that affect our daily lives, the appreciation of this same process within our cells has opened an unexplored arena for research enquiry. As cellular messengers, radical molecules seem whimsically designed: they are evanescent, rapidly and apparently indiscriminately reactive, and barely detectable by most biological methods. Yet, our initial probing of these reactive agents in cells and organisms has led us to postulate a virtually undescribed system of communication within and among cells which may have significant effects in multiple organs. In bone, radical reactants have been attributed with an important role in the control of bone resorption.

  11. Function and regulation of primary cilia and intraflagellar transport proteins in the skeleton

    PubMed Central

    Yuan, Xue; Serra, Rosa A.; Yang, Shuying

    2014-01-01

    Primary cilia are microtubule-based organelles that project from the cell surface to enable transduction of various developmental signaling pathways. The process of intraflagellar transport (IFT) is crucial for the building and maintenance of primary cilia. Ciliary dysfunction has been found in a range of disorders called ciliopathies, some of which display severe skeletal dysplasias. In recent years, interest has grown in uncovering the function of primary cilia/IFT proteins in bone development, mechanotransduction, and cellular regulation. We summarize recent advances in understanding the function of cilia and IFT proteins in the regulation of cell differentiation in osteoblasts, osteocytes, chondrocytes, and mesenchymal stem cells (MSCs). We also discuss the mechanosensory function of cilia and IFT proteins in bone cells, cilia orientation, and other functions of cilia in chondrocytes. PMID:24961486

  12. Function and regulation of primary cilia and intraflagellar transport proteins in the skeleton.

    PubMed

    Yuan, Xue; Serra, Rosa A; Yang, Shuying

    2015-01-01

    Primary cilia are microtubule-based organelles that project from the cell surface to enable transduction of various developmental signaling pathways. The process of intraflagellar transport (IFT) is crucial for the building and maintenance of primary cilia. Ciliary dysfunction has been found in a range of disorders called ciliopathies, some of which display severe skeletal dysplasias. In recent years, interest has grown in uncovering the function of primary cilia/IFT proteins in bone development, mechanotransduction, and cellular regulation. We summarize recent advances in understanding the function of cilia and IFT proteins in the regulation of cell differentiation in osteoblasts, osteocytes, chondrocytes, and mesenchymal stem cells (MSCs). We also discuss the mechanosensory function of cilia and IFT proteins in bone cells, cilia orientation, and other functions of cilia in chondrocytes.

  13. The Radical S-Adenosyl-l-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds*

    PubMed Central

    Nakai, Tadashi; Ito, Hiroto; Kobayashi, Kazuo; Takahashi, Yasuhiro; Hori, Hiroshi; Tsubaki, Motonari; Tanizawa, Katsuyuki; Okajima, Toshihide

    2015-01-01

    The bacterial enzyme designated QhpD belongs to the radical S-adenosyl-l-methionine (SAM) superfamily of enzymes and participates in the post-translational processing of quinohemoprotein amine dehydrogenase. QhpD is essential for the formation of intra-protein thioether bonds within the small subunit (maturated QhpC) of quinohemoprotein amine dehydrogenase. We overproduced QhpD from Paracoccus denitrificans as a stable complex with its substrate QhpC, carrying the 28-residue leader peptide that is essential for the complex formation. Absorption and electron paramagnetic resonance spectra together with the analyses of iron and sulfur contents suggested the presence of multiple (likely three) [4Fe-4S] clusters in the purified and reconstituted QhpD. In the presence of a reducing agent (sodium dithionite), QhpD catalyzed the multiple-turnover reaction of reductive cleavage of SAM into methionine and 5′-deoxyadenosine and also the single-turnover reaction of intra-protein sulfur-to-methylene carbon thioether bond formation in QhpC bound to QhpD, producing a multiknotted structure of the polypeptide chain. Homology modeling and mutagenic analysis revealed several conserved residues indispensable for both in vivo and in vitro activities of QhpD. Our findings uncover another challenging reaction catalyzed by a radical SAM enzyme acting on a ribosomally translated protein substrate. PMID:25778402

  14. Human prion protein-induced autophagy flux governs neuron cell damage in primary neuron cells.

    PubMed

    Moon, Ji-Hong; Lee, Ju-Hee; Nazim, Uddin Md; Lee, You-Jin; Seol, Jae-Won; Eo, Seong-Kug; Lee, John-Hwa; Park, Sang-Youel

    2016-05-24

    An unusual molecular structure of the prion protein, PrPsc is found only in mammals with transmissible prion diseases. Prion protein stands for either the infectious pathogen itself or a main component of it. Recent studies suggest that autophagy is one of the major functions that keep cells alive and has a protective effect against the neurodegeneration. In this study, we investigated that the effect of human prion protein on autophagy-lysosomal system of primary neuronal cells. The treatment of human prion protein induced primary neuron cell death and decreased both LC3-II and p62 protein amount indicating autophagy flux activation. Electron microscope pictures confirmed the autophagic flux activation in neuron cells treated with prion protein. Inhibition of autophagy flux using pharmacological and genetic tools prevented neuron cell death induced by human prion protein. Autophagy flux induced by prion protein is more activated in prpc expressing cells than in prpc silencing cells. These data demonstrated that prion protein-induced autophagy flux is involved in neuron cell death in prion disease and suggest that autophagy flux might play a critical role in neurodegenerative diseases including prion disease.

  15. Predictability of gene ontology slim-terms from primary structure information in Embryophyta plant proteins

    PubMed Central

    2013-01-01

    Background Proteins are the key elements on the path from genetic information to the development of life. The roles played by the different proteins are difficult to uncover experimentally as this process involves complex procedures such as genetic modifications, injection of fluorescent proteins, gene knock-out methods and others. The knowledge learned from each protein is usually annotated in databases through different methods such as the proposed by The Gene Ontology (GO) consortium. Different methods have been proposed in order to predict GO terms from primary structure information, but very few are available for large-scale functional annotation of plants, and reported success rates are much less than the reported by other non-plant predictors. This paper explores the predictability of GO annotations on proteins belonging to the Embryophyta group from a set of features extracted solely from their primary amino acid sequence. Results High predictability of several GO terms was found for Molecular Function and Cellular Component. As expected, a lower degree of predictability was found on Biological Process ontology annotations, although a few biological processes were easily predicted. Proteins related to transport and transcription were particularly well predicted from primary structure information. The most discriminant features for prediction were those related to electric charges of the amino-acid sequence and hydropathicity derived features. Conclusions An analysis of GO-slim terms predictability in plants was carried out, in order to determine single categories or groups of functions that are most related with primary structure information. For each highly predictable GO term, the responsible features of such successfulness were identified and discussed. In addition to most published studies, focused on few categories or single ontologies, results in this paper comprise a complete landscape of GO predictability from primary structure encompassing 75 GO

  16. Protein oxidation: identification and utilisation of molecular markers to differentiate singlet oxygen and hydroxyl radical-mediated oxidative pathways.

    PubMed

    Plowman, Jeffrey E; Deb-Choudhury, Santanu; Grosvenor, Anita J; Dyer, Jolon M

    2013-11-01

    The effect of reactive oxidation species (ROS) on tryptophan or tyrosine was investigated by qualitatively determining the major detectable oxidation products generated by hydroxyl radicals, produced by the Fenton process, or singlet oxygen, generated by exposure to green light in the presence of Rose Bengal, on these photosensitive amino acids in synthetic pentapeptides. Based on mass spectrometric analysis it would appear that the hydroxyl radical favours a pathway leading to the formation of tryptophandione-based products from tryptophan. In contrast singlet oxygen attack appears to favour the formation of kynurenine-type products from tryptophan. Specific oxidative products observed proteomically are therefore potentially able to discriminate between predominant ROS-mediated pathways. To validate these findings, a keratin-enriched extract was exposed to UVB light under aqueous conditions. The observation of the conversion of tryptophan to hydroxytryptophan in marker peptides, and the absence of singlet-oxygen specific modifications, suggested that under these conditions oxidative degradation occurred primarily via hydroxyl radical attack. These observations provide the first direct proteomic evidence of the dominant photodegradation pathways in wet wool.

  17. Spectroscopic Studies of the Iron and Manganese Reconstituted Tyrosyl Radical in Bacillus Cereus Ribonucleotide Reductase R2 Protein

    PubMed Central

    Tomter, Ane B.; Zoppellaro, Giorgio; Bell, Caleb B.; Barra, Anne-Laure; Andersen, Niels H.; Solomon, Edward I.; Andersson, K. Kristoffer

    2012-01-01

    Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g1-value of 2.0090 for the tyrosyl radical was extracted. This g1-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν7a = 1500 cm−1) was found to be insensitive to deuterium-oxide exchange. Additionally, the 18O-sensitive Fe-O-Fe symmetric stretching (483 cm−1) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g1-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053–33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher

  18. Induction of heat shock proteins in response to primary alcohols in Acinetobacter calcoaceticus.

    PubMed

    Benndorf, D; Loffhagen, N; Babel, W

    1999-01-01

    Cells of Acinetobacter calcoaceticus 69-V, a species able to metabolize a range of aliphatic hydrocarbons and alcohols, were confronted with ethanol, butanol, hexanol or heat shock during growth on acetate as sole source of carbon and energy. The primary alcohols and the heat shock led to the synthesis of new proteins or amplified expression of specific, common and general proteins, which were detected by silver staining after two-dimensional gel electrophoresis. Some of the alcohol-inducible proteins were identified as heat shock proteins by comparing protein patterns of alcohol-shocked cells with those of heat-shocked cells, and by N-terminal amino acid sequencing. DnaK was found to be amplified after all treatments, but GroEI only after heat shock and ethanol treatment. The N-terminal amino acid sequence of the protein, which was considerably amplified after alcohol treatment and heat shock, shows homology to HtpG (high temperature protein G). Some of the heat shock proteins induced by ethanol differ from those induced by butanol and hexanol, suggesting there are at least two different signals for the induction of some heat shock proteins by primary alcohols. This could be due to the different localization of ethanol, butanol and hexanol in the membrane, or because higher cytoplasmic concentrations of ethanol than of butanol or hexanol were applied in these tests in order to keep concentrations of the alcohols in the membrane roughly similar. Besides heat shock proteins, a group of proteins were observed which were only induced by butanol and hexanol, possibly indicating the existence of a further defense mechanism against high concentrations of hydrophobic substrates preventing protein denaturation and membrane damage.

  19. Carbohydrase inhibition and anti-cancerous and free radical scavenging properties along with DNA and protein protection ability of methanolic root extracts of Rumex crispus

    PubMed Central

    Shiwani, Supriya; Singh, Naresh Kumar

    2012-01-01

    The study elucidated carbohydrase inhibition, anti-cancerous, free radical scavenging properties and also investigated the DNA and protein protection abilities of methanolic root extract of Rumex crispus (RERC). For this purpose, pulverized roots of Rumex crispus was extracted in methanol (80% and absolute conc.) for 3 hrs for 60℃ and filtered and evaporated with vacuum rotary evaporator. RERC showed high phenolic content (211 µg/GAE equivalent) and strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging (IC50 = 42.86 (absolute methanol) and 36.91 µg/mL (80% methanolic extract)) and reduced power ability. Furthermore, RERC exhibited significant protective ability in H2O2/Fe3+/ascorbic acid-induced protein or DNA damage and percentage inhibition of the HT-29 cell growth rate following 80% methanolic RERC exposure at 400 µg/mL was observed to be highest (10.2% ± 1.03). Moreover, methanolic RERC inhibited α-glucosidase and amylase effectively and significantly (P < 0.05). Conclusively, RERC could be considered as potent carbohydrase inhibitor, anti-cancerous and anti-oxidant. PMID:23198017

  20. Carbohydrase inhibition and anti-cancerous and free radical scavenging properties along with DNA and protein protection ability of methanolic root extracts of Rumex crispus.

    PubMed

    Shiwani, Supriya; Singh, Naresh Kumar; Wang, Myeong Hyeon

    2012-10-01

    The study elucidated carbohydrase inhibition, anti-cancerous, free radical scavenging properties and also investigated the DNA and protein protection abilities of methanolic root extract of Rumex crispus (RERC). For this purpose, pulverized roots of Rumex crispus was extracted in methanol (80% and absolute conc.) for 3 hrs for 60℃ and filtered and evaporated with vacuum rotary evaporator. RERC showed high phenolic content (211 µg/GAE equivalent) and strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging (IC(50) = 42.86 (absolute methanol) and 36.91 µg/mL (80% methanolic extract)) and reduced power ability. Furthermore, RERC exhibited significant protective ability in H(2)O(2)/Fe(3+)/ascorbic acid-induced protein or DNA damage and percentage inhibition of the HT-29 cell growth rate following 80% methanolic RERC exposure at 400 µg/mL was observed to be highest (10.2% ± 1.03). Moreover, methanolic RERC inhibited α-glucosidase and amylase effectively and significantly (P < 0.05). Conclusively, RERC could be considered as potent carbohydrase inhibitor, anti-cancerous and anti-oxidant.

  1. Primary Blast Injury Depressed Hippocampal Long-Term Potentiation through Disruption of Synaptic Proteins.

    PubMed

    Vogel, Edward W; Rwema, Steve H; Meaney, David F; Bass, Cameron R Dale; Morrison, Barclay

    2017-03-01

    Blast-induced traumatic brain injury (bTBI) is a major threat to United States service members in military conflicts worldwide. The effects of primary blast, caused by the supersonic shockwave interacting with the skull and brain, remain unclear. Our group has previously reported that in vitro primary blast exposure can reduce long-term potentiation (LTP), the electrophysiological correlate of learning and memory, in rat organotypic hippocampal slice cultures (OHSCs) without significant changes to cell viability or basal, evoked neuronal function. We investigated the time course of primary blast-induced deficits in LTP and the molecular mechanisms that could underlie these deficits. We found that pure primary blast exposure induced LTP deficits in a delayed manner, requiring longer than 1 hour to develop, and that these deficits spontaneously recovered by 10 days following exposure depending on blast intensity. Additionally, we observed that primary blast exposure reduced total α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor 1 (GluR1) subunit expression and phosphorylation of the GluR1 subunit at the serine-831 site. Blast also reduced the expression of postsynaptic density protein-95 (PSD-95) and phosphorylation of stargazin protein at the serine-239/240 site. Finally, we found that modulation of the cyclic adenosine monophosphate (cAMP) pathway ameliorated electrophysiological and protein-expression changes caused by blast. These findings could inform the development of novel therapies to treat blast-induced loss of neuronal function.

  2. Identification of Surface-Exposed Protein Radicals and A Substrate Oxidation Site in A-Class Dye-Decolorizing Peroxidase from Thermomonospora curvata

    SciTech Connect

    Shrestha, Ruben; Chen, Xuejie; Ramyar, Kasra X.; Hayati, Zahra; Carlson, Eric A.; Bossmann, Stefan H.; Song, Likai; Geisbrecht, Brian V.; Li, Ping

    2016-12-12

    Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases in which a catalytic distal aspartate is involved in H2O2 activation to catalyze oxidations under acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial TcDyP from Thermomonospora curvata is among the most active DyPs and shows activity toward phenolic lignin model compounds. On the basis of the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Blue 19 (RB19), and formation of compound II like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the TcDyP catalyzes oxidation of large-size substrates via multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in TcDyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of the surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the cross-linking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class TcDyP is minimal, possibly due to its extremely small solvent-accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding the DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin.

  3. Shotgun proteomics reveals specific modulated protein patterns in tears of patients with primary open angle glaucoma naïve to therapy.

    PubMed

    Pieragostino, Damiana; Agnifili, Luca; Fasanella, Vincenzo; D'Aguanno, Simona; Mastropasqua, Rodolfo; Di Ilio, Carmine; Sacchetta, Paolo; Urbani, Andrea; Del Boccio, Piero

    2013-06-01

    Primary open angle glaucoma (POAG) is one of the main causes of irreversible blindness worldwide. The pathogenesis of POAG is still unclear. Alteration and sclerosis of trabecular meshwork with changes in aqueous humor molecular composition seem to play the key role. Increased intraocular pressure is widely known to be the main risk factor for the onset and progression of the disease. Unfortunately, the early diagnosis of POAG still remains the main challenge. In order to provide insight into the patho-physiology of glaucoma, here we report a shotgun proteomics approach to tears of patients with POAG naïve to therapy. Our proteomics results showed 27 differential tear proteins in POAG vs. CTRL comparison (25 up regulated proteins in the POAG group and two unique proteins in the CTRL group), 16 of which were associated with inflammatory response, free radical scavenging, cell-to-cell signaling and interaction. Overall the protein modulation shown in POAG tears proves the involvement of biochemical networks linked to inflammation. Among all regulated proteins, a sub-group of 12 up-regulated proteins in naïve POAG patients were found to be down-regulated in medically controlled POAG patients treated with prostanoid analogues (PGA), as reported in our previous work (i.e., lipocalin-1, lysozyme C, lactotransferrin, proline-rich-protein 4, prolactin-inducible protein, zinc-alpha-2-glycoprotein, polymeric immunoglobulin receptor, cystatin S, Ig kappa chain C region, Ig alpha-2 chain C region, immunoglobulin J chain, Ig alpha-1 chain C region). In summary, our findings indicate that the POAG tears protein expression is a mixture of increased inflammatory proteins that could be potential biomarkers of the disease, and their regulation may be involved in the mechanism by which PGA are able to decrease the intraocular pressure in glaucoma patients.

  4. A Primary Sequence Analysis of the ARGONAUTE Protein Family in Plants

    PubMed Central

    Rodríguez-Leal, Daniel; Castillo-Cobián, Amanda; Rodríguez-Arévalo, Isaac; Vielle-Calzada, Jean-Philippe

    2016-01-01

    Small RNA (sRNA)-mediated gene silencing represents a conserved regulatory mechanism controlling a wide diversity of developmental processes through interactions of sRNAs with proteins of the ARGONAUTE (AGO) family. On the basis of a large phylogenetic analysis that includes 206 AGO genes belonging to 23 plant species, AGO genes group into four clades corresponding to the phylogenetic distribution proposed for the ten family members of Arabidopsis thaliana. A primary analysis of the corresponding protein sequences resulted in 50 sequences of amino acids (blocks) conserved across their linear length. Protein members of the AGO4/6/8/9 and AGO1/10 clades are more conserved than members of the AGO5 and AGO2/3/7 clades. In addition to blocks containing components of the PIWI, PAZ, and DUF1785 domains, members of the AGO2/3/7 and AGO4/6/8/9 clades possess other consensus block sequences that are exclusive of members within these clades, suggesting unforeseen functional specialization revealed by their primary sequence. We also show that AGO proteins of animal and plant kingdoms share linear sequences of blocks that include motifs involved in posttranslational modifications such as those regulating AGO2 in humans and the PIWI protein AUBERGINE in Drosophila. Our results open possibilities for exploring new structural and functional aspects related to the evolution of AGO proteins within the plant kingdom, and their convergence with analogous proteins in mammals and invertebrates. PMID:27635128

  5. A Primary Sequence Analysis of the ARGONAUTE Protein Family in Plants.

    PubMed

    Rodríguez-Leal, Daniel; Castillo-Cobián, Amanda; Rodríguez-Arévalo, Isaac; Vielle-Calzada, Jean-Philippe

    2016-01-01

    Small RNA (sRNA)-mediated gene silencing represents a conserved regulatory mechanism controlling a wide diversity of developmental processes through interactions of sRNAs with proteins of the ARGONAUTE (AGO) family. On the basis of a large phylogenetic analysis that includes 206 AGO genes belonging to 23 plant species, AGO genes group into four clades corresponding to the phylogenetic distribution proposed for the ten family members of Arabidopsis thaliana. A primary analysis of the corresponding protein sequences resulted in 50 sequences of amino acids (blocks) conserved across their linear length. Protein members of the AGO4/6/8/9 and AGO1/10 clades are more conserved than members of the AGO5 and AGO2/3/7 clades. In addition to blocks containing components of the PIWI, PAZ, and DUF1785 domains, members of the AGO2/3/7 and AGO4/6/8/9 clades possess other consensus block sequences that are exclusive of members within these clades, suggesting unforeseen functional specialization revealed by their primary sequence. We also show that AGO proteins of animal and plant kingdoms share linear sequences of blocks that include motifs involved in posttranslational modifications such as those regulating AGO2 in humans and the PIWI protein AUBERGINE in Drosophila. Our results open possibilities for exploring new structural and functional aspects related to the evolution of AGO proteins within the plant kingdom, and their convergence with analogous proteins in mammals and invertebrates.

  6. [Quantitative monitoring targeted proteins and intermediate metabolites in Escherichia coli primary metabolic pathways].

    PubMed

    Cheng, Yongbo; Deng, Zixin; Liu, Tiangang

    2015-11-04

    This study aimed to monitor targeted protein expression levels and changes in intermediate metabolites in the primary metabolic pathways of Escherichia coli to obtain basic data and to develop testing methods that can be used in metabolic engineering. We used the Skyline software to design a label-free method (multiple reaction monitoring) for relative quantitative monitoring of proteins from primary metabolic pathways (i. e., glycolytic pathway, pentose phosphate pathway, mixed acid fermentation, tricarboxylic acid cycle, and fatty acid synthesis pathway). We used the same mass spectrometry platform (Triple Quad 4500) for liquid chromatography-tandem mass spectrometry (multiple reaction monitoring) analysis of targeted intermediate metabolites during absolute quantitative monitoring. Protein expression in the primary metabolic pathways of E. coli showed four different phenomena in the different growth periods (exponential phase, stationary phase, and decline phase). Expression levels of a single protein cannot provide accurate information regarding the status of these pathways. More proteins in the pentose phosphate pathway, mixed acid fermentation, and the tricarboxylic acid cycle showed the highest expression in the decline phase, but accumulation of several targeted intermediate metabolites (ATP, ADP, AMP, NAD+, NADH, NADP+, NADPH, CoA, and acetyl-CoA) in the stationary phase and decline phase correspondingly decreased compared to the levels in the exponential phase (in addition to acetyl-CoA). The detection methods used in this study can help determine the basic conditions of E. coli metabolism in vivo.

  7. Exploiting the diversity of the heat-shock protein family for primary and secondary tauopathy therapeutics.

    PubMed

    Abisambra, Jose F; Jinwal, Umesh K; Jones, Jeffrey R; Blair, Laura J; Koren, John; Dickey, Chad A

    2011-12-01

    The heat shock protein (Hsp) family is an evolutionarily conserved system that is charged with preventing unfolded or misfolded proteins in the cell from aggregating. In Alzheimer's disease, extracellular accumulation of the amyloid β peptide (Aβ) and intracellular aggregation of the microtubule associated protein tau may result from mechanisms involving chaperone proteins like the Hsps. Due to the ability of Hsps to regulate aberrantly accumulating proteins like Aβ and tau, therapeutic strategies are emerging that target this family of chaperones to modulate their pathobiology. This article focuses on the use of Hsp-based therapeutics for treating primary and secondary tauopathies like Alzheimer's disease. It will particularly focus on the pharmacological targeting of the Hsp70/90 system and the value of manipulating Hsp27 for treating Alzheimer's disease.

  8. Photochemical tyrosine oxidation in the structurally well-defined α3Y protein: proton-coupled electron transfer and a long-lived tyrosine radical.

    PubMed

    Glover, Starla D; Jorge, Christine; Liang, Li; Valentine, Kathleen G; Hammarström, Leif; Tommos, Cecilia

    2014-10-08

    Tyrosine oxidation-reduction involves proton-coupled electron transfer (PCET) and a reactive radical state. These properties are effectively controlled in enzymes that use tyrosine as a high-potential, one-electron redox cofactor. The α3Y model protein contains Y32, which can be reversibly oxidized and reduced in voltammetry measurements. Structural and kinetic properties of α3Y are presented. A solution NMR structural analysis reveals that Y32 is the most deeply buried residue in α3Y. Time-resolved spectroscopy using a soluble flash-quench generated [Ru(2,2'-bipyridine)3](3+) oxidant provides high-quality Y32-O• absorption spectra. The rate constant of Y32 oxidation (kPCET) is pH dependent: 1.4 × 10(4) M(-1) s(-1) (pH 5.5), 1.8 × 10(5) M(-1) s(-1) (pH 8.5), 5.4 × 10(3) M(-1) s(-1) (pD 5.5), and 4.0 × 10(4) M(-1) s(-1) (pD 8.5). k(H)/k(D) of Y32 oxidation is 2.5 ± 0.5 and 4.5 ± 0.9 at pH(D) 5.5 and 8.5, respectively. These pH and isotope characteristics suggest a concerted or stepwise, proton-first Y32 oxidation mechanism. The photochemical yield of Y32-O• is 28-58% versus the concentration of [Ru(2,2'-bipyridine)3](3+). Y32-O• decays slowly, t1/2 in the range of 2-10 s, at both pH 5.5 and 8.5, via radical-radical dimerization as shown by second-order kinetics and fluorescence data. The high stability of Y32-O• is discussed relative to the structural properties of the Y32 site. Finally, the static α3Y NMR structure cannot explain (i) how the phenolic proton released upon oxidation is removed or (ii) how two Y32-O• come together to form dityrosine. These observations suggest that the dynamic properties of the protein ensemble may play an essential role in controlling the PCET and radical decay characteristics of α3Y.

  9. Atom transfer radical polymerization to fabricate monodisperse poly[glycidyl methacrylate-co-poly (ethylene glycol) methacrylate] microspheres and its application for protein affinity purification.

    PubMed

    Yu, Ling; Shi, Zhuan Zhuan; Li, Chang Ming

    2015-09-01

    Poly[glycidyl methacrylate-co-poly (ethylene glycol) methacrylate] microspheres for the first time were successfully synthesized by atom transfer radical polymerization (ATRP) method at room temperature. The co-polymerization approach was investigated to delicately control the microsphere morphology and size-distribution by reaction conditions including solvent percentage, monomer loading and rotation speed. The results show that the average size of the microspheres is ∼5.7 μm with coexistence of epoxy, hydroxyl and ether groups, which provide plentiful functional sites for protein anchoring. The mechanism of the microsphere formation is proposed. The microsphere successfully demonstrates its unique application for affinity purification of proteins, in which the functional epoxy group facilitates a simple and efficient protein covalent immobilization to purify immunoglobulin G on the microspheres, while the hydrophilic poly (ethylene glycol) motif can repulse nonspecific protein adsorption for good specificity. This microspheres can be used in broad protein biosensors due to their abundant functional groups and high surface to volume ratio.

  10. Epigenetic regulation of human hedgehog interacting protein in glioma cell lines and primary tumor samples

    PubMed Central

    Shahi, Mehdi H.; Zazpe, Idoya; Afzal, Mohammad; Sinha, Subrata; Rebhun, Robert B.; Meléndez, Bárbara; Rey, Juan A.

    2016-01-01

    Glioma constitutes one of the most common groups of brain tumors, and its prognosis is influenced by different genetic and epigenetic modulations. In this study, we demonstrated low or no expression of hedgehog interacting protein (HHIP) in most of the cell lines and primary glioma tumor samples. We further proceeded to promoter methylation study of this gene in the same cell lines and primary tumor samples and found 87 % (7/8) HHIP methylation in glioblastoma cell lines and 75 % (33/44) in primary tumor samples. These methylation pattern correlates with low or unexpressed HHIP in both cell lines and primary tumor samples. Our results suggest the possibility of epigenetic regulation of this gene in glioma, similarly to medulloblastoma, gastric, hepatic, and pancreatic cancers. Also, HHIP might be a diagnostic or prognostic marker in glioma and help to the detection of these tumors in early stages of disease. PMID:25416442

  11. Angelman syndrome protein UBE3A interacts with primary microcephaly protein ASPM, localizes to centrosomes and regulates chromosome segregation.

    PubMed

    Singhmar, Pooja; Kumar, Arun

    2011-01-01

    Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

  12. Angelman Syndrome Protein UBE3A Interacts with Primary Microcephaly Protein ASPM, Localizes to Centrosomes and Regulates Chromosome Segregation

    PubMed Central

    Singhmar, Pooja; Kumar, Arun

    2011-01-01

    Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome. PMID:21633703

  13. Folding by Numbers: Primary Sequence Statistics and Their Use in Studying Protein Folding

    PubMed Central

    Wathen, Brent; Jia, Zongchao

    2009-01-01

    The exponential growth over the past several decades in the quantity of both primary sequence data available and the number of protein structures determined has provided a wealth of information describing the relationship between protein primary sequence and tertiary structure. This growing repository of data has served as a prime source for statistical analysis, where underlying relationships between patterns of amino acids and protein structure can be uncovered. Here, we survey the main statistical approaches that have been used for identifying patterns within protein sequences, and discuss sequence pattern research as it relates to both secondary and tertiary protein structure. Limitations to statistical analyses are discussed, and a context for their role within the field of protein folding is given. We conclude by describing a novel statistical study of residue patterning in β-strands, which finds that hydrophobic (i,i+2) pairing in β-strands occurs more often than expected at locations near strand termini. Interpretations involving β-sheet nucleation and growth are discussed. PMID:19468326

  14. Radical induced disulfide bond cleavage within peptides via ultraviolet irradiation of an electrospray plume.

    PubMed

    Stinson, Craig A; Xia, Yu

    2013-05-21

    Radical induced disulfide bond cleavage in peptides was demonstrated by ultraviolet (UV) radiation of the electrospray ionization (ESI) plume using a low pressure mercury (LP-Hg) lamp. Tandem mass spectrometry and accurate mass measurements confirmed that the primary reaction products were due to disulfide bond cleavage to form thiol (-SH) and sulfinyl radical (-SO˙). Mechanistic studies showed that the 185 nm emission from a LP-Hg lamp was responsible for UV photolysis of atmospheric O2, which further initiated secondary radical formation and subsequent disulfide bond cleavage by radical attack. The radical induced disulfide bond cleavage was found to be analytically useful in providing rich sequence information for naturally occurring peptides containing intrachain disulfide bonds. The utility of this method was also demonstrated for facile disulfide peptide identification and characterization from protein digests.

  15. Atypical protein kinase C regulates primary dendrite specification of cerebellar Purkinje cells by localizing Golgi apparatus.

    PubMed

    Tanabe, Koji; Kani, Shuichi; Shimizu, Takashi; Bae, Young-Ki; Abe, Takaya; Hibi, Masahiko

    2010-12-15

    Neurons have highly polarized structures that determine what parts of the soma elaborate the axon and dendrites. However, little is known about the mechanisms that establish neuronal polarity in vivo. Cerebellar Purkinje cells extend a single primary dendrite from the soma that ramifies into a highly branched dendritic arbor. We used the zebrafish cerebellum to investigate the mechanisms by which Purkinje cells acquire these characteristics. To examine dendritic morphogenesis in individual Purkinje cells, we marked the cell membrane using a Purkinje cell-specific promoter to drive membrane-targeted fluorescent proteins. We found that zebrafish Purkinje cells initially extend multiple neurites from the soma and subsequently retract all but one, which becomes the primary dendrite. In addition, the Golgi apparatus specifically locates to the root of the primary dendrite, and its localization is already established in immature Purkinje cells that have multiple neurites. Inhibiting secretory trafficking through the Golgi apparatus reduces dendritic growth, suggesting that the Golgi apparatus is involved in the dendritic morphogenesis. We also demonstrated that in a mutant of an atypical protein kinase C (aPKC), Prkci, Purkinje cells retain multiple primary dendrites and show disrupted localization of the Golgi apparatus. Furthermore, a mosaic inhibition of Prkci in Purkinje cells recapitulates the aPKC mutant phenotype. These results suggest that the aPKC cell autonomously controls the Golgi localization and thereby regulates the specification of the primary dendrite of Purkinje cells.

  16. Tyrosine-lipid peroxide adducts from radical termination: para coupling and intramolecular Diels-Alder cyclization.

    PubMed

    Shchepin, Roman; Möller, Matias N; Kim, Hye-young H; Hatch, Duane M; Bartesaghi, Silvina; Kalyanaraman, Balaraman; Radi, Rafael; Porter, Ned A

    2010-12-15

    Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogues of tyrosine gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The novel adducts were isolated and characterized by 1D and 2D NMR spectroscopy as well as by mass spectrometry (MS). The spectral data suggest that the polyunsaturated lipid peroxyl radicals give stable peroxide coupling products exclusively at the para position of the tyrosyl (phenoxy) radicals. These adducts have characteristic (13)C chemical shifts at 185 ppm due to the cross-conjugated carbonyl of the phenol-derived cyclohexadienone. The primary peroxide adducts subsequently undergo intramolecular Diels-Alder (IMDA) cyclization, affording a number of diastereomeric tricyclic adducts that have characteristic carbonyl (13)C chemical shifts at ~198 ppm. All of the NMR HMBC and HSQC correlations support the structure assignments of the primary and Diels-Alder adducts, as does MS collision-induced dissociation data. Kinetic rate constants and activation parameters for the IMDA reaction were determined, and the primary adducts were reduced with cuprous ion to give a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products from adduction of peroxyls at the phenolic ortho position were found in either the primary or cuprous reduction product mixtures. These studies provide a framework for understanding the nature of lipid-protein adducts formed by peroxyl-tyrosyl radical-radical termination processes. Coupling of lipid peroxyl radicals with tyrosyl radicals leads to cyclohexenone and cyclohexadienone adducts, which are of interest in and of themselves since, as electrophiles, they are likely targets for protein nucleophiles. One consequence of lipid peroxyl reactions with tyrosyls may therefore be protein-protein cross-links via interprotein Michael adducts.

  17. 43-kilodalton protein of Torpedo nicotinic postsynaptic membranes: purification and determination of primary structure

    SciTech Connect

    Carr, C.; McCourt, D.; Cohen, J.B.

    1987-11-03

    The primary structure of the 43-kilodalton peripheral membrane protein (43-kDa protein) of Torpedo nicotinic postsynaptic membrane has been determined. The /sup 14/C-labelled 43-kDa protein, which was isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has an amino terminus resistant to Edman degradation, while the sequence at the carboxyl terminus is Tyr-Val. An amino acid sequence of 405 residues was obtained by NH/sub 2/-terminal sequence analysis of complementary peptides generated by digestion with trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and endoproteinase Lys-C, as well as by chemical cleavage at methionine. This sequence of molecular mass 45,618 daltons lacks the amino terminus but extends to the carboxyl terminus of the 43-kDa protein. Unusual structural features of the 43-kDa protein include two regions of approx. 80 residues, each containing 10% cysteine, as well as stretches predicted to exist as amphipathic ..cap alpha..-helices. Other than the group blocking the amino terminus, no evidence was found for posttranslational modification of amino acids. The 43-kDa protein may represent a novel protein family because a computer search of this sequence with the National Biomedical Research Foundation data base (Release 12.0) did not reveal any significant homology to known protein sequences.

  18. Comparison of gel properties and biochemical characteristics of myofibrillar protein from bighead carp (Aristichthys nobilis) affected by frozen storage and a hydroxyl radical-generation oxidizing system.

    PubMed

    Lu, Han; Zhang, Longteng; Li, Qingzheng; Luo, Yongkang

    2017-05-15

    We wanted to clarify whether gel properties can be affected by in vivo or in vitro myofibrillar protein oxidation and, thus, to provide relevant information and a scientific foundation for the processing of gel products. To accomplish this, we measured the changes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), total disulfide (SS) content, surface hydrophobicity (So-ANS), carbonyl content, and gel texture and water-holding capacity (WHC) of isolated myofibrillar protein from bighead carp fillets during frozen storage and under different H2O2 concentrations, which were used to represent in vivo and in vitro conditions, respectively. The results indicated that a certain range in content of disulfide crosslinks (0.91mol/10(5)g protein) would promote gel hardness. Mild protein oxidation caused by a certain degree of frozen storage and hydroxyl radicals can promote gel texture and WHC. Based on those results, freezing bighead carp for a certain period can be used to produce gel products. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. BAG3 protein regulates caspase-3 activation in HIV-1-infected human primary microglial cells

    PubMed Central

    Rosati, Alessandra; Khalili, Kamel; Deshmane, Satish L.; Radhakrishnan, Sujatha; Pascale, Maria; Turco, M. Caterina; Marzullo, Liberato

    2015-01-01

    BAG3, a member of the BAG co-chaperones family, is expressed in several cell types subjected to stressful conditions, such as exposure to high temperature, heavy metals, drugs. Furthermore, it is constitutively expressed in some tumors. Among the biological activities of the protein, there is apoptosis downmodulation; this appears to be exerted through BAG3 interaction with the heat shock protein (Hsp) 70, that influences cell apoptosis at several levels. We recently reported that BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV-1-associated encephalopathy biopsies. Here we report that downmodulation of BAG3 protein levels allows caspase-3 activation by HIV-1 infection in human primary microglial cells. This is the first reported evidence of a role for BAG3 in the balance of death versus survival during viral infection. PMID:18821563

  20. A new hybrid coding for protein secondary structure prediction based on primary structure similarity.

    PubMed

    Li, Zhong; Wang, Jing; Zhang, Shunpu; Zhang, Qifeng; Wu, Wuming

    2017-03-16

    The coding pattern of protein can greatly affect the prediction accuracy of protein secondary structure. In this paper, a novel hybrid coding method based on the physicochemical properties of amino acids and tendency factors is proposed for the prediction of protein secondary structure. The principal component analysis (PCA) is first applied to the physicochemical properties of amino acids to construct a 3-bit-code, and then the 3 tendency factors of amino acids are calculated to generate another 3-bit-code. Two 3-bit-codes are fused to form a novel hybrid 6-bit-code. Furthermore, we make a geometry-based similarity comparison of the protein primary structure between the reference set and the test set before the secondary structure prediction. We finally use the support vector machine (SVM) to predict those amino acids which are not detected by the primary structure similarity comparison. Experimental results show that our method achieves a satisfactory improvement in accuracy in the prediction of protein secondary structure.

  1. Interaction of Tissue Engineering Substrates with Serum Proteins and Its Influence on Human Primary Endothelial Cells.

    PubMed

    Mohan, Tamilselvan; Niegelhell, Katrin; Nagaraj, Chandran; Reishofer, David; Spirk, Stefan; Olschewski, Andrea; Stana Kleinschek, Karin; Kargl, Rupert

    2017-02-13

    Polymer-based biomaterials particularly polycaprolactone (PCL) are one of the most promising substrates for tissue engineering. The surface chemistry of these materials plays a major role since it governs protein adsorption, cell adhesion, viability, degradation, and biocompatibility in the first place. This study correlates the interaction of the most abundant serum proteins (albumin, immunoglobulins, fibrinogen) with the surface properties of PCL and its influence on the morphology and metabolic activity of primary human arterial endothelial cells that are seeded on the materials. Prior to that, thin films of PCL are manufactured by spin-coating and characterized in detail. A quartz crystal microbalance with dissipation (QCM-D), a multiparameter surface plasmon resonance spectroscopy instrument (MP-SPR), wettability data, and atomic force microscopy are combined to elucidate the pH-dependent protein adsorption on the PCL substrates. Primary endothelial cells are cultured on the protein modified polymer, and conclusions are drawn on the significant impact of type and form of proteins coatings on cell morphology and metabolic activity.

  2. Quantitative Correlation Between the Protein Primary Sequences and Secondary Structures in Spider Dragline Silks

    PubMed Central

    Jenkins, Janelle E.; Creager, Melinda S.; Lewis, Randolph V.; Holland, Gregory P.; Yarger, Jeffery L.

    2009-01-01

    Synthetic spider silk holds great potential for use in various applications spanning medical uses to ultra lightweight armor, however producing synthetic fibers with mechanical properties comparable to natural spider silk has eluded the scientific community. Natural dragline spider silks are commonly made from proteins that contain highly repetitive amino acid motifs, adopting an array of secondary structures. Before further advances can be made in the production of synthetic fibers based on spider silk proteins, it is imperative to know the percentage of each amino acid in the protein that forms a specific secondary structure. Linking these percentages to the primary amino acid sequence of the protein will establish a structural foundation for synthetic silk. In this study, Nuclear Magnetic Resonance (NMR) techniques are used to quantify the percentage of Ala, Gly, and Ser that form both β-sheet and helical secondary structures. The fraction of these three amino acids and their secondary structure are quantitatively correlated to the primary amino acid sequence for the proteins that comprise major and minor ampullate silk from the Nephila clavipes spider providing a blueprint for synthetic spider silks. PMID:20000730

  3. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

    PubMed

    Rao, Vasudev R; Eugenin, Eliseo A; Prasad, Vinayaka R

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

  4. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures

    PubMed Central

    Rao, Vasudev R.; Eugenin, Eliseo A.; Prasad, Vinayaka R.

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood–brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes. PMID:26714725

  5. The diagnosis and management of cow milk protein intolerance in the primary care setting.

    PubMed

    Ewing, Whitney Merrill; Allen, Patricia Jackson

    2005-01-01

    Cow milk protein intolerance (CMPI) affects 3% of infants under the age of 12 months and is often misdiagnosed as GERD or colic, risking dangerous exposure to antigens. Most infants out grow CMPI by 12 months; however, those with IgE-mediated reactions usually continue to be intolerant to cow's milk proteins and also develop other allergens including environmental allergens that cause asthmatic symptoms. Clinical manifestations of CMPI include diarrhea, bloody stools, vomiting, feeding refusal, eczema, atopic dermatitis, urticaria, angioedema, allergic rhinitis, coughing, wheezing, failure to thrive, and anaphylaxis. The research and literature showed that CMPI is easily missed in the primary care setting and needs to be considered as a cause of infant distress and clinical symptoms. This article focuses on correctly diagnosing CMPI and managing it in the primary care setting.

  6. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

    PubMed

    Storey, Stephen M; McIntosh, Avery L; Huang, Huan; Landrock, Kerstin K; Martin, Gregory G; Landrock, Danilo; Payne, H Ross; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2012-04-15

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2

  7. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes

    PubMed Central

    Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Landrock, Kerstin K.; Martin, Gregory G.; Landrock, Danilo; Payne, H. Ross; Atshaves, Barbara P.; Kier, Ann B.

    2012-01-01

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2

  8. Protein-protein interactions among components of the Drosophila primary sex determination signal.

    PubMed

    Liu, Y; Belote, J M

    1995-07-28

    Sex determination in Drosophila melanogaster is initiated in the early embryo by a signal provided by three types of genes: (1) X-linked numerator elements [e.g., sisterless-a (sis-a) and sisterless-b (sis-b)], (2) autosomally linked denominator elements [e.g., deadpan (dpn)], and (3) maternal factors [e.g., daughterless (da)]. This signal acts to stimulate transcription from an embryo-specific promoter of the master regulatory gene Sex-lethal (Sxl) in embryos that have two X chromosomes (females), while it fails to activate Sxl in those with only one X (males). It has been previously proposed that competitive dimerizations among the components of this signal might provide the molecular basis for this sex specificity. Here, we use the yeast two-hybrid system to demonstrate specific protein-protein interactions among the above-mentioned factors, and to delimit their interacting domains. These results support and extend the model of the molecular basis of the X/A ratio signal.

  9. Photolysis of CH₃CHO at 248 nm: evidence of triple fragmentation from primary quantum yield of CH₃ and HCO radicals and H atoms.

    PubMed

    Morajkar, Pranay; Bossolasco, Adriana; Schoemaecker, Coralie; Fittschen, Christa

    2014-06-07

    Radical quantum yields have been measured following the 248 nm photolysis of acetaldehyde, CH3CHO. HCO radical and H atom yields have been quantified by time resolved continuous wave Cavity Ring Down Spectroscopy in the near infrared following their conversion to HO2 radicals by reaction with O2. The CH3 radical yield has been determined using the same technique following their conversion into CH3O2. Absolute yields have been deduced for HCO radicals and H atoms through fitting of time resolved HO2 profiles, obtained under various O2 concentrations, to a complex model, while the CH3 yield has been determined relative to the CH3 yield from 248 nm photolysis of CH3I. Time resolved HO2 profiles under very low O2 concentrations suggest that another unknown HO2 forming reaction path exists in this reaction system besides the conversion of HCO radicals and H atoms by reaction with O2. HO2 profiles can be well reproduced under a large range of experimental conditions with the following quantum yields: CH3CHO + hν(248nm) → CH3CHO*, CH3CHO* → CH3 + HCO ϕ(1a) = 0.125 ± 0.03, CH3CHO* → CH3 + H + CO ϕ(1e) = 0.205 ± 0.04, CH3CHO*[Formula: see text]CH3CO + HO2 ϕ(1f) = 0.07 ± 0.01. The CH3O2 quantum yield has been determined in separate experiments as ϕ(CH₃) = 0.33 ± 0.03 and is in excellent agreement with the CH3 yields derived from the HO2 measurements considering that the triple fragmentation (R1e) is an important reaction path in the 248 nm photolysis of CH3CHO. From arithmetic considerations taking into account the HO2 and CH3 measurements we deduce a remaining quantum yield for the molecular pathway: CH3CHO* → CH4 + CO ϕ(1b) = 0.6. All experiments can be consistently explained with absence of the formerly considered pathway: CH3CHO* → CH3CO + H ϕ(1c) = 0.

  10. Immortalization of primary human keratinocytes by the helix–loop–helix protein, Id-1

    PubMed Central

    Alani, Rhoda M.; Hasskarl, Jens; Grace, Miranda; Hernandez, Maria-Clementia; Israel, Mark A.; Münger, Karl

    1999-01-01

    Basic helix–loop–helix (bHLH) DNA-binding proteins have been demonstrated to regulate tissue-specific transcription within multiple cell lineages. The Id family of helix–loop–helix proteins does not possess a basic DNA-binding domain and functions as a negative regulator of bHLH proteins. Overexpression of Id proteins within a variety of cell types has been shown to inhibit their ability to differentiate under appropriate conditions. We demonstrate that ectopic expression of Id-1 leads to activation of telomerase activity and immortalization of primary human keratinocytes. These immortalized cells have a decreased capacity to differentiate as well as activate phosphorylation of the retinoblastoma protein. Additionally, these cells acquire an impaired p53-mediated DNA-damage response as a late event in immortalization. We conclude that bHLH proteins play a pivotal role in regulating normal keratinocyte growth and differentiation, which can be disrupted by the immortalizing functions of Id-1 through activation of telomerase activity and inactivation of the retinoblastoma protein. PMID:10449746

  11. Human Immunodeficiency Virus Type 1 Coat Protein Neurotoxicity Mediated by Nitric Oxide in Primary Cortical Cultures

    NASA Astrophysics Data System (ADS)

    Dawson, Valina L.; Dawson, Ted M.; Uhl, George R.; Snyder, Solomon H.

    1993-04-01

    The human immunodeficiency virus type 1 coat protein, gp120, kills neurons in primary cortical cultures at low picomolar concentrations. The toxicity requires external glutamate and calcium and is blocked by glutamate receptor antagonists. Nitric oxide (NO) contributes to gp120 toxicity, since nitroarginine, an inhibitor of NO synthase, prevents toxicity as does deletion of arginine from the incubation medium and hemoglobin, which binds NO. Superoxide dismutase also attenuates toxicity, implying a role for superoxide anions.

  12. Human hedgehog interacting protein expression and promoter methylation in medulloblastoma cell lines and primary tumor samples

    PubMed Central

    Shahi, Mehdi H.; Afzal, Mohammad; Sinha, Subrata; Eberhart, Charles G.; Rey, Juan A.; Fan, Xing

    2015-01-01

    Medulloblastoma is the most common pediatric brain tumor and its development is affected by genetic and epigenetic factors. In this study we found there is low or no expression of the hedgehog interacting protein (HHIP), a negative regulator of the sonic hedgehog pathway, in most medulloblastoma cell lines and primary samples explored. We proceeded to promoter methylation assays of this gene by MCA-Meth, and found that HHIP was hypermethylated in all medulloblastoma cell lines, but only in 2 out of 14 (14%) primary tumor samples. Methylation correlated with low or unexpressed HHIP in cell lines but not in primary tumor samples. These results suggest the possibility of epigenetic regulation of HHIP in medulloblastoma, similarly to gastric, hepatic and pancreatic cancer. However, HHIP seems to be not only under regulation of promoter methylation, but under other factors involved in the control of its low levels of expression in medulloblastoma. PMID:20853133

  13. Exploiting sulphur-carrier proteins from primary metabolism for 2-thiosugar biosynthesis

    PubMed Central

    Sasaki, Eita; Zhang, Xuan; Sun, He G.; Lu, Mei-Yeh Jade; Liu, Tsung-lin; Ou, Albert; Li, Jeng-yi; Chen, Yu-hsiang; Ealick, Steven E.; Liu, Hung-wen

    2014-01-01

    Sulphur is an essential element for life and exists ubiquitously in living systems1,2. Yet, how the sulphur atom is incorporated in many sulphur-containing secondary metabolites remains poorly understood. For C-S bond formation in primary metabolites, the major ionic sulphur sources are the protein-persulphide and protein-thiocarboxylate3,4. In each case, the persulphide and thiocarboxylate group on these sulphur-carrier (donor) proteins are post-translationally generated through the action of a specific activating enzyme. In all bacterial cases reported thus far, the genes encoding the enzyme that catalyzes the actual C-S bond formation reaction and its cognate sulphur-carrier protein co-exist in the same gene cluster5. To study 2-thiosugar production in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action appear similar to those of ThiG, the enzyme catalyzing thiazole formation in thiamin biosynthesis6,7. However, no sulphur-carrier protein gene could be located in the BE-7585A cluster. Subsequent genome sequencing revealed the presence of a few sulphur-carrier proteins likely involved in the biosynthesis of primary metabolites, but surprisingly only a single activating enzyme gene in the entire genome of A. orientalis. Further experiments showed that this activating enzyme is capable of adenylating each of these sulphur-carrier proteins, and likely also catalyzing the subsequent thiolation taking advantage of its rhodanese activity. A proper combination of these sulphur delivery systems is effective for BexX-catalyzed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. These studies represent the first complete characterization of a thiosugar formation in nature and also demonstrate the receptor promiscuity of the sulphur-delivery system in A. orientalis. Our

  14. Analysis of Soluble Protein Entry into Primary Cilia Using Semi-Permeabilized Cells

    PubMed Central

    Breslow, David K.; Nachury, Maxence V.

    2016-01-01

    The primary cilium is a protrusion from the cell surface that serves as a specialized compartment for signal transduction. Many signaling factors are known to be dynamically concentrated within cilia and to require cilia for their function. Yet protein entry into primary cilia remains poorly understood. To enable a mechanistic analysis of soluble protein entry into cilia, we developed a method for semi-permeabilization of mammalian cells in which the plasma membrane is permeabilized while the ciliary membrane remains intact. Using semi-permeabilized cells as the basis for an in vitro diffusion-to-capture assay, we uncovered a size-dependent diffusion barrier that restricts soluble protein exchange between the cytosol and the cilium. The manipulability of this in vitro system enabled an extensive characterization of the ciliary diffusion barrier and led us to show that the barrier is mechanistically distinct from those at the axon initial segment and the nuclear pore complex. Because semi-permeabilized cells enable a range of experimental perturbations that would not be easily feasible in intact cells, we believe this methodology will provide a unique resource for investigating primary cilium function in development and disease. PMID:25837393

  15. High 1,3-trans stereoselectivity in nucleophilic substitution at the anomeric position and β-fragmentation of the primary alkoxyl radical in 3-amino-3-deoxy-ribofuranose derivatives: application to the synthesis of 2-epi-(-)-jaspine B.

    PubMed

    Sánchez-Eleuterio, Alma; Quintero, Leticia; Sartillo-Piscil, Fernando

    2011-07-01

    The high inverse stereoselectivity in the nucleophilic substitution at the anomeric position of 3-amino-3-deoxy-ribofuranose derivatives is reported. This unprecedented stereoselectivity is explained in terms of preferential nucleophilic attack on the "inside face" of the respective five-membered ring oxocarbenium ion that orients pseudoequatorially to the benzylamine group placed at the C-3 position. In addition, an unusual β-fragmentation of a primary alkoxyl radical generated from its corresponding N-phthalimide derivative was achieved, and thus taking advantages of both reactions, the total synthesis of 2-epi-(-)-jaspine B was completed.

  16. Identification of novel DNA repair proteins via primary sequence, secondary structure, and homology.

    PubMed

    Brown, J B; Akutsu, Tatsuya

    2009-01-20

    DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM). We identify that SVM techniques are capable of identifying portions of DNA repair protein datasets without admitting false positives; at low levels of false positive tolerance, homology can also identify and classify proteins with good performance. Secondary structure information provides improved performance compared to using primary structure alone. Furthermore, we observe that machine learning methods incorporating homology information perform best when data is filtered by some clustering technique. Analysis by applying these methodologies to the scanning of multiple vertebrate genomes confirms a positive correlation between the size of a genome and the number of DNA repair protein transcripts it is likely to contain, and simultaneously suggests that all organisms have a non-zero minimum number of repair genes. In addition, the scan result clusters several organisms' repair abilities in an evolutionarily consistent fashion. Analysis also identifies several functionally unconfirmed proteins that are highly

  17. Identification of novel DNA repair proteins via primary sequence, secondary structure, and homology

    PubMed Central

    Brown, JB; Akutsu, Tatsuya

    2009-01-01

    Background DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM). Results We identify that SVM techniques are capable of identifying portions of DNA repair protein datasets without admitting false positives; at low levels of false positive tolerance, homology can also identify and classify proteins with good performance. Secondary structure information provides improved performance compared to using primary structure alone. Furthermore, we observe that machine learning methods incorporating homology information perform best when data is filtered by some clustering technique. Analysis by applying these methodologies to the scanning of multiple vertebrate genomes confirms a positive correlation between the size of a genome and the number of DNA repair protein transcripts it is likely to contain, and simultaneously suggests that all organisms have a non-zero minimum number of repair genes. In addition, the scan result clusters several organisms' repair abilities in an evolutionarily consistent fashion. Analysis also identifies several functionally unconfirmed

  18. Modulation of genotoxic effects in asbestos-exposed primary human mesothelial cells by radical scavengers, metal chelators and a glutathione precursor.

    PubMed

    Poser, Ina; Rahman, Qamar; Lohani, Mohtashim; Yadav, Santosh; Becker, Hans-Henner; Weiss, Dieter G; Schiffmann, Dietmar; Dopp, Elke

    2004-04-11

    The genotoxicity of asbestos fibers is generally mediated by reactive oxygen species (ROS) and by insufficient antioxidant protection. To further elucidate which radicals are involved in asbestos-mediated genotoxicity and to which extent, we have carried out experiments with the metal chelators deferoxamine (DEF) and phytic acid (PA), and with the radical scavengers superoxide dismutase (SOD), dimethylthiourea (DMTU) and the glutathione precursor Nacystelyn trade mark (NAL). We investigated the influence of these compounds on the potency of crocidolite, an amphibole asbestos fiber with a high iron content (27%), and chrysotile, a serpentine asbestos fiber with a low iron content (2%), to induce micronuclei (MN) in human mesothelial cells (HMC) after an exposure time of 24-72 h. Our results show that the number of crocidolite-induced MN is significantly reduced after pretreatment of fibers with PA and DEF. This effect was not observed with chrysotile. In contrast, simultaneous treatment of cells with asbestos and the OH*scavenging DMTU or the O2- -scavenging SOD significantly decreased the number of MN induced by chrysotile and crocidolite. In particular, DMTU almost completely suppressed micronucleus induction by both fiber types. A similar effect was observed in the presence of the H(2)O(2)-scavenging NAL after chrysotile treatment of HMC. By means of kinetochore analysis, it could be shown that the number of clastogenic events is decreased after PA and DEF pretreatment of fibers as well as after application of the above-mentioned scavengers. Our results show that chrysotile asbestos induces an increased release of H(2)O(2) in contrast to crocidolite. Also, the iron content of the fiber plays an important role in radical formation, but nevertheless, chrysotile produces oxy radicals to a similar extent as crocidolite, probably by phagocytosis-mediated oxidative bursting.

  19. Detection of radicals produced by reaction of hydroperoxides with rat liver microsomal fractions.

    PubMed

    Greenley, T L; Davies, M J

    1992-04-22

    EPR spin trapping using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 3,5-dibromo-4-nitrosobenzene sulphonic acid (DBNBS) has been employed to examine the generation of radicals produced on reaction of a number of primary, secondary and lipid hydroperoxides with rat liver microsomal fractions in both the presence and absence of reducing equivalents. Two major mechanisms of radical generation have been elucidated. In the absence of NADPH or NADH, oxidative degradation of the hydroperoxide occurs to give initially a peroxyl radical which in the majority of cases can be detected as a spin adduct to DMPO; these radicals can undergo further reactions which result in the generation of alkoxyl and carbon-centered radicals. In the presence of NADPH (and to a lesser extent NADH) alkoxyl radicals are generated directly via reductive cleavage of the hydroperoxide. These alkoxyl radicals undergo further fragmentation and rearrangement reactions to give carbon-centered species which can be identified by trapping with DBNBS. The type of transformation that occurs is highly dependent on the structure of the alkoxyl radical with species arising from beta-scission, 1,2-hydrogen shifts and ring closure reactions being identified; these processes are in accord with previous chemical studies and are characteristic of alkoxyl radicals present in free solution. Studies using specific enzyme inhibitors and metal-ion chelators suggest that most of the radical generation occurs via a catalytic process involving haem proteins and in particular cytochrome P-450. An unusual species (an acyl radical) is observed with lipid hydroperoxides; this is believed to arise via a cage reaction after beta-scission of an initial alkoxyl radical.

  20. Mechanisms of hydroxyl radical-induced contraction of rat aorta.

    PubMed

    Li, Jianfeng; Li, Wenyan; Liu, Weimin; Altura, Bella T; Altura, Burton M

    2004-09-19

    The present study was designed to investigate the effects of hydroxyl radicals (*OH), generated via the Fe2+-mediated Fenton reaction, on isolated rat aortic rings with and without endothelium. In the absence of any vasoactive agent, generation of *OH alone elicited an endothelium-independent contraction in rat aortic rings in a concentration-dependent manner. Hydroxyl radical-induced contractions of denuded rat aortic rings appeared, however, to be slightly stronger than those on intact rat aortic rings. The contractile responses to *OH were neither reversible nor reproducible in the same ring; even small concentrations of *OH radicals resulted in tachyphylaxis. Removal of extracellular calcium ions (Ca2+) or buffering intracellular Ca2+ with 10 microM acetyl methyl ester of bis(o-aminophenoxy) ethane-N,N,N',N',-tetraacetic acid (BAPTA-AM) significantly attenuated the contractile actions of *OH radicals. The presence of 1 microM staurosporine, 1 microM bisindolylmaleimide I, 1 microM Gö6976 [inhibitor of protein kinase C (PKC)], 2 microM PD-980592 (inhibitor of ERK), 10 microM genistein, and 1 microM wortmannin significantly inhibited the contractions induced by *OH. Proadifen (10 microM), on the other hand, significantly potentiated the hydroxyl radical-induced contractions. Exposure of primary cultured aortic smooth muscle cells to *OH produced significant, rapid rises of intracellular free Ca2+ ([Ca2+]i). Several, specific antagonists of possible endogenously formed vasoconstrictors did not inhibit or attenuate either hydroxyl radical-induced contractions or the elevation of [Ca2+]i. Our new results suggest that hydroxyl radical-triggered contractions on rat aortic rings are Ca2+-dependent. Several intracellular signal transduction systems seem to play some role in hydroxyl radical-induced vasoconstriction of rat aortic rings.

  1. Preparation and characterization of protein-resistant hydrogels for soft contact lens applications via radical copolymerization involving a zwitterionic sulfobetaine comonomer.

    PubMed

    Zhang, Wanlu; Li, Guangji; Lin, Yinlei; Wang, Liying; Wu, Shuqing

    2017-11-01

    We aimed to introduce hydrophilic sulfobetaine-type zwitterionic groups to macromolecular chains of copolymers to construct novel copolymer hydrogels with anti-protein-fouling performance that could be used as soft contact lens (SCL) materials. Using hydroxyethyl methacrylate (HEMA), N-vinyl pyrrolidinone (NVP) and sulfobetaine methacrylate (SBMA) as comonomers, several copolymer hydrogels with different SBMA contents, poly(HEMA-NVP-SBMA), are synthesized via radical copolymerization in an aqueous phase. Surface chemistry, structural morphologies, water contact angle (WCA), equilibrium water content (EWC), visible light transmittance and tensile mechanical properties are investigated. The prepared hydrogels exhibit a closed-type porous structure. With increasing SBMA content in the comonomer mixture, the prepared copolymer hydrogel pore size gradually increases up to the micron level, WCA tends to decrease, EWC tends to increase, and visible light transmittance slightly increases, but their tensile mechanical properties decline. The amounts of protein Lyz and BSA adsorbed on the copolymer hydrogels and on commercially available EASY DAY(®) SCLs as a control are also determined by protein adsorption tests. The amount of protein adsorbed on the copolymer hydrogel decreases with increasing SBMA content. For the hydrogel prepared using the comonomer mixture with 5.0 wt % SBMA, the amount of adsorbed Lyz is 0.91 μg/cm(2), which corresponds to only 56.8% of the amount adsorbed on EASY DAY(®) SCLs. Thus, novel SCL materials with high water content and excellent anti-protein-fouling performance were efficiently constructed by introducing sulfobetaine-type zwitterionic groups into a traditional polymer hydrogel system.

  2. [Prediction of protein thermostability from their primary structure: the current state and development factors].

    PubMed

    Grishin, D V; Pokrovskaya, M V; Podobed, O V; Gladilina, Ju A; Pokrovsky, V S; Aleksandrova, S S; Sokolov, N N

    2017-03-01

    The construction of proteins and peptides with desired properties, including resistance to high temperatures, as well as optimization of their amino acid composition, is an important and complex task, which attracts much attention in various branches of the basic sciences, and also in biomedicine and biotechnology. This raises the question: what method is more relevant for the at the pilot stage of research in order to estimate the influence of the planned amino acid substitutions on the thermostability of the resultant protein construct? In this brief review we have classified existing basic practical and theoretical approaches used in studies and predicting the thermal stability of native and recombinant polypeptides. Particular attention has been paid to the predictive potential of statistical methods for studying the thermodynamic parameters of the primary protein structure and prospects of their use.

  3. Determination of the primary structure of two lipid transfer proteins from apricot (Prunus armeniaca).

    PubMed

    Conti, A; Fortunato, D; Ortolani, C; Giuffrida, M G; Pravettoni, V; Napolitano, L; Farioli, L; Perono Garoffo, L; Trambaioli, C; Pastorello, E A

    2001-05-25

    It has been recently demonstrated that the major allergen of apricot is a protein of molecular mass (Mr) 9000 belonging to the family of Lipid Transfer Protein. The aim of this study was the determination of the primary structure of apricot LTP by micro-sequencing and mass spectrometric analyses. Apricot LTP is a 91 amino acids protein like peach and almond LTPs with a sequence identity of 91% and 94%, respectively. Like for the peach LTP, out of the 25 amino acids forming the inner surface of the tunnel-like hydrophobic cavity in maize ns-LTP, 16 are identical and 7 similar in the apricot LTP, supporting the hypothesis of a similar function.

  4. Differential protein expression in tears of patients with primary open angle and pseudoexfoliative glaucoma.

    PubMed

    Pieragostino, Damiana; Bucci, Sonia; Agnifili, Luca; Fasanella, Vincenzo; D'Aguanno, Simona; Mastropasqua, Alessandra; Ciancaglini, Marco; Mastropasqua, Leonardo; Di Ilio, Carmine; Sacchetta, Paolo; Urbani, Andrea; Del Boccio, Piero

    2012-04-01

    Primary open angle (POAG) and pseudoexfoliative glaucoma (PXG) are the most common primary and secondary forms of glaucoma, respectively. Even though the patho-physiology, aqueous humor composition, risk factors, clinical features, therapy and drug induced ocular surface changes in POAG and PXG have been widely studied, to date information concerning tear protein characterization is lacking. Tears are a source of nourishment for ocular surface tissues and a vehicle to remove local waste products, metabolized drugs and inflammatory mediators produced in several ophthalmic diseases. In glaucoma, the proteomic definition of tears may provide insights concerning patho-physiology of the disease and ocular surface modifications induced by topical therapy. Our study aimed at characterizing protein patterns in tears of patients with medically controlled POAG and PXG. A comparative tears proteomic analysis by label-free LC-MS(E) highlighted differences in the expression of several proteins in the two glaucoma sub-types and control subjects, highlighting inflammation pathways expressed in both diseases. Results were independently reconfirmed by SDS-PAGE and linear MALDI-TOF MS, validating altered levels of Lysozyme C, Lipocalin-1, Protein S100, Immunoglobulins and Prolactin Inducible Protein. Moreover, we found a differential pattern of phosphorylated Cystatin-S that distinguishes the two pathologies. The most relevant results suggest that in both pathologies there may be active inflammation pathways related to the disease and/or induced by therapy. We show, for the first time, tear protein patterns expressed under controlled intraocular pressure conditions in POAG and PXG subjects. These findings could help in the understanding of molecular machinery underlying these ophthalmologic diseases, resulting in early diagnosis and more specific therapy.

  5. Prediction of RNA-binding proteins from primary sequence by a support vector machine approach

    PubMed Central

    HAN, LIAN YI; CAI, CONG ZHONG; LO, SIEW LIN; CHUNG, MAXEY C.M.; CHEN, YU ZONG

    2004-01-01

    Elucidation of the interaction of proteins with different molecules is of significance in the understanding of cellular processes. Computational methods have been developed for the prediction of protein–protein interactions. But insufficient attention has been paid to the prediction of protein–RNA interactions, which play central roles in regulating gene expression and certain RNA-mediated enzymatic processes. This work explored the use of a machine learning method, support vector machines (SVM), for the prediction of RNA-binding proteins directly from their primary sequence. Based on the knowledge of known RNA-binding and non-RNA-binding proteins, an SVM system was trained to recognize RNA-binding proteins. A total of 4011 RNA-binding and 9781 non-RNA-binding proteins was used to train and test the SVM classification system, and an independent set of 447 RNA-binding and 4881 non-RNA-binding proteins was used to evaluate the classification accuracy. Testing results using this independent evaluation set show a prediction accuracy of 94.1%, 79.3%, and 94.1% for rRNA-, mRNA-, and tRNA-binding proteins, and 98.7%, 96.5%, and 99.9% for non-rRNA-, non-mRNA-, and non-tRNA-binding proteins, respectively. The SVM classification system was further tested on a small class of snRNA-binding proteins with only 60 available sequences. The prediction accuracy is 40.0% and 99.9% for snRNA-binding and non-snRNA-binding proteins, indicating a need for a sufficient number of proteins to train SVM. The SVM classification systems trained in this work were added to our Web-based protein functional classification software SVMProt, at http://jing.cz3.nus.edu.sg/cgi-bin/svmprot.cgi. Our study suggests the potential of SVM as a useful tool for facilitating the prediction of protein–RNA interactions. PMID:14970381

  6. Prognostic significance of the bcl-2 apoptotic family of proteins in primary and recurrent cervical cancer.

    PubMed Central

    Crawford, R. A.; Caldwell, C.; Iles, R. K.; Lowe, D.; Shepherd, J. H.; Chard, T.

    1998-01-01

    bcl-2 is one of a family of genes that control the apoptotic threshold of a cell. bcl-2 protein and its anti-apoptotic homologue, mcl-1, with the pro-apoptotic protein, bax, are thought to function by forming homo- and heterotypic dimers that then control the progression to apoptosis. p53 is also involved as a down-regulator of bcl-2 and a promoter of bax. To determine the effect of these apoptotic mechanisms, we used immunohistochemistry to determine the prognostic significance of the expression of bcl-2, mcl-1, bax and p53 in primary and recurrent cervical cancer. Tissues from 46 patients with primary cervical cancer and 28 women with recurrent carcinoma were stained for bcl-2, mcl-1, bax and p53. Kaplan-Meier survival analysis was performed using the log-rank test for differences between groups. In the primary disease group, positive staining for bcl-2 was associated with a better 5-year survival (bcl-2 +ve, 84% vs bcl-2 -ve, 53%, P = 0.03). Positive staining for p53 was associated with a survival disadvantage (p53 +ve, 4-year survival 38% vs p53 -ve, 4-year survival 78%, P = 0.02). mcl-1 and bax staining were not useful as prognostic indicators in primary disease. No marker was prognostic in recurrent disease. Positive bcl-2 staining defines a group of patients with primary disease with a good prognosis. p53, an activator of the bax promoter, identifies a group with a worse outcome. In recurrent disease, none of the markers reflected prognosis. PMID:9683295

  7. Co-opting sulphur-carrier proteins from primary metabolic pathways for 2-thiosugar biosynthesis.

    PubMed

    Sasaki, Eita; Zhang, Xuan; Sun, He G; Lu, Mei-yeh Jade; Liu, Tsung-lin; Ou, Albert; Li, Jeng-yi; Chen, Yu-hsiang; Ealick, Steven E; Liu, Hung-wen

    2014-06-19

    Sulphur is an essential element for life and is ubiquitous in living systems. Yet how the sulphur atom is incorporated into many sulphur-containing secondary metabolites is poorly understood. For bond formation between carbon and sulphur in primary metabolites, the major ionic sulphur sources are the persulphide and thiocarboxylate groups on sulphur-carrier (donor) proteins. Each group is post-translationally generated through the action of a specific activating enzyme. In all reported bacterial cases, the gene encoding the enzyme that catalyses the carbon-sulphur bond formation reaction and that encoding the cognate sulphur-carrier protein exist in the same gene cluster. To study the production of the 2-thiosugar moiety in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action seem similar to those of ThiG, the enzyme that catalyses thiazole formation in thiamine biosynthesis. However, no gene encoding a sulphur-carrier protein could be located in the BE-7585A cluster. Subsequent genome sequencing uncovered a few genes encoding sulphur-carrier proteins that are probably involved in the biosynthesis of primary metabolites but only one activating enzyme gene in the A. orientalis genome. Further experiments showed that this activating enzyme can adenylate each of these sulphur-carrier proteins and probably also catalyses the subsequent thiolation, through its rhodanese domain. A proper combination of these sulphur-delivery systems is effective for BexX-catalysed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. This study is, to our knowledge, the first complete characterization of thiosugar formation in nature and also demonstrates the receptor promiscuity of the A. orientalis sulphur-delivery system. Our results also show that co-opting the sulphur-delivery machinery

  8. Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF*♦

    PubMed Central

    Li, Zhu; Kitanishi, Kenichi; Twahir, Umar T.; Cracan, Valentin; Chapman, Derrell; Warncke, Kurt; Banerjee, Ruma

    2017-01-01

    IcmF is a 5′-deoxyadenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the carbon skeleton rearrangement of isobutyryl-CoA to butyryl-CoA. It is a bifunctional protein resulting from the fusion of a G-protein chaperone with GTPase activity and the cofactor- and substrate-binding mutase domains with isomerase activity. IcmF is prone to inactivation during catalytic turnover, thus setting up its dependence on a cofactor repair system. Herein, we demonstrate that the GTPase activity of IcmF powers the ejection of the inactive cob(II)alamin cofactor and requires the presence of an acceptor protein, adenosyltransferase, for receiving it. Adenosyltransferase in turn converts cob(II)alamin to AdoCbl in the presence of ATP and a reductant. The repaired cofactor is then reloaded onto IcmF in a GTPase-gated step. The mechanistic details of cofactor loading and offloading from the AdoCbl-dependent IcmF are distinct from those of the better characterized and homologous methylmalonyl-CoA mutase/G-protein chaperone system. PMID:28130442

  9. Ultrastructural Localization of a Bean Glycine-Rich Protein in Unlignified Primary Walls of Protoxylem Cells.

    PubMed Central

    Ryser, U; Keller, B

    1992-01-01

    A polyclonal antibody was used to localize a glycine-rich cell wall protein (GRP 1.8) in French bean hypocotyls with the indirect immunogold method. GRP 1.8 could be localized mainly in the unlignified primary cell walls of the oldest protoxylem elements and also in cell corners of both proto- and metaxylem elements. In addition, GRP 1.8 was detected in phloem using tissue printing. The labeled primary walls of dead protoxylem cells showed a characteristically dispersed ultrastructure, resulting from the action of hydrolases during the final steps of cell maturation and from mechanical stress due to hypocotyl growth. Primary walls of living protoxylem and adjacent parenchyma cells were only weakly labeled. This was true also for the secondary walls of proto- and metaxylem cells, which in addition showed high background labeling. Inhibition of lignification with a specific and potent inhibitor of phenylalanine ammonia-lyase did not lead to enhanced labeling of secondary walls, showing that lignin does not mask the presence of GRP 1.8 in these walls. Dictyosomes of living proto- and metaxylem cells were not labeled, but dictyosomes of xylem parenchyma cells without secondary walls, adjacent to strongly labeled protoxylem elements, were clearly labeled. These observations suggest that GRP 1.8 is not produced by xylem vessels but by xylem parenchyma cells that export the protein to the wall of protoxylem vessels. PMID:12297662

  10. Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development.

    PubMed

    Bhattacharyya, Sohinee; Rainey, Mark A; Arya, Priyanka; Dutta, Samikshan; George, Manju; Storck, Matthew D; McComb, Rodney D; Muirhead, David; Todd, Gordon L; Gould, Karen; Datta, Kaustubh; Gelineau-van Waes, Janee; Band, Vimla; Band, Hamid

    2016-02-17

    Members of the four-member C-terminal EPS15-Homology Domain-containing (EHD) protein family play crucial roles in endocytic recycling of cell surface receptors from endosomes to the plasma membrane. In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal. Ehd1-null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube. We found that Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5). Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1-null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH. Using Ehd1-null MEFS we found that EHD1 protein co-localizes with the SHH receptor Smoothened in the primary cilia upon ligand stimulation. Under the same conditions, EHD1 was shown to co-traffic with Smoothened into the developing primary cilia and we identify EHD1 as a direct binding partner of Smoothened. Overall, our studies identify the endocytic recycling regulator EHD1 as a novel regulator of the primary cilium-associated trafficking of Smoothened and Hedgehog signaling.

  11. Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

    PubMed Central

    Bhattacharyya, Sohinee; Rainey, Mark A; Arya, Priyanka; Dutta, Samikshan; George, Manju; Storck, Matthew D.; McComb, Rodney D.; Muirhead, David; Todd, Gordon L.; Gould, Karen; Datta, Kaustubh; Waes, Janee Gelineau-van; Band, Vimla; Band, Hamid

    2016-01-01

    Members of the four-member C-terminal EPS15-Homology Domain-containing (EHD) protein family play crucial roles in endocytic recycling of cell surface receptors from endosomes to the plasma membrane. In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal. Ehd1-null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube. We found that Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5). Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1-null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH. Using Ehd1-null MEFS we found that EHD1 protein co-localizes with the SHH receptor Smoothened in the primary cilia upon ligand stimulation. Under the same conditions, EHD1 was shown to co-traffic with Smoothened into the developing primary cilia and we identify EHD1 as a direct binding partner of Smoothened. Overall, our studies identify the endocytic recycling regulator EHD1 as a novel regulator of the primary cilium-associated trafficking of Smoothened and Hedgehog signaling. PMID:26884322

  12. Benidipine reduces myocardial infarct size involving reduction of hydroxyl radicals and production of protein kinase C-dependent nitric oxide in rabbits.

    PubMed

    Wang, Ningyuan; Minatoguchi, Shinya; Chen, Xue-Hai; Arai, Masazumi; Uno, Yoshihiro; Lu, ChuanJiang; Misao, Yu; Nagai, Hiroshi; Takemura, Genzou; Fujiwara, Hisayoshi

    2004-06-01

    Japanese white rabbits underwent 30 minutes of ischemia and 48 hours of reperfusion. Benidipine (3 or 10 microg/kg, i.v.) was administered 10 minutes before ischemia with and without pretreatment with L-NAME (10 mg/kg, i.v., a NOS inhibitor), chelerythrine (5 mg/kg, i.v., a PKC blocker) or 5-HD (5 mg/kg, i.v. a mitochondrial KATP channel blocker), genistein (5 mg/kg, i.v. a protein tyrosin kinase blocker). SNAP (2.5 mg/kg/min x 70 minutes, i.v., an NO donor) was also administered 10 minutes before ischemia. Benidipine significantly reduced the infarct size in a dose-dependent manner (3 microg/kg: 29.0 +/- 2.7%, n = 8, 10 microg/kg: 23.0 +/- 2.4%, n = 10) compared with the control (41.6 +/- 3.3%, n = 10). This effect was completely blocked by L-NAME (39.9 +/- 3.6%, n = 8) and chelerythrine (35.5 +/- 2.4%, n = 8) but not by 5-HD (23.0 +/- 2.4%, n = 10) or genistein (24.6 +/- 3.1%, n = 10). SNAP also reduced the infarct size (24.6 +/- 3.1%, n = 8). Benidipine significantly increased the expression of eNOS mRNA at 30 minutes after reperfusion and significantly increased the expression of eNOS protein at 3 hours after reperfusion in the ischemic area of the left ventricle. Benidipine and SNAP significantly decreased myocardial interstitial 2,5-DHBA levels, an indicator of hydroxyl radicals, during ischemia and reperfusion. Benidipine increased myocardial interstitial NOx levels, which effect was blocked by chelerythrine, during 0 to 30 minutes and 150 to 180 minutes after reperfusion. Benidipine reduces the infarct size through PKC-dependent production of nitric oxide and decreasing hydroxyl radicals but not through involving protein tyrosine kinase or mitochondrial KATP channels in rabbits.

  13. Molecular Recognition of PTS-1 Cargo Proteins by Pex5p: Implications for Protein Mistargeting in Primary Hyperoxaluria

    PubMed Central

    Mesa-Torres, Noel; Tomic, Nenad; Albert, Armando; Salido, Eduardo; Pey, Angel L.

    2015-01-01

    Peroxisomal biogenesis and function critically depends on the import of cytosolic proteins carrying a PTS1 sequence into this organelle upon interaction with the peroxin Pex5p. Recent structural studies have provided important insights into the molecular recognition of cargo proteins by Pex5p. Peroxisomal import is a key feature in the pathogenesis of primary hyperoxaluria type 1 (PH1), where alanine:glyoxylate aminotransferase (AGT) undergoes mitochondrial mistargeting in about a third of patients. Here, we study the molecular recognition of PTS1 cargo proteins by Pex5p using oligopeptides and AGT variants bearing different natural PTS1 sequences, and employing an array of biophysical, computational and cell biology techniques. Changes in affinity for Pex5p (spanning over 3–4 orders of magnitude) reflect different thermodynamic signatures, but overall bury similar amounts of molecular surface. Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation. Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities. Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state. PMID:25689234

  14. Molecular recognition of PTS-1 cargo proteins by Pex5p: implications for protein mistargeting in primary hyperoxaluria.

    PubMed

    Mesa-Torres, Noel; Tomic, Nenad; Albert, Armando; Salido, Eduardo; Pey, Angel L

    2015-02-13

    Peroxisomal biogenesis and function critically depends on the import of cytosolic proteins carrying a PTS1 sequence into this organelle upon interaction with the peroxin Pex5p. Recent structural studies have provided important insights into the molecular recognition of cargo proteins by Pex5p. Peroxisomal import is a key feature in the pathogenesis of primary hyperoxaluria type 1 (PH1), where alanine:glyoxylate aminotransferase (AGT) undergoes mitochondrial mistargeting in about a third of patients. Here, we study the molecular recognition of PTS1 cargo proteins by Pex5p using oligopeptides and AGT variants bearing different natural PTS1 sequences, and employing an array of biophysical, computational and cell biology techniques. Changes in affinity for Pex5p (spanning over 3-4 orders of magnitude) reflect different thermodynamic signatures, but overall bury similar amounts of molecular surface. Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation. Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities. Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

  15. Turnover of thylakoid membrane proteins during senescence of primary bean leaves. [Phaseolus vulgaris

    SciTech Connect

    Roberts, D.R.; Dumbroff, E.B.; Mattoo, A.K.; Thompson, J.E.

    1986-04-01

    Pulse-labelling of primary bean leaves (Phaseolus vulgaris) with /sup 35/S-methionine has revealed differential changes in the rates at which proteins in thylakoid membranes are synthesized during senescence. In particular, synthesis of the 32 Kd herbicide-binding protein remains highly active throughout senescence, whereas turnover of the ..cap alpha.. and ..beta.. subunits of ATPase and of the LHCP declines. During a 24-h pulse chase experiment with unlabelled methionine, only the 32 KD protein showed evidence of degradation. Degradation of the 32 Kd unit and, to a lesser extent, of other thylakoid proteins was also observed when the membranes were aged in vitro. The latter process resembled that observed in vivo in that it was light dependent, sensitive to DCMU, and it was inhibited by spermine and Ca/sup 2 +/, both of which alter membrane fluidity. Collectively, these observations suggest that in vitro aging of thylakoid membranes is a useful model system for studying the characteristics of the thylakoid protein degradation.

  16. Phylogenetic and immunological definition of four lipoylated proteins from Novosphingobium aromaticivorans, implications for primary biliary cirrhosis.

    PubMed

    Padgett, Kerstien A; Selmi, Carlo; Kenny, Thomas P; Leung, Patrick S C; Balkwill, David L; Ansari, Aftab A; Coppel, Ross L; Gershwin, M Eric

    2005-05-01

    Novosphingobium aromaticivorans, a unique ubiquitous bacterium that metabolizes xenobiotics and activates environmental estrogens, has been suggested as a pathogenic factor in the development of primary biliary cirrhosis (PBC). To define the molecular basis of PBC sera reactivity, we investigated the characteristic of the bacterial antigens involved. We cloned and sequenced four genes from N. aromaticivorans coding for immunoreactive proteins, arbitrarily named Novo 1 through Novo 4. We subsequently analyzed these proteins for their homology to known mitochondrial proteins and defined their reactivity using monoclonal antibodies (mAbs), rabbit anti-lipoic acid antibody, and PBC/control sera. Moreover, we studied their phylogenetic relation with the known PBC autoantigens. Novo proteins have an extraordinary degree of amino acid homology with all of the major human mitochondrial autoantigens PDC-E2 (Novo 1 and 2), OGDC-E2 (Novo 3), and BCOADC-E2 (Novo 4). Moreover, Novo 1-4 contain a lipoylated domain, are recognized by AMA-positive sera, and react with specific mAbs to mitochondrial antigens. Interestingly, the phylogenetic relation of the proteins emphasizes the conservation of the lipoylated domain. In conclusion, our data provide a high degree of confidence that N. aromaticivorans may potentiate the breakdown of self tolerance in genetically susceptible individuals.

  17. Why whey? Camel whey protein as a new dietary approach to the management of free radicals and for the treatment of different health disorders.

    PubMed

    Badr, Gamal; Ramadan, Nancy K; Sayed, Leila H; Badr, Badr M; Omar, Hossam M; Selamoglu, Zeliha

    2017-04-01

    The balance between free radicals and antioxidants is an important factor for maintaining health and slowing disease progression. The use of antioxidants, particularly natural antioxidants, has become an important strategy for dealing with this cause of widespread diseases. Natural antioxidants have been used as therapeutic tools against many diseases because they are safe, effective, and inexpensive and are among the most commonly used adjuvants in the treatment of several diseases. Camel whey protein (CWP) is considered a strong natural antioxidant because it decreases oxidative stress, enhances immune system function, and increases glutathione levels. The structure of CWP is very similar to that of other types of whey protein from different types of milk. CWP contains many components, such as lactoferrin (LF), lactalbumin, lactoglobulins, lactoperoxidase, and lysozyme, and is rich in immunoglobulins. However, in contrast to other WPs, CWP lacks β-lactoglobulin, the main cause of milk allergies in children. The components of CWP have many beneficial effects, including stimulation of both innate and adaptive immunity and anti-inflammatory, anticancer, antibacterial, and antiviral activities. Recently, it has been shown that CWP and its unique components can facilitate the treatment of impaired diabetic wound healing. However, the molecular mechanisms underlying the protective effects of CWP in human and other animal disorders are not fully understood. Therefore, the current review presents a concise summary of the scientific evidence of the beneficial effects of CWP to support its therapeutic use in disease treatment and nutritional intervention.

  18. Why whey? Camel whey protein as a new dietary approach to the management of free radicals and for the treatment of different health disorders

    PubMed Central

    Badr, Gamal; Ramadan, Nancy K; Sayed, Leila H; Badr, Badr M; Omar, Hossam M; Selamoglu, Zeliha

    2017-01-01

    The balance between free radicals and antioxidants is an important factor for maintaining health and slowing disease progression. The use of antioxidants, particularly natural antioxidants, has become an important strategy for dealing with this cause of widespread diseases. Natural antioxidants have been used as therapeutic tools against many diseases because they are safe, effective, and inexpensive and are among the most commonly used adjuvants in the treatment of several diseases. Camel whey protein (CWP) is considered a strong natural antioxidant because it decreases oxidative stress, enhances immune system function, and increases glutathione levels. The structure of CWP is very similar to that of other types of whey protein from different types of milk. CWP contains many components, such as lactoferrin (LF), lactalbumin, lactoglobulins, lactoperoxidase, and lysozyme, and is rich in immunoglobulins. However, in contrast to other WPs, CWP lacks β-lactoglobulin, the main cause of milk allergies in children. The components of CWP have many beneficial effects, including stimulation of both innate and adaptive immunity and anti-inflammatory, anticancer, antibacterial, and antiviral activities. Recently, it has been shown that CWP and its unique components can facilitate the treatment of impaired diabetic wound healing. However, the molecular mechanisms underlying the protective effects of CWP in human and other animal disorders are not fully understood. Therefore, the current review presents a concise summary of the scientific evidence of the beneficial effects of CWP to support its therapeutic use in disease treatment and nutritional intervention. PMID:28804604

  19. Neuronal inclusion protein TDP-43 has no primary genetic role in FTD and ALS.

    PubMed

    Gijselinck, Ilse; Sleegers, Kristel; Engelborghs, Sebastiaan; Robberecht, Wim; Martin, Jean-Jacques; Vandenberghe, Rik; Sciot, Raf; Dermaut, Bart; Goossens, Dirk; van der Zee, Julie; De Pooter, Tim; Del-Favero, Jurgen; Santens, Patrick; De Jonghe, Peter; De Deyn, Peter P; Van Broeckhoven, Christine; Cruts, Marc

    2009-08-01

    The nuclear TAR DNA binding protein (TDP-43) is deposited in ubiquitin-positive inclusions in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two clinicopathologically overlapping neurodegenerative diseases. In this study we excluded mutations and copy number variations in the gene encoding TDP-43 (TARDBP) from an extended series of 173 FTD and 237 ALS patients. Further, we did not identify association of common genetic variants in these patients. Our data implicate that TDP-43 has no primary genetic role in the pathophysiological mechanisms underlying central nervous system neurodegeneration in these diseases.

  20. Methionine restriction decreases mitochondrial oxygen radical generation and leak as well as oxidative damage to mitochondrial DNA and proteins.

    PubMed

    Sanz, Alberto; Caro, Pilar; Ayala, Victoria; Portero-Otin, Manuel; Pamplona, Reinald; Barja, Gustavo

    2006-06-01

    Previous studies have consistently shown that caloric restriction (CR) decreases mitochondrial reactive oxygen species (ROS) (mitROS) generation and oxidative damage to mtDNA and mitochondrial proteins, and increases maximum longevity, although the mechanisms responsible for this are unknown. We recently found that protein restriction (PR) also produces these changes independent of energy restriction. Various facts link methionine to aging, and methionine restriction (MetR) without energy restriction increases, like CR, maximum longevity. We have thus hypothesized that MetR is responsible for the decrease in mitROS generation and oxidative stress in PR and CR. In this investigation we subjected male rats to exactly the same dietary protocol of MetR that is known to increase their longevity. We have found, for the first time, that MetR profoundly decreases mitROS production, decreases oxidative damage to mtDNA, lowers membrane unsaturation, and decreases all five markers of protein oxidation measured in rat heart and liver mitochondria. The concentration of complexes I and IV also decreases in MetR. The decrease in mitROS generation occurs in complexes I and III in liver and in complex I in heart mitochondria, and is due to an increase in efficiency of the respiratory chain in avoiding electron leak to oxygen. These changes are strikingly similar to those observed in CR and PR, suggesting that the decrease in methionine ingestion is responsible for the decrease in mitochondrial ROS production and oxidative stress, and possibly part of the decrease in aging rate, occurring during caloric restriction.

  1. Enhanced proliferation of primary rat type II pneumocytes by Jaagsiekte sheep retrovirus envelope protein

    SciTech Connect

    Johnson, Chassidy; Jahid, Sohail; Voelker, Dennis R.; Fan Hung

    2011-04-10

    Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep. The envelope protein (Env) is the oncogene, as it can transform cell lines in culture and induce tumors in animals, although the mechanisms for transformation are not yet clear because a system to perform transformation assays in differentiated type II pneumocytes does not exist. In this study we report culture of primary rat type II pneumocytes in conditions that favor prolonged expression of markers for type II pneumocytes. Env-expressing cultures formed more colonies that were larger in size and were viable for longer periods of time compared to vector control samples. The cells that remained in culture longer were confirmed to be derived from type II pneumocytes because they expressed surfactant protein C, cytokeratin, displayed alkaline phosphatase activity and were positive for Nile red. This system will be useful to study JSRV Env in the targets of transformation.

  2. Novel protein transfection of primary rat cortical neurons using an antibody that penetrates living cells.

    PubMed

    Weisbart, R H; Baldwin, R; Huh, B; Zack, D J; Nishimura, R

    2000-06-01

    An Ab-based system to deliver functional proteins into neurons was developed using the murine mAb, mAb 3E10. This was achieved by covalently conjugating catalase to the Ab so that the conjugate retained high activity for the degradation of hydrogen peroxide. Three-dimensional fluorescence microscopy was used to demonstrate penetration of the Ab into the nucleus of living primary cortical neurons. The Ab conjugate localized in both the cytoplasm and nucleus. Retention of catalase activity after penetration and distribution of conjugate was demonstrated by reduction in cell death following exposure of treated neurons to hydrogen peroxide. These studies illustrate the potential of this method for the intracellular delivery of therapeutic proteins.

  3. Role of peptide primary sequence in polyphenol-protein recognition: an example with neurotensin.

    PubMed

    Richard, T; Vitrac, X; Merillon, J M; Monti, J P

    2005-11-30

    Polyphenols are known for their impact on health and one of their major properties is the formation of complexes with proteins. To investigate the involvement of polyphenol-protein complexes in health, the interactions between bioactive polyphenols and neurotensin were examined by structural NMR and molecular modeling. Neurotensin is a linear bioactive tridecapeptide and polyphenols seem to affect the NT metabolism. We studied the polyphenols resveratrol and its glucoside the piceid in order to observe the possible role of glucose group and the penta-O-galloyl-D-glucopyranose (PGG). NMR data and molecular modeling showed that interaction occurred with the three polyphenols involving hydrophobic stacking and hydrogen bonds. Moreover, the peptide primary sequence plays a role in the specificity of complex formation.

  4. Immuno-spin trapping detection of antioxidant/pro-oxidant properties of zinc or selenium on DNA and protein radical formation via hydrogen peroxide.

    PubMed

    Deletioglu, Vedia; Tuncay, Erkan; Toy, Aysegul; Atalay, Mustafa; Turan, Belma

    2015-11-01

    Trace elements can participate in the catalysis of group-transfer reactions and can serve as their structural components. However, most of them including zinc and selenium have multifunctional roles in biological environments such as antioxidant and/or pro-oxidant effects, as concentration-dependent manner. Although it has been demonstrated the antioxidant actions of either selenium or zinc compounds, there are several documents pointing out their pro-oxidant/oxidant roles in biological systems. Here we have used ELISA-based immuno-spin trapping, a method for detection of free radical formation, to detect whether or not a zinc compound, Zn3(PO4)2, or a selenium compound, Na2SeO3, has antioxidant and/or pro-oxidant effect on 5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO)-DNA nitrone adducts induced with Cu(II)-H2O2-oxidizing system in in vitro preparations. Second, we examined whether this technique is capable to demonstrate the different DMPO-protein nitrone adduct productions in isolated protein crude of hearts from normal rats (CON) or rats with metabolic syndrome (MetS). Our data demonstrated that either Zn(2+) (100 µM) or SeO3(-2) (50 nM) has very strong antioxidant action against 200 µM H2O2-induced DMPO-DNA nitrone adduct production, whereas their higher concentrations have apparent pro-oxidant actions. We also used verification by Western blotting analysis whether immuno-spin trapping can be used to assess H2O2-induced DMPO-protein nitrone adducts in heart protein crudes. Our Western blot data further confirmed the ELISA-data from proteins and demonstrated how Zn(2+) or SeO3(-2) are dual-functioning ions such as antioxidant at lower concentrations while pro-oxidant at higher concentrations. Particularly, our present data with SeO3(-2) in DMPO-protein nitrone adducts, being in line with our previous observation on its dual-actions in ischemia/reperfusion-induced damaged heart, have shown that this ion has higher pro-oxidant actions over 50 nM in Met

  5. Potential cytoprotection: antioxidant defence by caffeic acid phenethyl ester against free radical-induced damage of lipids, DNA, and proteins.

    PubMed

    Wang, Ting; Chen, Lixiang; Wu, Weimin; Long, Yuan; Wang, Rui

    2008-05-01

    Oxidative stress is considered to be a major cause of cellular injuries in a variety of chronic health problems, such as carcinogenesis and neurodegenerative disorders. Caffeic acid phenethyl ester (CAPE), derived from the propolis of honeybee hives, possesses a variety of biological and pharmacological properties including antioxidant and anticancer activity. In the present study, we focused on the diverse antioxidative functionalities of CAPE and its related polyphenolic acid esters on cellular macromolecules in vitro. The effects on human erythrocyte membrane ghost lipid peroxidation, plasmid pBR322 DNA, and protein damage initiated by the water-soluble initiator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) and hydrogen peroxide (H(2)O(2)) were monitored by formation of hydroperoxides and by DNA nicking assay, single-cell alkaline electrophoresis, and SDS-polyacrylamide gel electrophoresis. Our results showed that CAPE and its related polyphenolic acid esters elicited remarkable inhibitory effects on erythrocyte membrane lipid peroxidation, cellular DNA strand breakage, and protein fragmentation. The results suggest that CAPE is a potent exogenous cytoprotective and antigenotoxic agent against cell oxidative damage that could be used as a template for designing novel drugs to combat diseases induced by oxidative stress components, such as various types of cancer.

  6. Inhibitory properties of bambara groundnut protein hydrolysate and peptide fractions against angiotensin-converting enzymes, renin and free radicals.

    PubMed

    Arise, Abimbola K; Alashi, Adeola M; Nwachukwu, Ifeanyi D; Malomo, Sunday A; Aluko, Rotimi E; Amonsou, Eric O

    2017-07-01

    An increased rate of high blood pressure has led to critical human hypertensive conditions in most nations. In the present study, bambara protein hydrolysates (BPHs) obtained using three different proteases (alcalase, trypsin and pepsin) and their peptide fractions (molecular weight: 10, 5, 3 and 1 kDa) were investigated for antihypertensive and antioxidant activities. Alcalase hydrolysate contained the highest amount of low molecular weight (LMW) peptides compared to pepsin and trypsin hydrolysates. LMW peptides fractions (<1 kDa) exhibited the highest inhibitory activity against angiotensin-converting enzyme (ACE) for all the enzymes hydrolysates. For renin inhibition, alcalase hydrolysate showed the highest inhibition at 59% compared to other hydrolysates and their corresponding membrane fractions. The antioxidant power of bambara protein hydrolysates and peptide fractions was evaluated through the inhibition of linoleic acid peroxidation and ABTS scavenging activity. Among the hydrolysates, alcalase exhibited the highest inhibition of linoleic acid oxidation. Furthermore, all BPHs were able to scavenge ABTS(•+) to a three-fold greater extent compared to the isolate. BPH and LMW peptide fractions could potentially serve as useful ingredients in the formulation of functional foods and nutraceuticals against high blood pressure and oxidative stress. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  7. ToF-SIMS Analysis of Adsorbed Proteins: Principal Component Analysis of the Primary Ion Species Effect on the Protein Fragmentation Patterns

    PubMed Central

    Muramoto, Shin; Graham, Daniel J.; Wagner, Matthew S.; Lee, Tae Geol; Moon, Dae Won; Castner, David G.

    2011-01-01

    In time-of-flight secondary ion mass spectrometry (ToF-SIMS), the choice of primary ion used for analysis can influence the resulting mass spectrum. This is because different primary ion types can produce different fragmentation pathways. In this study, analysis of single-component protein monolayers were performed using monatomic, tri-atomic, and polyatomic primary ion sources. Eight primary ions (Cs+, Au+, Au3+, Bi+, Bi3+, Bi3++, C60+) were used to examine to the low mass (m/z < 200) fragmentation patterns from five different proteins (bovine serum albumin, bovine serum fibrinogen, bovine immunoglobulin G and chicken egg white lysozyme) adsorbed onto mica surfaces. Principal component analysis (PCA) processing of the ToF-SIMS data showed that variation in peak intensity caused by the primary ions was greater than differences in protein composition. The spectra generated by Cs+, Au+ and Bi+ primary ions were similar, but the spectra generated by monatomic, tri-atomic and polyatomic primary ion ions varied significantly. C60 primary ions increased fragmentation of the adsorbed proteins in the m/z < 200 region, resulting in more intense low m/z peaks. Thus, comparison of data obtained by one primary ion species with that obtained by another primary ion species should be done with caution. However, for the spectra generated using a given primary ion beam, discrimination between the spectra of different proteins followed similar trends. Therefore, a PCA model of proteins created with a given ion source should only be applied to datasets obtained using the same ion source. The type of information obtained from PCA depended on the peak set used. When only amino acid peaks were used, PCA was able to identify the relationship between proteins by their amino acid composition. When all peaks from m/z 12-200 were used, PCA separated proteins based on a ratio of C4H8N+ to K+ peak intensities. This ratio correlated with the thickness of the protein films and Bi1+ primary ions

  8. ToF-SIMS Analysis of Adsorbed Proteins: Principal Component Analysis of the Primary Ion Species Effect on the Protein Fragmentation Patterns.

    PubMed

    Muramoto, Shin; Graham, Daniel J; Wagner, Matthew S; Lee, Tae Geol; Moon, Dae Won; Castner, David G

    2011-12-15

    In time-of-flight secondary ion mass spectrometry (ToF-SIMS), the choice of primary ion used for analysis can influence the resulting mass spectrum. This is because different primary ion types can produce different fragmentation pathways. In this study, analysis of single-component protein monolayers were performed using monatomic, tri-atomic, and polyatomic primary ion sources. Eight primary ions (Cs(+), Au(+), Au(3) (+), Bi(+), Bi(3) (+), Bi(3) (++), C(60) (+)) were used to examine to the low mass (m/z < 200) fragmentation patterns from five different proteins (bovine serum albumin, bovine serum fibrinogen, bovine immunoglobulin G and chicken egg white lysozyme) adsorbed onto mica surfaces. Principal component analysis (PCA) processing of the ToF-SIMS data showed that variation in peak intensity caused by the primary ions was greater than differences in protein composition. The spectra generated by Cs(+), Au(+) and Bi(+) primary ions were similar, but the spectra generated by monatomic, tri-atomic and polyatomic primary ion ions varied significantly. C(60) primary ions increased fragmentation of the adsorbed proteins in the m/z < 200 region, resulting in more intense low m/z peaks. Thus, comparison of data obtained by one primary ion species with that obtained by another primary ion species should be done with caution. However, for the spectra generated using a given primary ion beam, discrimination between the spectra of different proteins followed similar trends. Therefore, a PCA model of proteins created with a given ion source should only be applied to datasets obtained using the same ion source. The type of information obtained from PCA depended on the peak set used. When only amino acid peaks were used, PCA was able to identify the relationship between proteins by their amino acid composition. When all peaks from m/z 12-200 were used, PCA separated proteins based on a ratio of C(4)H(8)N(+) to K(+) peak intensities. This ratio correlated with the thickness

  9. Structural Analysis of the Glycosylated Intact HIV-1 gp120–b12 Antibody Complex Using Hydroxyl Radical Protein Footprinting

    PubMed Central

    2017-01-01

    Glycoprotein gp120 is a surface antigen and virulence factor of human immunodeficiency virus 1. Broadly neutralizing antibodies (bNAbs) that react to gp120 from a variety of HIV isolates offer hope for the development of broadly effective immunogens for vaccination purposes, if the interactions between gp120 and bNAbs can be understood. From a structural perspective, gp120 is a particularly difficult system because of its size, the presence of multiple flexible regions, and the large amount of glycosylation, all of which are important in gp120–bNAb interactions. Here, the interaction of full-length, glycosylated gp120 with bNAb b12 is probed using high-resolution hydroxyl radical protein footprinting (HR-HRPF) by fast photochemical oxidation of proteins. HR-HRPF allows for the measurement of changes in the average solvent accessible surface area of multiple amino acids without the need for measures that might alter the protein conformation, such as mutagenesis. HR-HRPF of the gp120–b12 complex coupled with computational modeling shows a novel extensive interaction of the V1/V2 domain, probably with the light chain of b12. Our data also reveal HR-HRPF protection in the C3 domain caused by interaction of the N330 glycan with the b12 light chain. In addition to providing information about the interactions of full-length, glycosylated gp120 with b12, this work serves as a template for the structural interrogation of full-length glycosylated gp120 with other bNAbs to better characterize the interactions that drive the broad specificity of the bNAb. PMID:28102671

  10. Primary Central Nervous System (CNS) Lymphoma B Cell Receptors Recognize CNS Proteins.

    PubMed

    Montesinos-Rongen, Manuel; Purschke, Frauke G; Brunn, Anna; May, Caroline; Nordhoff, Eckhard; Marcus, Katrin; Deckert, Martina

    2015-08-01

    Primary lymphoma of the CNS (PCNSL) is a diffuse large B cell lymphoma confined to the CNS. To elucidate its peculiar organ tropism, we generated recombinant Abs (recAbs) identical to the BCR of 23 PCNSLs from immunocompetent patients. Although none of the recAbs showed self-reactivity upon testing with common autoantigens, they recognized 1547 proteins present on a large-scale protein microarray, indicating polyreactivity. Interestingly, proteins (GRINL1A, centaurin-α, BAIAP2) recognized by the recAbs are physiologically expressed by CNS neurons. Furthermore, 87% (20/23) of the recAbs, including all Abs derived from IGHV4-34 using PCNSL, recognized galectin-3, which was upregulated on microglia/macrophages, astrocytes, and cerebral endothelial cells upon CNS invasion by PCNSL. Thus, PCNSL Ig may recognize CNS proteins as self-Ags. Their interaction may contribute to BCR signaling with sustained NF-κB activation and, ultimately, may foster tumor cell proliferation and survival. These data may also explain, at least in part, the affinity of PCNSL cells for the CNS.

  11. Protein Homeostasis Defects of Alanine-Glyoxylate Aminotransferase: New Therapeutic Strategies in Primary Hyperoxaluria Type I

    PubMed Central

    Pey, Angel L.; Albert, Armando; Salido, Eduardo

    2013-01-01

    Alanine-glyoxylate aminotransferase catalyzes the transamination between L-alanine and glyoxylate to produce pyruvate and glycine using pyridoxal 5′-phosphate (PLP) as cofactor. Human alanine-glyoxylate aminotransferase is a peroxisomal enzyme expressed in the hepatocytes, the main site of glyoxylate detoxification. Its deficit causes primary hyperoxaluria type I, a rare but severe inborn error of metabolism. Single amino acid changes are the main type of mutation causing this disease, and considerable effort has been dedicated to the understanding of the molecular consequences of such missense mutations. In this review, we summarize the role of protein homeostasis in the basic mechanisms of primary hyperoxaluria. Intrinsic physicochemical properties of polypeptide chains such as thermodynamic stability, folding, unfolding, and misfolding rates as well as the interaction of different folding states with protein homeostasis networks are essential to understand this disease. The view presented has important implications for the development of new therapeutic strategies based on targeting specific elements of alanine-glyoxylate aminotransferase homeostasis. PMID:23956997

  12. Mutations in exocyst complex subunit SEC6 gene impaired polar auxin transport and PIN protein recycling in Arabidopsis primary root.

    PubMed

    Tan, Xiaoyun; Feng, Yihong; Liu, Yulong; Bao, Yiqun

    2016-09-01

    Polar auxin transport, which is critical for land plant pattern formation and directional growth, is largely depended on asymmetric distribution of PIN proteins at the plasma membrane (PM). Endocytosis and recycling processes play important roles in regulating PIN protein distribution and abundance at the PM. Two subunits (SEC8, EXO70A1) of exocyst, an octameric vesicle-tethering complex, have been reported to be involved in PIN protein recycling in Arabidopsis. However, the function of exocyst complex in PIN protein recycling and polar auxin transport remains incompletely understood. In this study, we utilized two SEC6 down-regulation mutants (PRsec6-1 and PRsec6-2) to investigate the role of exocyst subunit SEC6 in the primary root development, polar auxin transport and PIN proteins recycling. We found that in PRsec6 mutants: 1. Primary root growth was retarded, and lateral root initiation were compromised. 2. Primary roots were sensitive to exogenous auxin 1-napthalene acetic acid (NAA) but not 2,4-dichlorophenoxy (2.4-D). 3. Recycling of PIN1 and PIN2 proteins from the Brefeldin A (BFA) compartment to the PM was delayed. 4. Vesicles accumulated in the primary root tip cells, especially accumulated in the cytosol closed to the PM. These results further demonstrated that the exocyst complex plays an important role in PIN protein recycling and polar auxin transport in Arabidopsis primary root. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. The Lowe syndrome protein OCRL1 is involved in primary cilia assembly.

    PubMed

    Coon, Brian G; Hernandez, Victor; Madhivanan, Kayalvizhi; Mukherjee, Debarati; Hanna, Claudia B; Barinaga-Rementeria Ramirez, Irene; Lowe, Martin; Beales, Philip L; Aguilar, R Claudio

    2012-04-15

    Lowe syndrome (LS) is a devastating, X-linked genetic disease characterized by the presence of congenital cataracts, profound learning disabilities and renal dysfunction. Unfortunately, children affected with LS often die early of health complications including renal failure. Although this syndrome was first described in the early 1950s and the affected gene, OCRL1, was identified more than 17 years ago, the mechanism by which Ocrl1 defects lead to LS's symptoms remains unknown. Here we show that LS display characteristics of a ciliopathy. Specifically, we found that patients' cells have defects in the assembly of primary cilia and this phenotype was reproduced in cell lines by knock-down of Ocrl1. Importantly, this defect could be rescued by re-introduction of WT Ocrl1 in both patient and Ocrl1 knock-down cells. In addition, a zebrafish animal model of LS exhibited cilia defects and multiple morphological and anatomical abnormalities typically seen in ciliopathies. Mechanistically, we show that Ocrl1 is involved in protein trafficking to the primary cilia in an Rab8-and IPIP27/Ses-dependent manner. Taking into consideration the relevance of the signaling pathways hosted by the primary cilium, our results suggest hitherto unrecognized mechanisms by which Ocrl1 deficiency may contribute to the phenotypic characteristics of LS. This conceptual change in our understanding of the disease etiology may provide an alternative avenue for the development of therapies.

  14. Tyrosine-Lipid Peroxide Adducts from Radical Termination: Para-Coupling and Intramolecular Diels-Alder Cyclization

    PubMed Central

    Shchepin, Roman; Möller, Matias N.; Kim, Hye-young H.; Hatch, Duane M.; Bartesaghi, Silvina; Kalyanaraman, Balaraman; Radi, Rafael

    2013-01-01

    Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogs of tyrosine gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The novel adducts were isolated and characterized by 1D and 2D NMR as well as by mass spectrometry. The spectral data suggest that the polyunsaturated lipid peroxyl radicals give stable peroxide coupling products exclusively at the para position of the tyrosyl (phenoxy) radicals. These adducts have characteristic 13C chemical shifts at 185 ppm due to the cross-conjugated carbonyl of the phenol-derived cyclohexadienone. The primary peroxide adducts subsequently undergo intramolecular Diels-Alder (IMDA) cyclization, affording a number of diastereomeric tricyclic adducts that have characteristic carbonyl 13C chemical shifts at ~198 ppm. All NMR HMBC and HSQC correlations support the structure assignment of the primary and Diels-Alder adducts, as does MS collision induced dissociation. Kinetic rate constants and activation parameters for the IMDA reaction were determined and the primary adducts were reduced with cuprous ion giving a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products from adduction of peroxyls at the phenolic ortho position were found either in the primary or the cuprous reduction product mixtures. These studies provide a framework for understanding the nature of lipid-protein adducts formed by peroxyl-tyrosyl radical-radical termination processes. Coupling of lipid peroxyl radicals with tyrosyl radicals leads to cyclohexenone and cyclohexadienone adducts which are of interest in and of themselves since, as electrophiles, they are likely targets for protein nucleophiles. One consequence of lipid peroxyl reactions with tyrosyls may therefore be protein-protein crosslinks via interprotein Michael adducts. PMID:21090613

  15. MicroRNA-210 regulates mitochondrial free radical response to hypoxia and krebs cycle in cancer cells by targeting iron sulfur cluster protein ISCU.

    PubMed

    Favaro, Elena; Ramachandran, Anassuya; McCormick, Robert; Gee, Harriet; Blancher, Christine; Crosby, Meredith; Devlin, Cecilia; Blick, Christopher; Buffa, Francesca; Li, Ji-Liang; Vojnovic, Borivoj; Pires das Neves, Ricardo; Glazer, Peter; Iborra, Francisco; Ivan, Mircea; Ragoussis, Jiannis; Harris, Adrian L

    2010-04-26

    Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis. In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis. Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

  16. Serum extracellular vesicles contain protein biomarkers for primary sclerosing cholangitis and cholangiocarcinoma.

    PubMed

    Arbelaiz, Ander; Azkargorta, Mikel; Krawczyk, Marcin; Santos-Laso, Alvaro; Lapitz, Ainhoa; Perugorria, Maria J; Erice, Oihane; Gonzalez, Esperanza; Jimenez-Agüero, Raul; Lacasta, Adelaida; Ibarra, Cesar; Sanchez-Campos, Alberto; Jimeno, Juan P; Lammert, Frank; Milkiewicz, Piotr; Marzioni, Marco; Macias, Rocio I R; Marin, Jose J G; Patel, Tushar; Gores, Gregory J; Martinez, Ibon; Elortza, Félix; Falcon-Perez, Juan M; Bujanda, Luis; Banales, Jesus M

    2017-10-01

    Cholangiocarcinoma (CCA) includes a heterogeneous group of biliary cancers with poor prognosis. Several conditions, such as primary sclerosing cholangitis (PSC), are risk factors. Noninvasive differential diagnosis between intrahepatic CCA and hepatocellular carcinoma (HCC) is sometimes difficult. Accurate noninvasive biomarkers for PSC, CCA, and HCC are not available. In the search for novel biomarkers, serum extracellular vesicles (EV) were isolated from CCA (n = 43), PSC (n = 30), or HCC (n = 29) patients and healthy individuals (control, n = 32); and their protein content was characterized. By using nanoparticle tracking analysis, serum EV concentration was found to be higher in HCC than in all the other groups. Round morphology (by transmission electron microscopy), size (∼180 nm diameter by nanoparticle tracking analysis), and markers (clusters of differentiation 9, 63, and 81 by immunoblot) indicated that most serum EV were exosomes. Proteome profiles (by mass spectrometry) revealed multiple differentially expressed proteins among groups. Several of these proteins showed high diagnostic values with maximum area under the receiver operating characteristic curve of 0.878 for CCA versus control, 0.905 for CCA stage I-II versus control, 0.789 for PSC versus control, 0.806 for noncirhottic PSC versus control, 0.796 for CCA versus PSC, 0.956 for CCA stage I-II versus PSC, 0.904 for HCC versus control, and 0.894 for intrahepatic CCA versus HCC. Proteomic analysis of EV derived from CCA human cells in vitro revealed higher abundance of oncogenic proteins compared to EV released by normal human cholangiocytes. Orthotopic implant of CCA human cells in the liver of immunodeficient mice resulted in the release to serum of EV containing some similar human oncogenic proteins. Proteomic signatures found in serum EV of CCA, PSC, and HCC patients show potential usefulness as diagnostic tools. (Hepatology 2017;66:1125-1143). © 2017 by the American Association for the Study

  17. Localization of sarcomeric proteins during myofibril assembly in cultured mouse primary skeletal myotubes.

    PubMed

    White, Jennifer; Barro, Marietta V; Makarenkova, Helen P; Sanger, Joseph W; Sanger, Jean M

    2014-09-01

    It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, nonmuscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that nonmuscle myosins II A and B are components of the Z-bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells. © 2014 Wiley Periodicals, Inc.

  18. Proinflammatory proteins in female and male patients with primary antiphospholipid syndrome: preliminary data.

    PubMed

    Bećarević, Mirjana; Ignjatović, Svetlana

    2016-10-01

    The latest classification criteria for the diagnosis of the antiphospholipid syndrome (APS, an autoimmune disease characterized by thromboses, miscarriages and presence of antiphospholipid antibodies (Abs)) emphasized that thrombotic manifestations of APS should be without any signs of an inflammatory process. However, atherosclerosis (a chronic inflammatory response to the accumulation of lipoproteins in the walls of arteries) and APS are characterized by some similar features. We evaluated whether proinflammatory proteins were associated with the features of the primary APS (PAPS). PAPS patients without obstetric complications and with impaired lipid profile were included in the study. Antiphospholipid antibodies, TNF-alpha, and apo(a) were determined by ELISA. Complement components and hsCRP were measured by immunonephelometry. Decreased C3c was observed in female patients with increased titers of IgG anti-β2gpI (χ(2) = 3.939, P = 0.047) and in male patients with increased IgM anticardiolipin Abs (χ(2) = 4.286, P = 0.038). Pulmonary emboli were associated with interleukin (IL)-6 in male (χ(2) = 6.519, P = 0.011) and in female (χ(2) = 10.405, P = 0.001) patients. Cerebrovascular insults were associated with LDL-cholesterol (P = 0.05, 95 % CI: 1.003 - 12.739) in female and with apo(a) (P = 0.016, 95 % CI: 0.000-0.003) in male patients. Older female patients had increased LDL-cholesterol levels and frequency of myocardial infarctions. Proinflammatory proteins were associated with features of primary APS. No real gender differences in regard to proinflammatory protein levels were observed. Premenopausal state of female PAPS patients confers lower cardiovascular risk.

  19. Tanshinone I increases CYP1A2 protein expression and enzyme activity in primary rat hepatocytes.

    PubMed

    Lee, Wayne Y W; Zhou, Xuelin; Or, Penelope M Y; Kwan, Yiu Wa; Yeung, John H K

    2012-01-15

    This study investigated the effects of Danshen and its active ingredients on the protein expression and enzymatic activity of CYP1A2 in primary rat hepatocytes. The ethanolic extract of Danshen roots (containing mainly tanshinones) inhibited CYP1A2-catalyzed phenacetin O-deethylation (IC(50)=24.6 μg/ml) in primary rat hepatocytes while the water extract containing mainly salvianolic acid B and danshenshu had no effect. Individual tanshinones such as cryptotanshinone, dihydrotanshinone, tanshinone IIA inhibited the CYP1A2-mediated metabolism with IC(50) values at 12.9, 17.4 and 31.9 μM, respectively. After 4-day treatment of the rat hepatocytes, the ethanolic extract of Danshen and tanshinone I increased rat CYP1A2 activity by 6.8- and 5.2-fold, respectively, with a concomitant up-regulation of CYP1A2 protein level by 13.5- and 6.5-fold, respectively. CYP1A2 induction correlated with the up-regulation of mRNA level of aryl hydrocarbon receptor (AhR), which suggested a positive feedback mechanism of tanshinone I-mediated CYP1A2 induction. A formulated Danshen pill (containing mainly danshensu and salvianolic acid B and the tanshinones) up-regulated CYP1A2 protein expression and enzyme activity, but danshensu and salvianolic acid B, when used individually, did not affect CYP1A2 activity. This study was the first report on the Janus action of the tanshinones on rat CYP1A2 activity.

  20. Primary structure and solution conditions determine conformational ensemble properties of intrinsically disordered proteins

    NASA Astrophysics Data System (ADS)

    Mao, Hsuan-Han Alberto

    Intrinsically disordered proteins (IDPs) are a class of proteins that do not exhibit well-defined three-dimensional structures. The absence of structure is intrinsic to their amino acid sequences, which are characterized by low hydrophobicity and high net charge per residue compared to folded proteins. Contradicting the classic structure-function paradigm, IDPs are capable of interacting with high specificity and affinity, often acquiring order in complex with protein and nucleic acid binding partners. This phenomenon is evident during cellular activities involving IDPs, which include transcriptional and translational regulation, cell cycle control, signal transduction, molecular assembly, and molecular recognition. Although approximately 30% of eukaryotic proteomes are intrinsically disordered, the nature of IDP conformational ensembles remains unclear. In this dissertation, we describe relationships connecting characteristics of IDP conformational ensembles to their primary structures and solution conditions. Using molecular simulations and fluorescence experiments on a set of base-rich IDPs, we find that net charge per residue segregates conformational ensembles along a globule-to-coil transition. Speculatively generalizing this result, we propose a phase diagram that predicts an IDP's average size and shape based on sequence composition and use it to generate hypotheses for a broad set of intrinsically disordered regions (IDRs). Simulations reveal that acid-rich IDRs, unlike their oppositely charged base-rich counterparts, exhibit disordered globular ensembles despite intra-chain repulsive electrostatic interactions. This apparent asymmetry is sensitive to simulation parameters for representing alkali and halide salt ions, suggesting that solution conditions modulate IDP conformational ensembles. We refine the ion parameters using a calibration procedure that relies exclusively on crystal lattice properties. Simulations with these parameters recover swollen

  1. Preparation of a thick polymer brush layer composed of poly(2-methacryloyloxyethyl phosphorylcholine) by surface-initiated atom transfer radical polymerization and analysis of protein adsorption resistance.

    PubMed

    Inoue, Yuuki; Onodera, Yuya; Ishihara, Kazuhiko

    2016-05-01

    The purpose of this study was to prepare a thick polymer brush layer composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) and assess its resistance to protein adsorption from the dissolved state of poly(MPC) chains in an aqueous condition. The thick poly(MPC) brush layer was prepared through the surface-initiated atom transfer radical polymerization (SI-ATRP) of MPC with a free initiator from an initiator-immobilized substrate at given [Monomer]/[Free initiator] ratios. The ellipsometric thickness of the poly(MPC) brush layers could be controlled by the polymerization degree of the poly(MPC) chains. The thickness of the poly(MPC) brush layer in an aqueous medium was larger than that in air, and this tendency became clearer when the polymerization degree of the poly(MPC) increased. The maximum thickness of the poly(MPC) brush layer in an aqueous medium was around 110 nm. The static air contact angle of the poly(MPC) brush layer in water indicated a reasonably hydrophilic nature, which was independent of the thickness of the poly(MPC) brush layer at the surface. This result occurred because the hydrated state of the poly(MPC) chains is not influenced by the environment surrounding them. Finally, as measured with a quartz crystal microbalance, the amount of protein adsorbed from a fetal bovine serum solution (10% in phosphate-buffered saline) on the original substrate was 420 ng/cm(2). However, the poly(MPC) brush layer reduced this value dramatically to less than 50 ng/cm(2). This effect was independent of the thickness of the poly(MPC) brush layer for thicknesses between 20 nm and about 110 nm. These results indicated that the surface covered with a poly(MPC) brush layer is a promising platform to avoid biofouling and could also be applied to analyze the reactions of biological molecules with a high signal/noise ratio.

  2. Methanol extract of Ocimum gratissimum protects murine peritoneal macrophages from nicotine toxicity by decreasing free radical generation, lipid and protein damage and enhances antioxidant protection

    PubMed Central

    Mahapatra, Santanu Kar; Chakraborty, Subhankari Prasad; Das, Subhasis

    2009-01-01

    In the present study, methanol extract of Ocimum gratissimum Linn (ME-Og) was tested against nicotine-induced murine peritoneal macrophage in vitro. Phytochemical analysis of ME-Og shown high amount of flavonoid and phenolic compound present in it. The cytotoxic effect of ME-Og was studied in murine peritoneal macrophages at different concentrations (0.1 to 100 µg/ml) using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) method. To establish the protective role of ME-Og against nicotine toxicity, peritoneal macrophages from mice were treated with nicotine (10 mM), nicotine + ME-Og (1 to 25 µg/ml) for 12 h in culture media. The significantly (p < 0.05) increased super oxide anion generation, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, myeloperoxidase (MPO) activity, lipid peroxidation, protein carbonyls, oxidized glutathione levels were observed in nicotine-treated group as compared to control group; those were significantly (p < 0.05) reduced in ME-Og supplemented groups in concentration dependent manner. More over, significantly (p < 0.05) reduced antioxidant status due to nicotine exposure was effectively ameliorated by ME-Og supplementation in murine peritoneal macrophages. Among the different concentration of ME-Og, maximum protective effect was observed by 25 µg/ml, which does not produce significant cell cytotoxicity in murine peritoneal macrophages. These findings suggest the potential use and beneficial role of O. gratissimum as a modulator of nicotine-induced free radical generation, lipid-protein damage and antioxidant status in important immune cell, peritoneal macrophages. PMID:20716908

  3. How protein targeting to primary plastids via the endomembrane system could have evolved? A new hypothesis based on phylogenetic studies

    PubMed Central

    2013-01-01

    Background It is commonly assumed that a heterotrophic ancestor of the supergroup Archaeplastida/Plantae engulfed a cyanobacterium that was transformed into a primary plastid; however, it is still unclear how nuclear-encoded proteins initially were imported into the new organelle. Most proteins targeted to primary plastids carry a transit peptide and are transported post-translationally using Toc and Tic translocons. There are, however, several proteins with N-terminal signal peptides that are directed to higher plant plastids in vesicles derived from the endomembrane system (ES). The existence of these proteins inspired a hypothesis that all nuclear-encoded, plastid-targeted proteins initially carried signal peptides and were targeted to the ancestral primary plastid via the host ES. Results We present the first phylogenetic analyses of Arabidopsis thaliana α-carbonic anhydrase (CAH1), Oryza sativa nucleotide pyrophosphatase/phosphodiesterase (NPP1), and two O. sativa α-amylases (αAmy3, αAmy7), proteins that are directed to higher plant primary plastids via the ES. We also investigated protein disulfide isomerase (RB60) from the green alga Chlamydomonas reinhardtii because of its peculiar dual post- and co-translational targeting to both the plastid and ES. Our analyses show that these proteins all are of eukaryotic rather than cyanobacterial origin, and that their non-plastid homologs are equipped with signal peptides responsible for co-translational import into the host ES. Our results indicate that vesicular trafficking of proteins to primary plastids evolved long after the cyanobacterial endosymbiosis (possibly only in higher plants) to permit their glycosylation and/or transport to more than one cellular compartment. Conclusions The proteins we analyzed are not relics of ES-mediated protein targeting to the ancestral primary plastid. Available data indicate that Toc- and Tic-based translocation dominated protein import into primary plastids from the

  4. How protein targeting to primary plastids via the endomembrane system could have evolved? A new hypothesis based on phylogenetic studies.

    PubMed

    Gagat, Przemysław; Bodył, Andrzej; Mackiewicz, Paweł

    2013-07-11

    It is commonly assumed that a heterotrophic ancestor of the supergroup Archaeplastida/Plantae engulfed a cyanobacterium that was transformed into a primary plastid; however, it is still unclear how nuclear-encoded proteins initially were imported into the new organelle. Most proteins targeted to primary plastids carry a transit peptide and are transported post-translationally using Toc and Tic translocons. There are, however, several proteins with N-terminal signal peptides that are directed to higher plant plastids in vesicles derived from the endomembrane system (ES). The existence of these proteins inspired a hypothesis that all nuclear-encoded, plastid-targeted proteins initially carried signal peptides and were targeted to the ancestral primary plastid via the host ES. We present the first phylogenetic analyses of Arabidopsis thaliana α-carbonic anhydrase (CAH1), Oryza sativa nucleotide pyrophosphatase/phosphodiesterase (NPP1), and two O. sativa α-amylases (αAmy3, αAmy7), proteins that are directed to higher plant primary plastids via the ES. We also investigated protein disulfide isomerase (RB60) from the green alga Chlamydomonas reinhardtii because of its peculiar dual post- and co-translational targeting to both the plastid and ES. Our analyses show that these proteins all are of eukaryotic rather than cyanobacterial origin, and that their non-plastid homologs are equipped with signal peptides responsible for co-translational import into the host ES. Our results indicate that vesicular trafficking of proteins to primary plastids evolved long after the cyanobacterial endosymbiosis (possibly only in higher plants) to permit their glycosylation and/or transport to more than one cellular compartment. The proteins we analyzed are not relics of ES-mediated protein targeting to the ancestral primary plastid. Available data indicate that Toc- and Tic-based translocation dominated protein import into primary plastids from the beginning. Only a handful of host

  5. Foxp3-dependent transformation of human primary CD4+ T lymphocytes by the retroviral protein tax.

    PubMed

    Chen, Li; Liu, Dan; Zhang, Yang; Zhang, Huan; Cheng, Hua

    2015-10-23

    The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells.

  6. Ganglioside inhibition of glutamate-mediated protein kinase C translocation in primary cultures of cerebellar neurons

    SciTech Connect

    Vaccarino, F.; Guidotti, A.; Costa, E.

    1987-12-01

    In primary cultures of cerebellar granule cells, protein kinase C (PKC) translocation and activation can be triggered by the stimulation of excitatory amino acid neurotransmitter receptors. Glutamate evokes a dose-related translocation of 4-..beta..-(/sup 3/H)phorbol 12,13-dibutyrate /(/sup 3/H)-P(BtO)/sub 2// binding sites from the cytosol to the neuronal membrane and stimulates the incorporation of /sup 32/P into a number of membrane proteins, particularly protein bands in the range of 80, 50, and 40 kDa. The glutamate-evoked PKC translocation is Mg/sup 2 +/ sensitive, is prevented by 2-amino-5-phosphonovalerate and phencyclidine, is not inhibited by nitrendipine (a voltage-dependent Ca/sup 2 +/-channel-blocker) but is abolished by the removal of Ca/sup 2 +/ from the incubation medium, suggesting that glutamate-mediated Ca/sup 2 +/ influx is operative in the redistribution of PKC. Exposure of granule cells to the gangliosides trisialosylgangliotetraglycosylceramide (GT1b) of monosialosylgangliotetraglycosylceramide (GM1) inhibits the translocation and activation of PKC evoked by glutamate. These glycosphingolipids fail to interfere with glutamate binding to its high-affinity recognition site of with the (/sup 3/H)P(BtO)/sub 2/ binding, nor do they affect the Ca/sup 2 +/ influx. These gangliosides may prevent PKC translocation by interfering with the PKC binding to the neuronal membrane phosphatidylserine.

  7. Automatic generation of primary sequence patterns from sets of related protein sequences.

    PubMed Central

    Smith, R F; Smith, T F

    1990-01-01

    We have developed a computer algorithm that can extract the pattern of conserved primary sequence elements common to all members of a homologous protein family. The method involves clustering the pairwise similarity scores among a set of related sequences to generate a binary dendrogram (tree). The tree is then reduced in a stepwise manner by progressively replacing the node connecting the two most similar termini by one common pattern until only a single common "root" pattern remains. A pattern is generated at a node by (i) performing a local optimal alignment on the sequence/pattern pair connected by the node with the use of an extended dynamic programming algorithm and then (ii) constructing a single common pattern from this alignment with a nested hierarchy of amino acid classes to identify the minimal inclusive amino acid class covering each paired set of elements in the alignment. Gaps within an alignment are created and/or extended using a "pay once" gap penalty rule, and gapped positions are converted into gap characters that function as 0 or 1 amino acid of any type during subsequent alignment. This method has been used to generate a library of covering patterns for homologous families in the National Biomedical Research Foundation/Protein Identification Resource protein sequence data base. We show that a covering pattern can be more diagnostic for sequence family membership than any of the individual sequences used to construct the pattern. Images PMID:2296575

  8. PERK regulates Gq protein-coupled intracellular Ca(2+) dynamics in primary cortical neurons.

    PubMed

    Zhu, Siying; McGrath, Barbara C; Bai, Yuting; Tang, Xin; Cavener, Douglas R

    2016-10-01

    PERK (EIF2AK3) is an ER-resident eIF2α kinase required for behavioral flexibility and metabotropic glutamate receptor-dependent long-term depression via its translational control. Motivated by the recent discoveries that PERK regulates Ca(2+) dynamics in insulin-secreting β-cells underlying glucose-stimulated insulin secretion, and modulates Ca(2+) signals-dependent working memory, we explored the role of PERK in regulating Gq protein-coupled Ca(2+) dynamics in pyramidal neurons. We found that acute PERK inhibition by the use of a highly specific PERK inhibitor reduced the intracellular Ca(2+) rise stimulated by the activation of acetylcholine, metabotropic glutamate and bradykinin-2 receptors in primary cortical neurons. More specifically, acute PERK inhibition increased IP3 receptor mediated ER Ca(2+) release, but decreased receptor-operated extracellular Ca(2+) influx. Impaired Gq protein-coupled intracellular Ca(2+) rise was also observed in genetic Perk knockout neurons. Taken together, our findings reveal a novel role of PERK in neurons, which is eIF2α-independent, and suggest that the impaired working memory in forebrain-specific Perk knockout mice may stem from altered Gq protein-coupled intracellular Ca(2+) dynamics in cortical pyramidal neurons.

  9. Picosecond Raman Study of Vibrational Cooling and Protein Dynamics in the Primary Photochemistry of Rhodopsin

    NASA Astrophysics Data System (ADS)

    Kim, Judy; Mathies, Richard

    2003-03-01

    Picosecond Stokes and anti-Stokes Raman spectra are used to probe the structural dynamics and reactive energy flow of both the chromophore and binding pocket residues in the primary cis-to-trans isomerization reaction of rhodopsin. The appearance of characteristic ethylenic, hydrogen out-of-plane (HOOP) and low-wavenumber photoproduct bands in the Stokes Raman spectra of the chromophore is instrument-response limited, consistent with a sub-picosecond product appearance time (1,2). Intense high and low-frequency anti-Stokes chromophore peaks demonstrate that the all-trans photoproduct, photorhodopsin, is produced vibrationally hot on the ground-state surface (2). Specifically, the low-frequency modes at 282, 350 and 477 cm-1 are highly vibrationally excited (T > 2000 K) immediately following isomerization, revealing that these low-frequency motions directly participate in the reactive curve-crossing process. The anti-Stokes modes are characterized by a ˜2.5 ps temporal decay that coincides with the conversion of photorhodopsin to bathorhodopsin. This correspondence shows that the photo-to-batho transition is a ground-state cooling process, and that energy storage in the primary visual photoproduct is complete on the picosecond time scale. The remarkable similarity between the room-temperature picosecond vibrational structure of photo- and bathorhodopsin and that of the low-temperature trapped primary photoproduct suggests that chromophore isomerization impulsively excites and drives changes in nearby protein residues. These amino acid changes within the binding pocket are probed by picosecond UV Raman spectroscopy of aromatic residues (3). Difference spectra reveal that at least one tryptophan (trp265) and one tyrosine (tyr191, 268 and/or 178) residue undergoes structural changes in < 5 ps, presumably due to steric interaction with the isomerizing chromophore as well as energy flow from chromophore to the binding pocket. This result indicates that the protein

  10. Measurement of HNE-protein adducts in human plasma and serum by ELISA-Comparison of two primary antibodies.

    PubMed

    Weber, Daniela; Milkovic, Lidija; Bennett, Stuart J; Griffiths, Helen R; Zarkovic, Neven; Grune, Tilman

    2013-01-01

    There is increasing evidence that non-enzymatic post-translational protein modifications might play key roles in various diseases. These protein modifications can be caused by free radicals generated during oxidative stress or by their products generated during lipid peroxidation. 4-Hydroxynonenal (HNE), a major biomarker of oxidative stress and lipid peroxidation, has been recognized as important molecule in pathology as well as in physiology of living organisms. Therefore, its detection and quantification can be considered as valuable tool for evaluating various pathophysiological conditions. The HNE-protein adduct ELISA is a method to detect HNE bound to proteins, which is considered as the most likely form of HNE occurrence in living systems. Since the earlier described ELISA has been validated for cell lysates and the antibody used for detection of HNE-protein adducts is non-commercial, the aim of this work was to adapt the ELISA to a commercial antibody and to apply it in the analysis of human plasma samples. AFTER MODIFICATION AND VALIDATION OF THE PROTOCOL FOR BOTH ANTIBODIES, SAMPLES OF TWO GROUPS WERE ANALYZED: apparently healthy obese (n=62) and non-obese controls (n=15). Although the detected absolute values of HNE-protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE-protein adducts in the obese group.

  11. Measurement of HNE-protein adducts in human plasma and serum by ELISA—Comparison of two primary antibodies☆

    PubMed Central

    Weber, Daniela; Milkovic, Lidija; Bennett, Stuart J.; Griffiths, Helen R.; Zarkovic, Neven; Grune, Tilman

    2013-01-01

    There is increasing evidence that non-enzymatic post-translational protein modifications might play key roles in various diseases. These protein modifications can be caused by free radicals generated during oxidative stress or by their products generated during lipid peroxidation. 4-Hydroxynonenal (HNE), a major biomarker of oxidative stress and lipid peroxidation, has been recognized as important molecule in pathology as well as in physiology of living organisms. Therefore, its detection and quantification can be considered as valuable tool for evaluating various pathophysiological conditions. The HNE-protein adduct ELISA is a method to detect HNE bound to proteins, which is considered as the most likely form of HNE occurrence in living systems. Since the earlier described ELISA has been validated for cell lysates and the antibody used for detection of HNE-protein adducts is non-commercial, the aim of this work was to adapt the ELISA to a commercial antibody and to apply it in the analysis of human plasma samples. After modification and validation of the protocol for both antibodies, samples of two groups were analyzed: apparently healthy obese (n=62) and non-obese controls (n=15). Although the detected absolute values of HNE–protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE–protein adducts in the obese group. PMID:24024156

  12. Troglitazone increases cytochrome P-450 3A protein and activity in primary cultures of human hepatocytes.

    PubMed

    Ramachandran, V; Kostrubsky, V E; Komoroski, B J; Zhang, S; Dorko, K; Esplen, J E; Strom, S C; Venkataramanan, R

    1999-10-01

    Troglitazone (TRO) is an insulin sensitizer used in the treatment of type II diabetes. TRO is known to increase the activity of cytochrome P-450 (CYP) 3A in vivo. We have investigated the effect of TRO on CYP3A protein content and the activity of CYP3A (as measured by the formation of 6beta-hydroxytestosterone formation) in primary cultures of human hepatocytes in comparison with rifampicin (RIF). Hepatocytes were isolated from four human livers by perfusion with collagenase, plated on collagen-coated plates, and maintained in William's E medium. After 48 h in culture, cells were exposed to RIF (10 microM) or TRO (0-50 microM) twice, each over a period of 24 h, and the activity of CYP3A was measured. TRO increased the activity of CYP3A in a concentration-dependent manner, reaching a maximal response at 5 microM. Pretreatment of the hepatocytes with 10 microM TRO or 10 microM RIF resulted in a 4- to 15-fold increase in the activity of CYP3A. Maximum increase in CYP3A protein was observed at 5 microM TRO. There was a significant correlation (R(2) = 0.89) between the content of immunoreactive CYP3A protein in the hepatocytes and the rate of formation of 6beta-hydroxytestosterone. These results indicate that TRO is a potent inducer of CYP3A and is similar to RIF in inducing CYP3A in human hepatocytes. At concentrations of 25 microM and above, TRO was toxic to the cells, as determined by a decrease in the activity of CYP3A, a reduction in the amount of immunoreactive protein, and changes in the morphology of the cells.

  13. On the Involvement of Single-Bond Rotation in the Primary Photochemistry of Photoactive Yellow Protein

    PubMed Central

    Stahl, Andreas D.; Hospes, Marijke; Singhal, Kushagra; van Stokkum, Ivo; van Grondelle, Rienk; Groot, Marie Louise; Hellingwerf, Klaas J.

    2011-01-01

    Prior experimental observations, as well as theoretical considerations, have led to the proposal that C4-C7 single-bond rotation may play an important role in the primary photochemistry of photoactive yellow protein (PYP). We therefore synthesized an analog of this protein's 4-hydroxy-cinnamic acid chromophore, (5-hydroxy indan-(1E)-ylidene)acetic acid, in which rotation across the C4-C7 single bond has been locked with an ethane bridge, and we reconstituted the apo form of the wild-type protein and its R52A derivative with this chromophore analog. In PYP reconstituted with the rotation-locked chromophore, 1), absorption spectra of ground and intermediate states are slightly blue-shifted; 2), the quantum yield of photochemistry is ∼60% reduced; 3), the excited-state dynamics of the chromophore are accelerated; and 4), dynamics of the thermal recovery reaction of the protein are accelerated. A significant finding was that the yield of the transient ground-state intermediate in the early phase of the photocycle was considerably higher in the rotation-locked samples than in the corresponding samples reconstituted with p-coumaric acid. In contrast to theoretical predictions, the initial photocycle dynamics of PYP were observed to be not affected by the charge of the amino acid residue at position 52, which was varied by 1), varying the pH of the sample between 5 and 10; and 2), site-directed mutagenesis to construct R52A. These results imply that C4-C7 single-bond rotation in PYP is not an alternative to C7=C8 double-bond rotation, in case the nearby positive charge of R52 is absent, but rather facilitates, presumably with a compensatory movement, the physiological Z/E isomerization of the blue-light-absorbing chromophore. PMID:21889456

  14. BRAFV600E protein expression in primary cutaneous malignant melanomas and paired metastases.

    PubMed

    Eriksson, Hanna; Zebary, Abdlsattar; Vassilaki, Ismini; Omholt, Katarina; Ghaderi, Mehran; Hansson, Johan

    2015-04-01

    BRAFV600E mutations are present in approximately 50% of cutaneous malignant melanomas (CMMs). The use of BRAFV600E mutation-specific monoclonal antibody VE1 immunohistochemical analysis may facilitate rapid detection of BRAFV600E mutations in CMMs and demonstrate heterogeneity among tumors. To characterize the pattern of BRAFV600E protein expression in primary CMMs with matched metastases and to analyze the use of VE1 immunohistochemical analysis in clinical practice using formalin-fixed, paraffin-embedded tumor tissue. In this retrospective cohort study performed at Karolinska University Hospital from September 2012 to September 2013, we examined CMMs (124 primary tumors and 76 metastases) with VE1 immunohistochemical analysis, and results were compared with DNA mutation analyses. Determination of intratumoral and intertumoral heterogeneity as well as the sensitivity and specificity of VE1 immunohistochemical analysis. Positive staining results with the VE1 antibody were detected in 94 of 200 tumors (47.0%). In general, VE1 staining was homogeneous. However, VE1 staining intensity varied among the primary tumors and corresponding metastases in 63 of 135 tumors (46.7%), but a change of mutational status based on DNA analysis was found in only 4 matched tumors (3.0%). Discordant findings between DNA mutation analysis and immunohistochemical analysis were observed in 12 tumors. The overall sensitivity and specificity of VE1 immunohistochemical analysis were 96.7% and 94.5%, respectively. A comparable sensitivity was obtained for primary and metastatic CMMs. The specificity was lower among primary CMMs (92.4%) compared with metastases (98.0%). We found VE1 immunohistochemical analysis to be a useful and rapid assay for BRAFV600E mutations that may contribute to the detection of intratumoral and intertumoral heterogenetic subclones. Tumors with positive results, including strong staining, should be expedited for confirmatory BRAF mutation testing. If this test result

  15. Immunolocalization of dentin matrix protein-1 in human primary teeth treated with different pulp capping materials.

    PubMed

    Lourenço Neto, Natalino; Marques, Nádia C T; Fernandes, Ana Paula; Rodini, Camila O; Sakai, Vivien T; Abdo, Ruy Cesar C; Machado, Maria Aparecida A M; Santos, Carlos F; Oliveira, Thais M

    2016-01-01

    The aim of this study was to evaluate the immunolocalization of dentin matrix protein (DMP)-1 in human primary teeth treated with different pulp capping materials. Twenty-five primary molars were divided into the following groups: formocresol (FC), calcium hydroxide (CH), mineral trioxide aggregate (MTA), corticosteroid/antibiotic solution + CH (O + CH), and Portland cement (PC), and all received conventional pulpotomy treatment. The teeth at the regular exfoliation period were extracted for histological analysis and immunolocalization of DMP-1. Statistical analysis was performed using the χ(2) test (p < 0.05). Histological analysis revealed statistically significant differences in the comparison among the groups through the use of a score system regarding the presence of hard tissue barrier, odontoblastic layer, and internal resorption, but not regarding pulp calcification. Immunohistochemical analysis showed immunostaining for DMP-1 in groups CH, MTA, O + CH, and PC. Internal resorption was observed in the groups FC and CH. MTA and PC showed pulp repair without inflammation and with the presence of hard tissue barrier. DMP-1 immunostaining was higher for MTA and PC, confirming the reparative and bioinductive capacity of these materials.

  16. Spatiotemporal Alterations in Primary Odorant Representations in Olfactory Marker Protein Knockout Mice

    PubMed Central

    Kass, Marley D.; Moberly, Andrew H.; McGann, John P.

    2013-01-01

    Olfactory marker protein (OMP) is highly and selectively expressed in primary olfactory sensory neurons (OSNs) across species, but its physiological function remains unclear. Previous studies in the olfactory epithelium suggest that it accelerates the neural response to odorants and may modulate the odorant-selectivity of OSNs. Here we used a line of gene-targeted mice that express the fluorescent exocytosis indicator synaptopHluorin in place of OMP to compare spatiotemporal patterns of odorant-evoked neurotransmitter release from OSNs in adult mice that were heterozygous for OMP or OMP-null. We found that these patterns, which constitute the primary neural representation of each odorant, developed more slowly during the odorant presentation in OMP knockout mice but eventually reached the same magnitude as in heterozygous mice. In the olfactory bulb, each glomerulus receives synaptic input from a subpopulation of OSNs that all express the same odor receptor and thus typically respond to a specific subset of odorants. We observed that in OMP knockout mice, OSNs innervating a given glomerulus typically responded to a broader range of odorants than in OMP heterozygous mice and thus each odorant evoked synaptic input to a larger number of glomeruli. In an olfactory habituation task, OMP knockout mice behaved differently than wild-type mice, exhibiting a delay in their onset to investigate an odor stimulus during its first presentation and less habituation to that stimulus over repeated presentations. These results suggest that the actions of OMP in olfactory transduction carry through to the primary sensory representations of olfactory stimuli in adult mice in vivo. PMID:23630588

  17. The tyrosyl free radical in ribonucleotide reductase.

    PubMed Central

    Gräslund, A; Sahlin, M; Sjöberg, B M

    1985-01-01

    The enzyme, ribonucleotide reductase, catalyses the formation of deoxyribonucleotides from ribonucleotides, a reaction essential for DNA synthesis in all living cells. The Escherichia coli ribonucleotide reductase, which is the prototype of all known eukaryotic and virus-coded enzymes, consists of two nonidentical subunits, proteins B1 and B2. The B2 subunit contains an antiferromagnetically coupled pair of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of an electron, has its unpaired spin density delocalized in the aromatic ring of tyrosine. Effects of iron-radical interaction indicate a relatively close proximity between the iron center and the radical. The EPR signal of the radical can be studied directly in frozen packed cells of E. coli or mammalian origin, if the cells are made to overproduce ribonucleotide reductase. The hypothetic role of the tyrosyl free radical in the enzymatic reaction is not yet elucidated, except in the reaction with the inhibiting substrate analogue 2'-azido-CDP. In this case, the normal tyrosyl radical is destroyed with concomitant appearance of a 2'-azido-CDP-localized radical intermediate. Attempts at spin trapping of radical reaction intermediates have turned out negative. In E. coli the activity of ribonucleotide reductase may be regulated by enzymatic activities that interconvert a nonradical containing form and the fully active protein B2. In synchronized mammalian cells, however, the cell cycle variation of ribonucleotide reductase, studied by EPR, was shown to be due to de novo protein synthesis. Inhibitors of ribonucleotide reductase are of medical interest because of their ability to control DNA synthesis. One example is hydroxyurea, used in cancer therapy, which selectively destroys the tyrosyl free radical. PMID:3007085

  18. Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and is required for primary cilia assembly

    PubMed Central

    Jurczyk, Agata; Gromley, Adam; Redick, Sambra; Agustin, Jovenal San; Witman, George; Pazour, Gregory J.; Peters, Dorien J.M.; Doxsey, Stephen

    2004-01-01

    Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. Here, we show that the centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella. PMID:15337773

  19. Protein Analysis of Glioblastoma Primary and Posttreatment Pairs Suggests a Mesenchymal Shift at Recurrence.

    PubMed

    Wood, Matthew D; Reis, Gerald F; Reuss, David E; Phillips, Joanna J

    2016-10-01

    Glioblastomas (GBM) are aggressive brain tumors that inevitably recur despite surgical resection, chemotherapy, and radiation. The degree to which recurrent GBM retains its initial immunophenotype is incompletely understood. We generated tissue microarrays of paired initial and posttreatment GBM (3 pairs positive and 17 negative for IDH1(R132H)) from the same patients and made comparisons in the IDH1(R132H)-negative group for immunohistochemical and gene expression differences between primary and recurrent tumors. In initial tumors, immunopositivity for Ki-67 in > 20% of tumor cells was associated with shorter progression-free and overall survival. Recurrent tumors showed decreased staining for CD34 suggesting lower vessel density. A subset of tumors showed increased staining for markers associated with the mesenchymal gene expression pattern, including CD44, phosphorylated STAT3, and YKL40. Recurrent tumors with the greatest increase in mesenchymal marker expression had rapid clinical progression, but no difference in overall survival after second surgery. Comparison of protein and gene expression data from the same samples revealed a poor correlation. A subset of tumors (15%) showed loss of neurofibromin protein in both initial and recurrent tumors. These data support the notion that GBM progression is associated with a shift toward a mesenchymal phenotype in a subset of tumors and this may portend a more aggressive behavior.

  20. Detection and quantification of apoptosis in primary cells using Taqman® protein assay.

    PubMed

    Pfister, Christina; Pfrommer, Heike; Tatagiba, Marcos S; Roser, Florian

    2015-01-01

    There are several methods to detect apoptosis using cleaved caspase-3 and each harbors its own advantages and disadvantages. When primary cell cultures are used, the disadvantages of the standard methods can make apoptosis detection difficult due to their slow growth rate and replicative senescence, thereby limiting the available cell number and experiment time span. In this chapter, we describe apoptosis detection and quantification using an innovative method named TaqMan(®) protein assay. TaqMan(®) protein assay uses antibodies and proximity ligation for quantitative real-time PCR. Biotinylated antibodies are labeled with oligonucleotides. When the labeled antibodies bind in close proximity, the oligonucleotides are connected using DNA ligase. The ligation product is amplified and detected using Taqman(®) based Real-Time PCR. Using this technique, we can not only detect apoptosis with a 1,000-fold higher sensitivity than western blot, but we can also exactly quantify cleaved caspase-3 expression. Thereby apoptosis can be determined and quantified in a fast reliable manner.

  1. Autoantibodies against integral membrane proteins of the nuclear envelope in patients with primary biliary cirrhosis.

    PubMed

    Nickowitz, R E; Wozniak, R W; Schaffner, F; Worman, H J

    1994-01-01

    Autoantibodies against nuclear membrane proteins have been identified in patients with primary biliary cirrhosis (PBC). The aim of the present study was to determine the incidence of these autoantibodies in patients with PBC and examine their significance. An assay using recombinant polypeptides was designed to unequivocally detect autoantibodies against gp210 and the lamin B receptor, integral proteins of the nuclear membranes. Autoantibodies against gp210 were detected in 15 of 159 patients with PBC and 0 of 46 controls. Autoantibodies against lamin B receptor were detected in 2 patients with PBC and 0 controls. The presence of these autoantibodies had a sensitivity of 11% and specificity of 100% for the diagnosis of PBC. Autoantibodies against gp210 were present in 4 of 19 (21%) patients with PBC who did not have detectable antimitochondrial antibodies. Patients with PBC and gp210 autoantibodies had a higher incidence of associated arthritis. Autoantibodies against gp210 and the lamin B receptor are present in approximately 10% of patients with PBC. These autoantibodies are highly specific for the diagnosis of PBC and may be useful in diagnosing individuals without antimitochondrial antibodies and in identifying a subgroup of patients with an increased incidence of associated arthritis.

  2. Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding

    PubMed Central

    Aremu, David A.; Ezomo, Ojeiru F.

    2010-01-01

    Objectives Aluminum is notorious as a neurotoxic metal. The aim of our study was to determine whether endoplasmic reticulum (ER) stress is involved in aluminum-induced apoptosis in astrocytes. Methods Mitochondrial RNA (mRNA) was analyzed by reverse transcription (RT)-PCR following pulse exposure of aluminum glycinate to primary cultured astrocytes. Tunicamycin was used as a positive control. Results Gene expression analysis revealed that Ire1β was up-regulated in astrocytes exposed to aluminum while Ire1α was up-regulated by tunicamycin. Exposure to aluminum glycinate, in contrast to tunicamycin, seemed to down-regulate mRNA expression of many genes, including the ER resident molecular chaperone BiP/Grp78 and Ca2+-binding chaperones (calnexin and calreticulin), as well as stanniocalcin 2 and OASIS. The down-regulation or non-activation of the molecular chaperons, whose expressions are known to be protective by increasing protein folding, may spell doom for the adaptive response. Exposure to aluminum did not have any significant effects on the expression of Bax and Bcl2 in astrocytes. Conclusions The results of this study demonstrate that aluminum may induce apoptosis in astrocytes via ER stress by impairing the protein-folding machinery. PMID:21432213

  3. Plasma steroid-binding proteins: primary gatekeepers of steroid hormone action

    PubMed Central

    2016-01-01

    Biologically active steroids are transported in the blood by albumin, sex hormone-binding globulin (SHBG), and corticosteroid-binding globulin (CBG). These plasma proteins also regulate the non-protein-bound or ‘free’ fractions of circulating steroid hormones that are considered to be biologically active; as such, they can be viewed as the ‘primary gatekeepers of steroid action’. Albumin binds steroids with limited specificity and low affinity, but its high concentration in blood buffers major fluctuations in steroid concentrations and their free fractions. By contrast, SHBG and CBG play much more dynamic roles in controlling steroid access to target tissues and cells. They bind steroids with high (~nM) affinity and specificity, with SHBG binding androgens and estrogens and CBG binding glucocorticoids and progesterone. Both are glycoproteins that are structurally unrelated, and they function in different ways that extend beyond their transportation or buffering functions in the blood. Plasma SHBG and CBG production by the liver varies during development and different physiological or pathophysiological conditions, and abnormalities in the plasma levels of SHBG and CBG or their abilities to bind steroids are associated with a variety of pathologies. Understanding how the unique structures of SHBG and CBG determine their specialized functions, how changes in their plasma levels are controlled, and how they function outside the blood circulation provides insight into how they control the freedom of steroids to act in health and disease. PMID:27113851

  4. Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone.

    PubMed

    Murphy, Michael P; Echtay, Karim S; Blaikie, Frances H; Asin-Cayuela, Jordi; Cocheme, Helena M; Green, Katherine; Buckingham, Julie A; Taylor, Ellen R; Hurrell, Fiona; Hughes, Gillian; Miwa, Satomi; Cooper, Christopher E; Svistunenko, Dimitri A; Smith, Robin A J; Brand, Martin D

    2003-12-05

    Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap alpha-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2'-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.

  5. Primary renal carcinoid tumor.

    PubMed

    Kanodia, K V; Vanikar, A V; Patel, R D; Suthar, K S; Kute, V B; Modi, P R; Trivedi, H L

    2013-09-01

    Primary renal carcinoid tumor is extremely rare and, therefore, its pathogenesis and prognosis is not well known. We report a primary renal carcinoid in a 26-year-old man treated by radical nephrectomy.

  6. The Fusion Protein Specificity of the Parainfluenza Virus Hemagglutinin-Neuraminidase Protein Is Not Solely Defined by the Primary Structure of Its Stalk Domain

    PubMed Central

    Ito, Morihiro; Ohtsuka, Junpei; Hara, Kenichiro; Komada, Hiroshi; Nishio, Machiko; Nosaka, Tetsuya

    2015-01-01

    ABSTRACT Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction presumably is mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that a simian virus 41 (SV41) F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, replacement of 13 amino acids of the SCA head domain, which are located at or around the dimer interface of the head domain, with SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retroreplacement of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein, similar to the SV41 HN protein. Thus, we conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain. IMPORTANCE It is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular

  7. Expression of c-erbB3 protein in primary breast carcinomas.

    PubMed Central

    Naidu, R.; Yadav, M.; Nair, S.; Kutty, M. K.

    1998-01-01

    Expression of c-erbB3 protein was investigated in 104 primary breast carcinomas comprising nine comedo ductal carcinoma in situ (DCIS), 91 invasive ductal carcinomas and four invasive lobular carcinomas using two monoclonal antibodies, RTJ1 and RTJ2. Of the 91 invasive ductal carcinomas, seven contained the comedo DCIS component adjacent to the invasive component. An immunohistochemical technique was used to evaluate the association between expression of c-erbB3 and clinical parameters and tumour markers such as epidermal growth factor receptor (EGFR), c-erbB2, cathepsin-D and p53 in archival formalin-fixed paraffin-embedded tumour tissues. Our results indicated that RTJ1 and RTJ2 gave identical staining patterns and concordant results. It was found that the overexpression of c-erbB3 protein was observed in 67% (6/9) of comedo DCIS, 52% (44/84) of invasive ductal carcinomas, 71% (5/7) of carcinomas containing both the in situ and invasive lesions and 25% (1/4) of invasive lobular carcinomas. A significant relationship (P < 0.05) was observed between strong immunoreactivity of c-erbB3 protein and histological grade, EGFR and cathepsin-D, but not with expression of c-erbB2, p53, oestrogen receptor status, lymph node metastases or age of patient. However, we noted that a high percentage of oestrogen receptor-negative tumours (59%), lymph node-positive tumours (63%) and c-erbB2 (63%) were strongly positive for c-erbB3 protein. We have also documented that a high percentage of EGFR (67%), c-erbB2 (67%), p53 (75%) and cathepsin-D-positive DCIS (60%) were strongly positive for c-erbB3. These observations suggest that overexpression of c-erbB3 protein could play an important role in tumour progression from non-invasive to invasive and, also, that it may have the potential to be used as a marker for poor prognosis of breast cancer. Images Figure 1 Figure 2 PMID:9823984

  8. Substance P suppresses GABAA receptor function via protein kinase C in primary sensory neurones of bullfrogs.

    PubMed Central

    Yamada, K; Akasu, T

    1996-01-01

    1. The effects of substance P (SP) and related tachykinins on the function of gamma-aminobutyric acid-A (GABAA) receptors were examined in acutely dissociated neurones of bullfrog dorsal root ganglia (DRG) by using whole-cell voltage-clamp techniques. 2. Application of SP (10 nM to 1 microM) depressed inward currents produced by GABAA receptor activation (IGABA). Neurokinin A (NKA) and neurokinin B (NKB) also depressed IGABA; the rank order of agonist potency was SP > NKA > NKB. Spantide ([D-Arg1, D-Trp7,9,Leu11]SP) and L-703,606, NK1 receptor antagonists, blocked the SP-induced depression of IGABA. 3. SP irreversibly depressed IGABA, when neurones were intracellularly dialysed with GTP gamma S. Intracellular application of GDP beta S prevented the SP-induced depression of IGABA. Pertussis toxin (PTX) did not block the inhibitory effect of SP on IGABA. 4. The depression of IGABA produced by SP was inhibited by H-7 and PKC(19-36), protein kinase C (PKC) inhibitors, but not by H-9 and HA-1004, protein kinase A inhibitors. IGABA was suppressed by application of sn-1,2-dioctanoyl glycerol (DOG), a PKC activator. 5. It is concluded that activation of neurokinin-1 (NK1) receptors downregulates the function of the GABAA receptor of primary sensory neurones through a PTX-insensitive G-protein. PKC may be involved in the transduction pathway of the tachykinin-induced inhibition of the GABAA receptor. PMID:8910228

  9. Mutations in STIL, encoding a pericentriolar and centrosomal protein, cause primary microcephaly.

    PubMed

    Kumar, Arun; Girimaji, Satish C; Duvvari, Mahesh R; Blanton, Susan H

    2009-02-01

    Primary microcephaly (MCPH) is an autosomal-recessive congenital disorder characterized by smaller-than-normal brain size and mental retardation. MCPH is genetically heterogeneous with six known loci: MCPH1-MCPH6. We report mapping of a novel locus, MCPH7, to chromosome 1p32.3-p33 between markers D1S2797 and D1S417, corresponding to a physical distance of 8.39 Mb. Heterogeneity analysis of 24 families previously excluded from linkage to the six known MCPH loci suggested linkage of five families (20.83%) to the MCPH7 locus. In addition, four families were excluded from linkage to the MCPH7 locus as well as all of the six previously known loci, whereas the remaining 15 families could not be conclusively excluded or included. The combined maximum two-point LOD score for the linked families was 5.96 at marker D1S386 at theta = 0.0. The combined multipoint LOD score was 6.97 between markers D1S2797 and D1S417. Previously, mutations in four genes, MCPH1, CDK5RAP2, ASPM, and CENPJ, that code for centrosomal proteins have been shown to cause this disorder. Three different homozygous mutations in STIL, which codes for a pericentriolar and centrosomal protein, were identified in patients from three of the five families linked to the MCPH7 locus; all are predicted to truncate the STIL protein. Further, another recently ascertained family was homozygous for the same mutation as one of the original families. There was no evidence for a common haplotype. These results suggest that the centrosome and its associated structures are important in the control of neurogenesis in the developing human brain.

  10. MGMT promoter methylation and correlation with protein expression in primary central nervous system lymphoma.

    PubMed

    Toffolatti, L; Scquizzato, E; Cavallin, S; Canal, F; Scarpa, M; Stefani, P M; Gherlinzoni, F; Dei Tos, A P

    2014-11-01

    The O (6)-methylguanine-DNA-methyltransferase (MGMT) gene encodes for a DNA repairing enzyme of which silencing by promoter methylation is involved in brain tumorigenesis. MGMT promoter methylation represents a favorable prognostic factor and has been associated with a better response to alkylating agents in glioma and systemic lymphoma. Primary central nervous system lymphoma (PCNSL) is a rare and aggressive extranodal malignant lymphoma. The current standard of care, based on high-dose methotrexate chemotherapy, has improved prognosis but outcome remains poor for a majority of patients. Therapeutic progress in this field is conditioned by limited biological and molecular knowledge about the disease. Temozolomide has recently emerged as an alternative option for PCNSL treatment. We aimed to analyze the MGMT gene methylation status in a series of 24 PCNSLs, to investigate the relationship between methylation status of the gene and immunohistochemical expression of MGMT protein and to evaluate the possible prognostic significance of these biomarkers. Our results confirm that methylation of the MGMT gene and loss of MGMT protein are frequent events in these lymphomas (54 % of our cases) and suggest that they are gender and age related. MGMT methylation showed high correlation with loss of protein expression (concordance correlation coefficient = -0.49; Fisher exact test: p < 0.01), different from what has been observed in other brain tumors. In the subgroup of ten patients who received high dose chemotherapy, the presence of methylated MGMT promoter (n = 4), seems to be associated with a prolonged overall survival (>60 months in three of four patients). The prognostic significance of these molecular markers in PCNSL needs to be further studied in groups of patients treated in a homogeneous way.

  11. [Effects of Electroacupuncture Intervention on Oxygen Free Radicals and Expression of Apoptosis-related Proteins in Rats with Ischemic Learning and Memory Disorder].

    PubMed

    Hou, Zhi-tao; Sun, Zhong-ren; Liu, Song-tao; Xiong, Sheng-biao; Liu, Yi-tian; Han, Xiao-xia; Sun, Hong-fang; Han, Yu-sheng; Yin, Hong-na; Xu, Jin-qiao; Li, Dong-dong

    2015-12-01

    To observe the effect of electroacupuncture (EA) therapy on levels of oxygen free radicals (OFR) and hippocampal apoptosis-related protein expression in ischemic learning-memory disorder rats so as to investigate its mechanisms underlying improvement of ischemic learning-memory impairment. A total of 60 SD rats were randomly divided into sham operation (sham), model, medication, and EA groups, with 15 rats in each group. The learning-memory disorder model was made by occlusion of bilateral carotid arteries. EA (2- 3 Hz, 2 mA) was applied to "Zhi San Zhen" ["Shenting" (GV 24) and bilateral "Benshen" (GB 13)] for 30 min, once a day for 3 weeks. The rats of the medication group were treated by lavage of Aricept (0.03 mg . kg(-1) . d(-1)), once daily for 3 weeks. The rats' learning-memory ability was detected by Morris water maze tests and the state of hippocampal apoptosis cells was observed by light microscope after TUNEL staining and the expression of hippocampal Bcl-2, Bax and Caspase-3 proteins was detected by immunohistochemistry. Serum and hippocampal superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) contents were detected by chemical colorimetric analysis. Compared with the sham group, the escape latencies (place-navigation) after modeling were evidently prolonged, and the times of target-platform crossing in 90 sec (spatial probe test) considerably reduced in the model group (P<0.01), suggesting an impairment of learning-memory ability. After the treatment for 21 d, the increased escape latency and the reduced target-platform crossing time in both EA and medication groups were reversed in comparison with the model group (P<0.01), suggesting an improvement of memory ability, and the effect of the EA group was significantly superior to that of the medication group (P<0.05). Compared with the sham group, the number of apoptotic cells in hippocampal CA 1- CA 3 regions, and the expression levels of hippocampal Bcl-2

  12. Prognostic value of insulin-like growth factor II mRNA binding protein 3 in patients treated with radical prostatectomy.

    PubMed

    Chromecki, Thomas F; Cha, Eugene K; Pummer, Karl; Scherr, Douglas S; Tewari, Ashutosh K; Sun, Maxine; Fajkovic, Harun; Roehrborn, Claus G; Ashfaq, Raheela; Karakiewicz, Pierre I; Shariat, Shahrokh F

    2012-07-01

    Study Type - Prognosis (case series) Level of Evidence 4 What's known on the subject? and What does the study add? Insulin-like growth factor II mRNA binding protein 3 (IMP3) is associated with poor outcomes in a variety of malignancies. The role of IMP3 in protate cancer remains poorly understood. IMP3 expression was associated with features of aggressive biology and aggressive prostate cancer recurrence after surgery. Although IMP3 is differentially expressed in patients with features of biologically aggressive prostate cancer, it does not have independent prognostic value in patients treated with RP. To evaluate the association of insulin-like growth factor II mRNA binding protein 3 (IMP3) with pathological features and outcomes in patients treated with radical prostatectomy (RP). Immunohistochemical staining for IMP3 was performed on archival tissue microarray specimens from 232 consecutive patients treated with RP for clinically localized disease. None of the patients received neoadjuvant or adjuvant radiation or hormone therapy. IMP3 expression was histologically categorized as normal or abnormal. Disease recurrence was classified as aggressive if metastases were present, post-recurrence prostate-specific antigen (PSA) doubling time was less than 10 months, or if the patients failed to respond to salvage local radiation therapy. The median follow-up was 69.8 months (interquartile range [IQR]: 40.1-99.5). IMP3 expression was abnormal in 42 (18.1%) of 232 patients. IMP3 expression was associated with extracapsular extension (P= 0.020), seminal vesicle invasion (P= 0.024), lymphovascular invasion (P= 0.036) and a high pathological Gleason score (P= 0.009). The 5-year PSA recurrence-free survival for IMP3-negative patients was 83% (standard error [SE]= 3) vs 67% (SE = 8) in IMP3-positive patients (log-rank test, P= 0.015). In a multivariable analysis that adjusted for the effects of surgical margins, extracapsular extension and seminal vesicle invasion, PSA

  13. Construction of gene/protein interaction networks for primary myelofibrosis and KEGG pathway-enrichment analysis of molecular compounds.

    PubMed

    Sun, C G; Cao, X J; Zhou, C; Liu, L J; Feng, F B; Liu, R J; Zhuang, J; Li, Y J

    2015-12-08

    The objective of this study was the development of a gene/protein interaction network for primary myelofibrosis based on gene expression, and the enrichment analysis of KEGG pathways underlying the molecular complexes in this network. To achieve this, genes involved in primary myelofibrosis were selected from the OMIM database. A gene/protein interaction network for primary myelofibrosis was obtained through Cytoscape with the literature mining performed using the Agilent Literature Search plugin. The molecular complexes in the network were detected by ClusterViz plugin and KEGG pathway enrichment of molecular complexes was performed using DAVID online. We found 75 genes associated with primary myelofibrosis in the OMIM database. The gene/protein interaction network of primary myelofibrosis contained 608 nodes, 2086 edges, and 4 molecular complexes with a correlation integral value greater than 4. Molecular complexes involved in KEGG pathways are related to cytokine regulation, immune function regulation, ECM-receptor interaction, focal adhesion, actin cytoskeleton regulation, cell adhesion molecules, and other biological behavior of tumors, which can provide a reliable direction for the treatment of primary myelofibrosis and the bioinformatic foundation for further understanding the molecular mechanisms of this disease.

  14. Towards a comprehensive view of the primary structure of venom proteins from the parasitoid wasp Pimpla hypochondriaca.

    PubMed

    Parkinson, Neil M; Conyers, Christine; Keen, Jeff; MacNicoll, Alan; Smith, Ian; Audsley, Neil; Weaver, Robert

    2004-06-01

    Venom from the parasitoid wasp Pimpla hypochondriaca has potent in vivo activity against insect haemocytes and disrupts host immune responses. Using hybridisation techniques, and more recently random sequence analysis, we had previously identified cDNAs encoding 10 venom proteins from this wasp and deduced their primary structures. We have now extended the random sequence analysis and discovered a further nine cDNAs encoding proteins with predicted signal sequences. The mature proteins were calculated to have masses of between 4 and 22 kDa. Post-signal sequence residues predicted from the cDNAs matched those derived by Edman degradation from venom proteins separated using gel filtration and reverse phase chromatography, confirming that the cloned cDNAs encode proteins which are secreted into the venom sac. Proteins containing at least six cysteine residues were abundant and seven of these cysteine-rich venom proteins, cvp1-7, were identified. The sequences of some of these proteins were similar, or contained similar cysteine arrangements, to Kunitz type protease inhibitors, pacifastin, the trypsin inhibitor domain protein family, atracotoxin and omega-conotoxin, respectively, which occur in a diverse range of animals including spiders, molluscs, humans and grasshoppers. Two small venom proteins, svp1 and svp2, as well as cvp7 did not have similar sequences to proteins in the GenBank protein database suggesting they may be highly specialised venom components. The random sequencing approach has provided a rapid means of determining the primary structure of the majority of Pimpla hypochondriaca venom proteins.

  15. Counting the Cost of Failed Spinal Fusion for Relief of Low Back Pain: Does Primary Fusion With Bone Morphogenetic Protein Make Economic Sense From a Primary Payer Perspective?

    PubMed

    Lloyd, Adam P

    2017-07-01

    A retrospective cohort study. To investigate the unknown direct costs of failed instrumented lumbar fusion using iliac crest bone graft (ICBG) and subsequent reoperation utilizing recombinant human bone morphogenetic protein-2 (rhBMP-2) from a primary payer perspective. Recent evidence has demonstrated increased rates of instrumented lumbar fusion and utilization of rhBMP-2 to treat a range of conditions causing lower back pain. For health care providers with finite financial resources, there is an increasing demand to evaluate economic costs of available treatment modalities. The high cost of rhBMP-2 has often been cited as a leading reason for delaying its universal acceptance as a preferred substitute to ICBG. It has been hypothesized that rhBMP-2 may demonstrate cost-effectiveness if pseudarthrosis and reoperation rates are decreased, thus avoiding subsequent expenditure. This was a retrospective cohort study of patients who underwent instrumented lumbar fusions utilizing rhBMP-2. Hospital finance records were used to calculate direct total expenditure incurred by the primary payer for the procedure using rhBMP-2. For patients who received rhBMP-2 in a secondary lumbar fusion, additional total expenditure related to the patients' failed primary instrumented fusion with ICBG was also sought. The mean total costs associated with failed instrumented lumbar fusion using ICBG and reoperation using rhBMP-2 totaled £47,734 per patient. The total direct costs of a policy of primary instrumented lumbar fusion with rhBMP-2 were less at £26,923 per patient; however, this was not significant. To date, this is the first study to report the costs of failed primary instrumented lumbar fusions using ICBG and subsequent secondary fusions using rhBMP-2 from a primary payer perspective. On the basis of this evidence, a policy of using rhBMP-2 in all patients undergoing a primary instrumented lumbar fusion cannot be recommended.

  16. Dynamics of Radical-Mediated Enzyme Catalyses

    NASA Astrophysics Data System (ADS)

    Warncke, Kurt

    1997-11-01

    An emergent class of enzymes harnesses the extreme reactivity of electron-deficient free radical species to perform some of the most difficult reactions in biology. The regio- and stereo-selectivity achieved by these enzymes defies long-held ideas that radical reactions are non-specific. The common primary step in these catalyses is metal- or metallocenter-assisted generation of an electron-deficient organic "initiator radical". The initiator radical abstracts a hydrogen atom from the substrate, opening a new reaction channel for rearrangement to the product. Our aim is to elucidate the detailed molecular mechanisms of the radical pair separation and radical rearrangement steps. Radical pair separation and substrate radical rearrangement are tracked by using time-resolved (10-7 to 10-3 s) techniques of pulsed-electron paramagnetic resonance spectroscopy (FT-EPR, ESEEM). Synchronous time-evolution of the reactions is attained by triggering with a visible laser pulse. Transient non-Boltzmann population of the states of the spin-coupled systems, and resultant electron spin polarization, facilitates study at or near room temperature under conditions where the enzymes are operative. The systems examined include ethanolamine deaminase, a vitamin B12 coenzyme-dependent enzyme, ribonucleotide reductase and photosynthetic reaction centers. The electronic and nuclear structural and kinetic information obtained from the pulsed-EPR studies is used to address how the initiator radicals are stabilized against deleterious recombination with the metal, and to distinguish the participation of concerted versus sequential rearrangement pathways.

  17. Mutations in Centrosomal Protein CEP152 in Primary Microcephaly Families Linked to MCPH4

    PubMed Central

    Guernsey, Duane L.; Jiang, Haiyan; Hussin, Julie; Arnold, Marc; Bouyakdan, Khalil; Perry, Scott; Babineau-Sturk, Tina; Beis, Jill; Dumas, Nadine; Evans, Susan C.; Ferguson, Meghan; Matsuoka, Makoto; Macgillivray, Christine; Nightingale, Mathew; Patry, Lysanne; Rideout, Andrea L.; Thomas, Aidan; Orr, Andrew; Hoffmann, Ingrid; Michaud, Jacques L.; Awadalla, Philip; Meek, David C.; Ludman, Mark; Samuels, Mark E.

    2010-01-01

    Primary microcephaly is a rare condition in which brain size is substantially diminished without other syndromic abnormalities. Seven autosomal loci have been genetically mapped, and the underlying causal genes have been identified for MCPH1, MCPH3, MCPH5, MCPH6, and MCPH7 but not for MCPH2 or MCPH4. The known genes play roles in mitosis and cell division. We ascertained three families from an Eastern Canadian subpopulation, each with one microcephalic child. Homozygosity analysis in two families using genome-wide dense SNP genotyping supported linkage to the published MCPH4 locus on chromosome 15q21.1. Sequencing of coding exons of candidate genes in the interval identified a nonconservative amino acid change in a highly conserved residue of the centrosomal protein CEP152. The affected children in these two families were both homozygous for this missense variant. The third affected child was compound heterozygous for the missense mutation plus a second, premature-termination mutation truncating a third of the protein and preventing its localization to centrosomes in transfected cells. CEP152 is the putative mammalian ortholog of Drosphila asterless, mutations in which affect mitosis in the fly. Published data from zebrafish are also consistent with a role of CEP152 in centrosome function. By RT-PCR, CEP152 is expressed in the embryonic mouse brain, similar to other MCPH genes. Like some other MCPH genes, CEP152 shows signatures of positive selection in the human lineage. CEP152 is a strong candidate for the causal gene underlying MCPH4 and may be an important gene in the evolution of human brain size. PMID:20598275

  18. The first demonstration of CYP1A and the ABC protein(s) gene expression and activity in European seabass (Dicentrarchus labrax) primary hepatocytes.

    PubMed

    Ferreira, Marta; Santos, Pedro; Rey-Salgueiro, Ledicia; Zaja, Roko; Reis-Henriques, Maria Armanda; Smital, Tvrtko

    2014-04-01

    Primary hepatocytes are a model for studying various effects of different xenobiotics, including detoxification strategies. In this study we have isolated and cultured European seabass (Dicentrarchus labrax) primary hepatocytes and assessed gene transcription and activity of CYP1A (phase I of cellular detoxification) and ABCC1 and ABCC2 (phase III) transport proteins after exposure to benzo(a)pyrene (BaP). A dose dependent increase in Abcc2 and Cyp1a mRNA transcripts was observed in seabass primary hepatocytes upon exposure to BaP. The activity of ABC proteins, as key mediators of the multixenobiotic resistance (MXR), was further confirmed by assessing the accumulation of the model fluorescence substrate rhodamine 123 in the absence and presence of model inhibitors. A weak interaction between BaP and ABC proteins was observed. CYP1A dependent ethoxyresorufin-O-deeethylase (EROD) activity was significantly induced by the presence of BaP. After the 24h exposure period only 10% of the initial BaP was present in the incubation medium, clearly demonstrating biotransformation potential of primary seabass hepatocytes. Furthermore, the presence of the 3-hydroxybenzo(a)pyrene, a BaP metabolite, in the medium implies its active efflux. In conclusion, we showed that seabass primary hepatocytes do express important elements of the cellular detoxification machinery and may be a useful in vitro model for studying basic cellular detoxification mechanisms and their interaction with environmental contaminants. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Reading the primary structure of a protein with 0.07 nm3 resolution using a subnanometre-diameter pore

    NASA Astrophysics Data System (ADS)

    Kennedy, Eamonn; Dong, Zhuxin; Tennant, Clare; Timp, Gregory

    2016-11-01

    The primary structure of a protein consists of a sequence of amino acids and is a key factor in determining how a protein folds and functions. However, conventional methods for sequencing proteins, such as mass spectrometry and Edman degradation, suffer from short reads and lack sensitivity, so alternative approaches are sought. Here, we show that a subnanometre-diameter pore, sputtered through a thin silicon nitride membrane, can be used to detect the primary structure of a denatured protein molecule. When a denatured protein immersed in electrolyte is driven through the pore by an electric field, measurements of a blockade in the current reveal nearly regular fluctuations, the number of which coincides with the number of residues in the protein. Furthermore, the amplitudes of the fluctuations are highly correlated with the volumes that are occluded by quadromers (four residues) in the primary structure. Each fluctuation, therefore, represents a read of a quadromer. Scrutiny of the fluctuations reveals that the subnanometre pore is sensitive enough to read the occluded volume that is related to post-translational modifications of a single residue, measuring volume differences of ∼0.07 nm3, but it is not sensitive enough to discriminate between the volumes of all twenty amino acids.

  20. The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function.

    PubMed

    Escobar-Álvarez, Elizabeth; Leinisch, Fabian; Araya, Gissela; Monasterio, Octavio; Lorentzen, Lasse G; Silva, Eduardo; Davies, Michael J; López-Alarcón, Camilo

    2017-11-01

    FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROO•) generated from AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROO•. Quantification of the number of moles of amino acid consumed per mole of ROO• shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROO•-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Two Dynamical Regimes of the Substrate Radical Rearrangement Reaction in B12-Dependent Ethanolamine Ammonia-Lyase Resolve Contributions of Native Protein Configurations and Collective Configurational Fluctuations to Catalysis.

    PubMed

    Kohne, Meghan; Zhu, Chen; Warncke, Kurt

    2017-06-27

    The kinetics of the substrate radical rearrangement reaction step in B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium are measured over a 92 K temperature range. The observed first-order rate constants display a piecewise-continuous Arrhenius dependence, with linear regions over 295 → 220 K (monoexponential) and 214 → 203 K (biexponential) that are delineated by a kinetic bifurcation and kinks at 219 and 217 K, respectively. The results are interpreted by using a free energy landscape model and derived microscopic kinetic mechanism. The bifurcation and kink transitions correspond to the effective quenching of two distinct sets of native collective protein configurational fluctuations that (1) reconfigure the protein within the substrate radical free energy minimum, in a reaction-enabling step, and (2) create the protein configurations associated with the chemical step. Below 217 K, the substrate radical decay reaction persists. Increases in activation enthalpy and entropy of both the microscopic enabling and reaction steps indicate that this non-native reaction coordinate is conducted by local, incremental fluctuations. Continuity in the Arrhenius relations indicates that the same sets of protein groups and interactions mediate the rearrangement over the 295 to 203 K range, but with a repertoire of configurations below 217 K that is restricted, relative to the native configurations accessible above 219 K. The experimental features of a culled reaction step, first-order kinetic measurements, and wide room-to-cryogenic temperature range, allow the direct demonstration and kinetic characterization of protein dynamical contributions to the core adiabatic, bond-making/bond-breaking reaction in EAL.

  2. TGF beta-induced growth inhibition in primary fibroblasts requires the retinoblastoma protein.

    PubMed

    Herrera, R E; Mäkelä, T P; Weinberg, R A

    1996-09-01

    Transforming growth factor beta (TGF beta) inhibits cell proliferation by inducing a G1 cell-cycle arrest. Cyclin/CDK complexes have been implicated in this arrest, because TGF beta treatment leads to inhibition of cyclin/CDK activity. We have investigated the role of the retinoblastoma protein (pRb) in TGF beta-induced growth arrest by using RB+/+ and RB-/- primary mouse embryo fibroblasts. In both of these cell types, TGF beta inhibits CDK4-associated kinase activity. However, whereas CDK2-associated kinase activity was completely inhibited by TGF beta in the wild-type cells, it was reduced only slightly in the RB mutant cells. In addition, at high-cell density the growth-inhibitory effects of TGF beta are no longer observed in the RB-/- cells; on the contrary, TGF beta treatment promotes the growth of these mutant fibroblasts. Thus, under certain cellular growth conditions, elimination of pRb transforms the growth-inhibitory effects of TGF beta into growth-stimulatory effects. These observations could help to explain why TGF beta is often found to enhance tumorigenicity in vivo and why inactivation of the RB gene leads to tumorigenesis.

  3. Regulation of protein kinase D during differentiation and proliferation of primary mouse keratinocytes.

    PubMed

    Ernest Dodd, M; Ristich, Vladimir L; Ray, Sagarika; Lober, Robert M; Bollag, Wendy B

    2005-08-01

    Diseased skin often exhibits a deregulated program of the keratinocyte maturation necessary for epidermal stratification and function. Protein kinase D (PKD), a serine/threonine kinase, is expressed in proliferating keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. We have proposed that PKD functions as a pro-proliferative and/or anti-differentiative signal in keratinocytes and hypothesized that differentiation inducers will downmodulate PKD to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation, and activity were analyzed upon stimulation of differentiation and proliferation in primary mouse keratinocytes. Elevated extracellular calcium and acute 12-O-tetradecanoylphorbol-13-acetate (TPA) treatments induced differentiation and triggered a downmodulation of PKD levels, autophosphorylation at serine 916, and activity. Chronic TPA treatment stimulated proliferation and resulted in a recovery of PKD levels, autophosphorylation, and activity. Immunohistochemical analysis demonstrated PKD localization predominantly in the proliferative basal layer of mouse epidermis. Co-expression studies revealed a pro-proliferative, anti-differentiative effect of PKD on keratinocyte maturation as monitored by increased and decreased promoter activities of keratin 5, a proliferative marker, and involucrin, a differentiative marker, respectively. This work describes the inverse regulation of PKD during keratinocyte differentiation and proliferation and the pro-proliferative/anti-differentiative effects of PKD co-expression on keratinocyte maturation.

  4. Cadmium induces apoptosis in primary rat osteoblasts through caspase and mitogen-activated protein kinase pathways

    PubMed Central

    Zhao, Hongyan; Liu, Wei; Wang, Yi; Dai, Nannan; Gu, Jianhong; Yuan, Yan; Liu, Xuezhong; Bian, Jianchun

    2015-01-01

    Exposure to cadmium (Cd) induces apoptosis in osteoblasts (OBs); however, little information is available regarding the specific mechanisms of Cd-induced primary rat OB apoptosis. In this study, Cd reduced cell viability, damaged cell membranes and induced apoptosis in OBs. We observed decreased mitochondrial transmembrane potentials, ultrastructure collapse, enhanced caspase-3 activity, and increased concentrations of cleaved PARP, cleaved caspase-9 and cleaved caspase-3 following Cd treatment. Cd also increased the phosphorylation of p38-mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK)1/2 and c-jun N-terminal kinase (JNK) in OBs. Pretreatment with the caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, ERK1/2 inhibitor (U0126), p38 inhibitor (SB203580) and JNK inhibitor (SP600125) abrogated Cd-induced cell apoptosis. Furthermore, Cd-treated OBs exhibited signs of oxidative stress protection, including increased antioxidant enzymes superoxide dismutase and glutathione reductase levels and decreased formation of reactive oxygen species. Taken together, the results of our study clarified that Cd has direct cytotoxic effects on OBs, which are mediated by caspase- and MAPK pathways in Cd-induced apoptosis of OBs. PMID:26425111

  5. Prognostic impact of GATA binding protein-3 expression in primary lung adenocarcinoma.

    PubMed

    Hashiguchi, Toshihiro; Miyoshi, Hiroaki; Nakashima, Kazutaka; Yokoyama, Shintaro; Matsumoto, Ryoichi; Murakami, Daigo; Mitsuoka, Masahiro; Takamori, Shinzo; Akagi, Yoshito; Ohshima, Koichi

    2017-05-01

    GATA binding protein-3 (GATA3) is a transcription factor that regulates cell differentiation and maintenance in some types of normal cells. This study aimed to investigate the association between GATA3 expression and primary lung adenocarcinoma and to clarify the clinical significance of GATA3 expression in lung adenocarcinoma. Immunohistochemical GATA3 expression was evaluated using completely resected lung adenocarcinoma samples from 95 cases. GATA3 immunohistochemical staining was performed and scored. Associations between clinicopathological factors and GATA3 expression were analyzed by using the χ(2) test and Fisher exact test. The Kaplan-Meier method was used to analyze overall survival (OS) and disease-free survival (DFS). Forty-nine cases expressed high levels of GATA3, which were associated with lymphatic invasion (P=.003). In univariate and multivariate analyses, vascular invasion (P<.001) and high GATA3 expression (P=.023) were identified as independent risk factors for OS. Higher pathological stages (P=.012), vascular invasion (P=.010), and high GATA3 expression (P=.009) were identified as independent risk factors for DFS. The high GATA3 expression group exhibited statistically worse OS (P=.031) and DFS (P=.011) than the low-expression group based on the Kaplan-Meier curves. In resected lung adenocarcinoma, high GATA3 expression is associated with poorer prognosis for both OS and DFS. Therefore, the immunohistochemical evaluation of GATA3 represents a potentially useful prognostic tool for postoperative patients. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Primary Thrombophilia in México XI: Activated Protein C Resistance Phenotypes are Multifactorial.

    PubMed

    Vallejo-Villalobos, Ma Fernanda; León-Peña, Andrés; León-González, Mónica; Núñez-Cortés, Ana Karen; Olivares-Gazca, Juan Carlos; Valdés-Tapia, Patricia; Garcés-Eisele, Javier; Ruiz-Argüelles, Alejandro; Ruiz-Argüelles, Guillermo J

    2017-09-01

    Activated protein C resistance (aPCR) phenotypes represent around 20% of the laboratory findings in Mexican Mestizos having suffered thrombosis and displaying clinical markers of thrombophilia. In a single institution for a 276-month period, 96 Mexican mestizos with a history of thrombosis and clinical markers of a primary thrombophilic state were prospectively studied to identify a thrombophilic condition. An abnormal aPCR phenotype was identified in 18 individuals. Evaluation of those with an abnormal aPCR phenotype, identified that 44% had factor V Leiden mutation, 22% increased levels of factor VIII, 16% anti-phospholipid antibodies and 6% a lupus anticoagulant. In the remaining 22%, the use of direct oral anticoagulants (DOACs) in the past period of 12-24 h was recorded. We found significant associations between abnormal aPCR phenotype and the factor V Leiden mutation (p = 0022), between abnormal aPCR phenotype and the use of DOACs (p = 0.006) and between antiphospholipid antibodies and lupus anticoagulant (p < 0.0001). These data are consonant with those observed in other populations and further identify that consideration be given to identifying whether individuals are being treated with the novel DOACs when conducting laboratory studies oriented to identify the etiology of thrombosis.

  7. Heat shock proteins and chronic fatigue in primary Sjögren's syndrome.

    PubMed

    Bårdsen, Kjetil; Nilsen, Mari Mæland; Kvaløy, Jan Terje; Norheim, Katrine Brække; Jonsson, Grete; Omdal, Roald

    2016-04-01

    Fatigue occurs frequently in patients with cancer, neurological diseases and chronic inflammatory diseases, but the biological mechanisms that lead to and regulate fatigue are largely unknown. When the innate immune system is activated, heat shock proteins (HSPs) are produced to protect cells. Some extracellular HSPs appear to recognize cellular targets in the brain, and we hypothesize that fatigue may be generated by specific HSPs signalling through neuronal or glial cells in the central nervous system. From a cohort of patients with primary Sjögren's syndrome, 20 patients with high and 20 patients with low fatigue were selected. Fatigue was evaluated with a fatigue visual analogue scale. Plasma concentrations of HSP32, HSP60, HSP72 and HSP90α were measured and analysed to determine if there were associations with the level of fatigue. Plasma concentrations of HSP90α were significantly higher in patients with high fatigue compared with those with low fatigue, and there was a tendency to higher concentrations of HSP72 in patients with high fatigue compared with patients with low fatigue. There were no differences in concentrations of HSP32 and HSP60 between the high- and low-fatigue groups. Thus, extracellular HSPs, particularly HSP90α, may signal fatigue in chronic inflammation. This supports the hypothesis that fatigue is generated by cellular defence mechanisms.

  8. EFFECT OF ARSENICALS ON THE EXPRESSION OF CELL CYCLE PROTEINS AND EARLY SIGNALING EVENTS IN PRIMARY HUMAN KERATINOCYTES.

    EPA Science Inventory

    Effect of Arsenicals on the Expression of Cell Cycle Proteins and Early Signaling Events in Primary Human Keratinocytes.

    Mudipalli, A, Owen R. D. and R. J. Preston, Environmental Carcinogenesis Division, USEPA, RTP, NC 27711.

    Environmental exposure to arsenic is a m...

  9. EFFECT OF ARSENICALS ON THE EXPRESSION OF CELL CYCLE PROTEINS AND EARLY SIGNALING EVENTS IN PRIMARY HUMAN KERATINOCYTES.

    EPA Science Inventory

    Effect of Arsenicals on the Expression of Cell Cycle Proteins and Early Signaling Events in Primary Human Keratinocytes.

    Mudipalli, A, Owen R. D. and R. J. Preston, Environmental Carcinogenesis Division, USEPA, RTP, NC 27711.

    Environmental exposure to arsenic is a m...

  10. Complex Biotransformations Catalyzed by Radical S-Adenosylmethionine Enzymes*

    PubMed Central

    Zhang, Qi; Liu, Wen

    2011-01-01

    The radical S-adenosylmethionine (AdoMet) superfamily currently comprises thousands of proteins that participate in numerous biochemical processes across all kingdoms of life. These proteins share a common mechanism to generate a powerful 5′-deoxyadenosyl radical, which initiates a highly diverse array of biotransformations. Recent studies are beginning to reveal the role of radical AdoMet proteins in the catalysis of highly complex and chemically unusual transformations, e.g. the ThiC-catalyzed complex rearrangement reaction. The unique features and intriguing chemistries of these proteins thus demonstrate the remarkable versatility and sophistication of radical enzymology. PMID:21771780

  11. Understanding the relationship between the primary structure of proteins and its propensity to be soluble on overexpression in Escherichia coli

    PubMed Central

    Idicula-Thomas, Susan; Balaji, Petety V.

    2005-01-01

    Solubility of proteins on overexpression in Escherichia coli is a manifestation of the net effect of several sequence-dependent and sequence-independent factors. This study aims to delineate the relationship between the primary structure and solubility on overexpression. The amino acid sequences of proteins reported to be soluble or to form inclusion bodies on overexpression in E. coli under normal growth conditions were analyzed. The results show a positive correlation between thermostability and solubility of proteins, and an inverse correlation between the in vivo half-life of proteins and solubility. The amino acid (Asn, Thr, Tyr) composition and the tripeptide frequency of the protein were also found to influence its solubility on overexpression. The amino acids that were seen to be present in a comparatively higher frequency in inclusion body-forming proteins have a higher sheet propensity, whereas those that are seen more in soluble proteins have a higher helix propensity; this is indicative of a possible correlation between sheet propensity and inclusion body formation. Thus, the present analysis shows that thermostability, in vivo half-life, Asn, Thr, and Tyr content, and tripeptide composition of a protein are correlated to the propensity of a protein to be soluble on overexpression in E. coli. The precise mechanism by which these properties affect the solubility status of the overexpressed protein remains to be understood. PMID:15689506

  12. Contemporary Radical Economics.

    ERIC Educational Resources Information Center

    Sherman, Howard J.

    1984-01-01

    The origins of contemporary radical economics are examined. Applications of radical economics to price and value theory, labor segmentation theory, business cycles, industrial organization, government and business, imperialism and development, and comparative systems are reviewed. (Author/RM)

  13. Contemporary Radical Economics.

    ERIC Educational Resources Information Center

    Sherman, Howard J.

    1984-01-01

    The origins of contemporary radical economics are examined. Applications of radical economics to price and value theory, labor segmentation theory, business cycles, industrial organization, government and business, imperialism and development, and comparative systems are reviewed. (Author/RM)

  14. TP53 Promoter Methylation in Primary Glioblastoma: Relationship with TP53 mRNA and Protein Expression and Mutation Status

    PubMed Central

    Szybka, Malgorzata; Malachowska, Beata; Fendler, Wojciech; Potemski, Piotr; Piaskowski, Sylwester; Jaskolski, Dariusz; Papierz, Wielislaw; Skowronski, Wieslaw; Och, Waldemar; Kordek, Radzislaw

    2014-01-01

    Reduced expression of TP53 by promoter methylation has been reported in several neoplasms. It remains unclear whether TP53 promoter methylation is associated with reduced transcriptional and protein expression in glioblastoma (GB). The aim of our work was to study the impact of TP53 methylation and mutations on TP53 mRNA level and protein expression in 42 molecularly characterized primary GB tumors. We also evaluate the impact of all molecular alterations on the overall patient survival. The frequency of TP53 promoter methylation was found in 21.4%. To the best of our knowledge, this is the first report showing such high frequency of TP53 promoter methylation in primary GB. There was no relation between TP53 promoter methylation and TP53 mRNA level (p=0.5722) and between TP53 promoter methylation and TP53 protein expression (p=0.2045). No significant associations were found between TP53 mRNA expression and mutation of TP53 gene (p=0.9076). However, significant association between TP53 mutation and TP53 protein expression was found (p=0.0016). Our data suggest that in primary GB TP53 promoter methylation does not play a role in silencing of TP53 transcriptional and protein expression and is probably regulated by other genetic and epigenetic mechanisms associated with genes involved in the TP53 pathway. PMID:24506545

  15. Primary, secondary, and tertiary structure of the core of a histone H1-like protein from the sperm of Mytilus.

    PubMed

    Jutglar, L; Borrell, J I; Ausió, J

    1991-05-05

    We have analyzed the structure of the trypsin-resistant core of the protein PL-II* of the sperm from Mytilus californianus. The peptide has a molecular mass of 8436 Da and its primary sequence is ATGGAKKP STLSMIVAAIQAMKNRKGSSVQAIRKYILANNKG INTSRLGSAMKLAFAKGLKSGVLVRPKTSAGA SGATGSFRVG. This sequence bears an enormous homology and fulfills the constraints of the consensus sequence of the trypsin-resistant peptides of the proteins of the histone H1 family. Secondary structure analysis using Fourier-transform infared spectroscopy as well as predictive methods indicate the presence of 20-30% beta-structure and approximately 25% alpha-helix for this peptide. As in the case of histone H1 proteins, the protein PL-II* core exhibits a compact globular structure as deduced from hydrodynamic measurements. The presence of a histone H1 protein with protamine-like features, seems to be thus, a common general feature of the chromatin composition in the sperm of the bivalve molluscs.

  16. Primary structure and subcellular localization of the knob-associated histidine-rich protein of Plasmodium falciparum.

    PubMed Central

    Pologe, L G; Pavlovec, A; Shio, H; Ravetch, J V

    1987-01-01

    Plasmodium falciparum-infected erythrocytes bind to venular endothelial cells by means of electron-dense deformations (knobs) on the parasitized erythrocyte surface. The primary structure of a parasite-derived histidine-rich protein associated with the knob structure was deduced from cDNA sequence analysis. The 634 amino acid sequence is rich in lysine and histidine and contains three distinct, tandemly repeated domains. Indirect immunofluorescence, using affinity-purified monospecific antibodies directed against recombinant protein synthesized in Escherichia coli, localized the knob-associated histidine-rich protein to the membrane of knobby infected erythrocytes. Immunoelectron microscopy established that the protein is clustered on the cytoplasmic side of the erythrocyte membrane and is associated with the electron-dense knobs. A role for this histidine-rich protein in knob structure and cytoadherence is suggested based upon these data. Images PMID:3313387

  17. Radical prostatectomy - discharge

    MedlinePlus

    ... prostatectomy - discharge; Laparoscopic radical prostatectomy - discharge; LRP - discharge; Robotic-assisted laparoscopic prostatectomy - discharge ; RALP - discharge; Pelvic lymphadenectomy - ...

  18. BLUF Domain Function Does Not Require a Metastable Radical Intermediate State

    PubMed Central

    2014-01-01

    BLUF (blue light using flavin) domain proteins are an important family of blue light-sensing proteins which control a wide variety of functions in cells. The primary light-activated step in the BLUF domain is not yet established. A number of experimental and theoretical studies points to a role for photoinduced electron transfer (PET) between a highly conserved tyrosine and the flavin chromophore to form a radical intermediate state. Here we investigate the role of PET in three different BLUF proteins, using ultrafast broadband transient infrared spectroscopy. We characterize and identify infrared active marker modes for excited and ground state species and use them to record photochemical dynamics in the proteins. We also generate mutants which unambiguously show PET and, through isotope labeling of the protein and the chromophore, are able to assign modes characteristic of both flavin and protein radical states. We find that these radical intermediates are not observed in two of the three BLUF domains studied, casting doubt on the importance of the formation of a population of radical intermediates in the BLUF photocycle. Further, unnatural amino acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines, thus modifying the driving force for the proposed electron transfer reaction; the rate changes observed are also not consistent with a PET mechanism. Thus, while intermediates of PET reactions can be observed in BLUF proteins they are not correlated with photoactivity, suggesting that radical intermediates are not central to their operation. Alternative nonradical pathways including a keto–enol tautomerization induced by electronic excitation of the flavin ring are considered. PMID:24579721

  19. The radical amplifier

    NASA Technical Reports Server (NTRS)

    Hastie, D. R.

    1994-01-01

    The radical amplifier as a method for measuring radical concentrations in the atmosphere has received renewed attention lately. In principle, it can measure the total concentration of HO(x) and RO(x) radicals by reacting ambient air with high concentrations of CO (3-10 percent) and NO (2-6 ppmv), and measuring the NO2 produced.

  20. Mitogen-activated protein kinase signaling pathways promote low-density lipoprotein receptor-related protein 1-mediated internalization of beta-amyloid protein in primary cortical neurons.

    PubMed

    Yang, Wei-Na; Ma, Kai-Ge; Qian, Yi-Hua; Zhang, Jian-Shui; Feng, Gai-Feng; Shi, Li-Li; Zhang, Zhi-Chao; Liu, Zhao-Hui

    2015-07-01

    Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD) are caused by the intraneuronal accumulation of beta-amyloid protein (Aβ). Reuptake of extracellular Aβ is believed to contribute significantly to the intraneuronal Aβ pool in the early stages of AD. Published reports have claimed that the low-density lipoprotein receptor-related protein 1 (LRP1) mediates Aβ1-42 uptake and lysosomal trafficking in GT1-7 neuronal cells and mouse embryonic fibroblast non-neuronal cells. However, there is no direct evidence supporting the role of LRP1 in Aβ internalization in primary neurons. Our recent study indicated that p38 MAPK and ERK1/2 signaling pathways are involved in regulating α7 nicotinic acetylcholine receptor (α7nAChR)-mediated Aβ1-42 uptake in SH-SY5Y cells. This study was designed to explore the regulation of MAPK signaling pathways on LRP1-mediated Aβ internalization in neurons. We found that extracellular Aβ1-42 oligomers could be internalized into endosomes/lysosomes and mitochondria in cortical neurons. Aβ1-42 and LRP1 were also found co-localized in neurons during Aβ1-42 internalization, and they could form Aβ1-42-LRP1 complex. Knockdown of LRP1 expression significantly decreased neuronal Aβ1-42 internalization. Finally, we identified that p38 MAPK and ERK1/2 signaling pathways regulated the internalization of Aβ1-42 via LRP1. Therefore, these results demonstrated that LRP1, p38 MAPK and ERK1/2 mediated the internalization of Aβ1-42 in neurons and provided evidence that blockade of LRP1 or inhibitions of MAPK signaling pathways might be a potential approach to lowering brain Aβ levels and served a potential therapeutic target for AD.

  1. Intra-individual comparison of (18)F-PSMA-1007-PET/CT, multi-parametric MRI and radical prostatectomy specimen in patients with primary prostate cancer - a retrospective, proof of concept study.

    PubMed

    Kesch, Claudia; Vinsensia, Maria; Radtke, Jan P; Schlemmer, Heinz P; Heller, Martina; Ellert, Elena; Holland-Letz, Tim; Duensing, Stefan; Grabe, Nils; Afshar-Oromieh, Ali; Wieczorek, Kathrin; Schäfer, Martin; Neels, Oliver C; Cardinale, Jens; Kratochwil, Clemens; Hohenfellner, Markus; Kopka, Klaus; Haberkorn, Uwe; Hadaschik, Boris A; Giesel, Frederik L

    2017-05-04

    Background:(68)Ga-prostate-specific membrane antigen (PSMA)-11-positron-emission-tomography (PET)/ computer-tomography (CT) represents an advanced method for staging of primary prostate cancer (PCa) and diagnosis of recurrent or metastatic PCa. However, due to the narrow availability of (68)Ga the development of alternative tracers is of high interest. Objective: To examine the value of the new PET tracer (18)F-PSMA-1007 for staging of local disease by comparing it to multiparametric magnetic resonance imaging (mpMRI) and radical prostatectomy (RP) histopathology. Methods: In 2016, (18)F-PSMA-1007-PET/CT was performed in 10 men with biopsy confirmed high-risk prostate cancer (PCa). 9 patients underwent mpMRI in the process of primary diagnosis. Consecutively, RP was performed in all 10 men. Agreement analysis was performed retrospectively. PSMA-staining was added for representative sections in RP specimen slices. Localization and agreement analysis of (18)F-PSMA-1007-PET/CT, mpMRI and RP specimen was done dividing the prostate into 38 sections as described in PI-RADS v2. Sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV) and accuracy were calculated for total and near total agreement. Results:(18)F-PSMA-1007-PET/CT had an NPV of 68% and 75% accuracy and mpMRI had an NPV of 88% and 73% accuracy for total agreement. Near total agreement analysis resulted in an NPV of 91% and an accuracy of 93% for (18)F-PSMA-1007-PET/CT and 90% and 87% for mpMRI, respectively. Retrospective combination of mpMRI and PET/CT had an accuracy of 81% for total and 93% for near total agreement. Conclusion: Comparison with RP histopathology demonstrates that (18)F-PSMA-1007 PET/CT is promising for accurate local staging of PCa. Copyright © 2017 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  2. Behavior of Primary Cilia and Tricellular Tight Junction Proteins during Differentiation in Temperature-Sensitive Mouse Cochlear Precursor Hair Cells.

    PubMed

    Kakuki, Takuya; Kaneko, Yakuto; Takano, Kenichi; Ninomiya, Takafumi; Kohno, Takayuki; Kojima, Takashi; Himi, Tetsuo

    2016-01-01

    In the sensory hair cells of the mammalian cochlea, the primary cilia in the planar cell polarity as well as the tight junctions in the epithelial cell polarity and the barrier are important to maintain normal hearing. Temperature-sensitive mouse cochlear precursor hair cells were used to investigate the behavior of primary cilia and tricellular tight junction proteins during the differentiation of sensory hair cells. In undifferentiated cells (incubated at 33°C), many acetylated tubulin-positive primary cilia were observed, and each was accompanied with an x03B3;-tubulin-positive basal body. The primary cilia had a '9 + 0' architecture with nine outer microtubule doublets but lacking a central pair of microtubules. In differentiated cells (incubated at 39°C), acetylated tubulin-positive primary cilia as well as acetylated tubulin-positive cilia-like structures were partially observed on the cell surface. In differentiated cells, the number of primary cilia was markedly reduced compared with undifferentiated cells, and innumerable cilia-like structures with no ciliary pockets were partially observed on the cell surface. In undifferentiated cells, few tricellulin molecules and lipolysis-stimulated lipoprotein receptors (LSRs) were observed in the cytoplasm. In differentiated cells, many tricellulin molecules and LSRs were observed on the membranes and within the cytoplasm. Conditional immortalized mouse cochlear precursor hair cells may be useful to investigate the roles of primary cilia and tricellular tight junctions during cellular differentiation and degeneration such as apoptosis.

  3. Weak cation magnetic separation technology and MALDI-TOF-MS in screening serum protein markers in primary type I osteoporosis.

    PubMed

    Shi, X L; Li, C W; Liang, B C; He, K H; Li, X Y

    2015-11-30

    We investigated weak cation magnetic separation technology and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) in screening serum protein markers of primary type I osteoporosis. We selected 16 postmenopausal women with osteoporosis and nine postmenopausal women as controls to find a new method for screening biomarkers and establishing a diagnostic model for primary type I osteoporosis. Serum samples were obtained from controls and patients. Serum protein was extracted with the WCX protein chip system; protein fingerprints were examined using MALDI-TOF-MS. The preprocessed and model construction data were handled by the ProteinChip system. The diagnostic models were established using a genetic arithmetic model combined with a support vector machine (SVM). The SVM model with the highest Youden index was selected. Combinations with the highest accuracy in distinguishing different groups of data were selected as potential biomarkers. From the two groups of serum proteins, 123 cumulative MS protein peaks were selected. Significant intensity differences in the protein peaks of 16 postmenopausal women with osteoporosis were screened. The difference in Youden index between the four groups of protein peaks showed that the highest peaks had mass-to-charge ratios of 8909.047, 8690.658, 13745.48, and 15114.52. A diagnosis model was established with these four markers as the candidates, and the model specificity and sensitivity were found to be 100%. Two groups of specimens in the SVM results on the scatterplot were distinguishable. We established a diagnosis model, and provided a new serological method for screening and diagnosis of osteoporosis with high sensitivity and specificity.

  4. VUV Photoionisation of hydrocarbon radicals

    NASA Astrophysics Data System (ADS)

    Alcaraz, C.; Noller, Bastian; Hemberger, Patrick; Fischer, Ingo; Gans, Bérenger; Boyé-Peronne, Séverine; Douin, Stéphane; Gauyacq, Dolorès; Soldi-Lose, Héloïse; Garcia, Gustavo

    2008-09-01

    Hydrocarbon radicals CxHy are constituents of various planetary atmospheres, in particular Titan, as a result of the methane photochemistry induced by the solar radiation. They contribute to the neutral chemistry, but are also important for the ionosphere through their photoionisation leading to their cations CxHy +. These cations are also produced by ion-molecule reactions starting from the reaction of the primary ions CH4 + and CH3 + which are created in the non-dissociative and dissociative photoionisation of CH4. This work aims at caracterizing the VUV photoionisation of small hydrocarbon radicals as a function of photon energy. The objective is to provide laboratory data for modelers on the spectroscopy, the thermochemistry, and the reactivity of the radicals and their cations. The hydrocarbon radicals are much less caracterized than stable molecules since they have to be produced in situ in the laboratory experiment. We have adapted at Orsay [1-3] a pyrolysis source (Figure 1) well suited to produce cold beams of hydrocarbon radicals to our experimental setups. Available now at Orsay, we have two new sources of VUV radiation, complementary in terms of tunability and resolution, that can be used for these studies. The first one is the DESIRS beamline [4] at the new french synchrotron, SOLEIL. The second one is the VUV laser developped at the Centre Laser de l'Université Paris-Sud (CLUPS) [5]. At SOLEIL, a photoelectron-photoion coincidence spectrometer is used to monitor the photoionisation on a large photon energy range. At the CLUPS, a pulsedfield ionisation (PFI-ZEKE) spectrometer allows studies at higher resolution on selected photon energies. The first results obtained with these new setups will be presented. References [1] Fischer, I., Schussler, T., Deyerl, H.J., Elhanine, M. & Alcaraz, C., Photoionization and dissociative photoionization of the allyl radical, C3H5. Int. J. Mass Spectrom., 261 (2-3), 227-233 (2007) [2] Schüßler, T., Roth, W., Gerber

  5. Semiconduction of proteins as an attribute of the living state: the ideas of Albert Szent-Györgyi revisited in light of the recent knowledge regarding oxygen free radicals.

    PubMed

    Nagy, I Z

    1995-01-01

    Oxyradicals have been considered as harmful byproducts causing molecular damage during aging. However, evidence is accumulating to show that the actual situation is more complex: the living state implicitly involves the production of oxyradicals. (1) Blast type cells produce much less oxyradicals than the differentiated ones, and an increased production of OH radicals induce differentiation of various lines of leukemia cells; meanwhile, their superoxide dismutase expression increases to a very high extent. (2) The supramolecular organization of the cells is developed by means of "useful" crosslinking effects OH radicals. (3) Repiratory inhibition of oxyradical production (KCN-intoxication, suffocation, etc.) would kill living organisms prior to the exhaustion of energy reserves. It is assumed that the continuous flux of OH radicals is a prerequisite for a electron delocalization on the proteins, which is a semiconduction of p-type, proposes already in 1941 by Albert Szent-Györgyi, and refuted on a "theoretical" basis. It has become clear by now that the carbon-based semiconduction is possible because diamond transistors are known to exist. The recently developed atomic force microscopy offers some real possibilities for experimental testing of this assumption. This concept may lead us to new horizons in interpretation of living functions, such as the basic memory mechanisms in brain cells and their impairment during aging.

  6. The Meckel-Gruber Syndrome proteins MKS1 and meckelin interact and are required for primary cilium formation.

    PubMed

    Dawe, Helen R; Smith, Ursula M; Cullinane, Andrew R; Gerrelli, Dianne; Cox, Phillip; Badano, Jose L; Blair-Reid, Sarah; Sriram, Nisha; Katsanis, Nicholas; Attie-Bitach, Tania; Afford, Simon C; Copp, Andrew J; Kelly, Deirdre A; Gull, Keith; Johnson, Colin A

    2007-01-15

    Meckel-Gruber syndrome (MKS) is an autosomal recessive lethal malformation syndrome characterized by renal cystic dysplasia, central nervous system malformations (typically, posterior occipital encephalocele), and hepatic developmental defects. Two MKS genes, MKS1 and MKS3, have been identified recently. The present study describes the cellular, sub-cellular and functional characterization of the novel proteins, MKS1 and meckelin, encoded by these genes. In situ hybridization studies for MKS3 in early human embryos showed transcript localizations in agreement with the tissue phenotype of MKS patients. Both MKS proteins predominantly localized to epithelial cells, including proximal renal tubules and biliary epithelial cells. MKS1 localized to basal bodies, while meckelin localized both to the primary cilium and to the plasma membrane in ciliated cell-lines and primary cells. Meckelin protein with the Q376P missense mutation was unable to localize at the cell membrane. siRNA-mediated reduction of Mks1 and Mks3 expression in a ciliated epithelial cell-line blocked centriole migration to the apical membrane and consequent formation of the primary cilium. Co-immunoprecipitation experiments show that wild-type meckelin and MKS1 interact and, in three-dimensional tissue culture assays, epithelial branching morphogenesis was severely impaired. These results suggest that MKS proteins mediate a fundamental developmental stage of ciliary formation and epithelial morphogenesis.

  7. [Lavoisier and radicals].

    PubMed

    Lafont, Olivier

    2007-01-01

    Lavoisier and his co-workers (Guyton de Morveau, Bertholet, Fourcroy) considered that acids were constituted of oxygen and of something else that they called radicals. These radicals were known in some cases, i.e. nitrogen for nitrous acid, carbon for carbonic acid, phosphorus for phosphoric acid. In the case of sulfur, the sulfuric radical could be associated with different quantities of oxigen leading to sulfuric or sulfurous acids. In other cases radicals remained unknown at the time i.e. muriatic radical for muriatic acid, or benzoyl radical for benzoic acid. It is interesting to notice that Lavoisier evoked the case of compound radicals constituted of different substances such as carbon and hydrogen.

  8. Access of hydrogen-radicals to the peptide-backbone as a measure for estimating the flexibility of proteins using matrix-assisted laser desorption/ionization mass spectrometry.

    PubMed

    Takayama, Mitsuo; Nagoshi, Keishiro; Iimuro, Ryunosuke; Inatomi, Kazuma

    2014-05-13

    A factor for estimating the flexibility of proteins is described that uses a cleavage method of "in-source decay (ISD)" coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The MALDI-ISD spectra of bovine serum albumin (BSA), myoglobin and thioredoxin show discontinuous intense ion peaks originating from one-side preferential cleavage at the N-Cα bond of Xxx-Asp, Xxx-Asn, Xxx-Cys and Gly-Xxx residues. Consistent with these observations, Asp, Asn and Gly residues are also identified by other flexibility measures such as B-factor, turn preference, protection and fluorescence decay factors, while Asp, Asn, Cys and Gly residues are identified by turn preference factor based on X-ray crystallography. The results suggest that protein molecules embedded in/on MALDI matrix crystals partly maintain α-helix and that the reason some of the residues are more susceptible to ISD (Asp, Asn, Cys and Gly) and others less so (Ile and Val) is because of accessibility of the peptide backbone to hydrogen-radicals from matrix molecules. The hydrogen-radical accessibility in MALDI-ISD could therefore be adopted as a factor for measuring protein flexibility.

  9. Access of Hydrogen-Radicals to the Peptide-Backbone as a Measure for Estimating the Flexibility of Proteins Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

    PubMed Central

    Takayama, Mitsuo; Nagoshi, Keishiro; Iimuro, Ryunosuke; Inatomi, Kazuma

    2014-01-01

    A factor for estimating the flexibility of proteins is described that uses a cleavage method of “in-source decay (ISD)” coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The MALDI-ISD spectra of bovine serum albumin (BSA), myoglobin and thioredoxin show discontinuous intense ion peaks originating from one-side preferential cleavage at the N-Cα bond of Xxx-Asp, Xxx-Asn, Xxx-Cys and Gly-Xxx residues. Consistent with these observations, Asp, Asn and Gly residues are also identified by other flexibility measures such as B-factor, turn preference, protection and fluorescence decay factors, while Asp, Asn, Cys and Gly residues are identified by turn preference factor based on X-ray crystallography. The results suggest that protein molecules embedded in/on MALDI matrix crystals partly maintain α-helix and that the reason some of the residues are more susceptible to ISD (Asp, Asn, Cys and Gly) and others less so (Ile and Val) is because of accessibility of the peptide backbone to hydrogen-radicals from matrix molecules. The hydrogen-radical accessibility in MALDI-ISD could therefore be adopted as a factor for measuring protein flexibility. PMID:24828203

  10. The Dps protein of Agrobacterium tumefaciens does not bind to DNA but protects it toward oxidative cleavage: x-ray crystal structure, iron binding, and hydroxyl-radical scavenging properties.

    PubMed

    Ceci, Pierpaolo; Ilari, Andrea; Falvo, Elisabetta; Chiancone, Emilia

    2003-05-30

    Agrobacterium tumefaciens Dps (DNA-binding proteins from starved cells), encoded by the dps gene located on the circular chromosome of this plant pathogen, was cloned, and its structural and functional properties were determined in vitro. In Escherichia coli Dps, the family prototype, the DNA binding properties are thought to be associated with the presence of the lysine-containing N-terminal tail that extends from the protein surface into the solvent. The x-ray crystal structure of A. tumefaciens Dps shows that the positively charged N-terminal tail, which is 11 amino acids shorter than in the E. coli protein, is blocked onto the protein surface. This feature accounts for the lack of interaction with DNA. The intersubunit ferroxidase center characteristic of Dps proteins is conserved and confers to the A. tumefaciens protein a ferritin-like activity that manifests itself in the capacity to oxidize and incorporate iron in the internal cavity and to release it after reduction. In turn, sequestration of Fe(II) correlates with the capacity of A. tumefaciens Dps to reduce the production of hydroxyl radicals from H2O2 through Fenton chemistry. These data demonstrate conclusively that DNA protection from oxidative damage in vitro does not require formation of a Dps-DNA complex. In vivo, the hydroxyl radical scavenging activity of A. tumefaciens Dps may be envisaged to act in concert with catalase A to counteract the toxic effect of H2O2, the major component of the plant defense system when challenged by the bacterium.

  11. Two-color SERS microscopy for protein co-localization in prostate tissue with primary antibody-protein A/G-gold nanocluster conjugates.

    PubMed

    Salehi, Mohammad; Schneider, Lilli; Ströbel, Philipp; Marx, Alexander; Packeisen, Jens; Schlücker, Sebastian

    2014-02-21

    SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates.

  12. Autoantibodies from patients with primary biliary cirrhosis recognize a region within the nucleoplasmic domain of inner nuclear membrane protein LBR.

    PubMed

    Lin, F; Noyer, C M; Ye, Q; Courvalin, J C; Worman, H J

    1996-01-01

    Autoantibodies from rare patients with primary biliary cirrhosis (PBC) recognize LBR, or lamin B receptor, an integral membrane protein of the inner nuclear membrane. Human LBR has a nucleoplasmic, amino-terminal domain of 208 amino acids followed by a carboxyl-terminal domain with eight putative transmembrane segments. Autoantibodies against LBR from four patients with PBC recognized the nucleoplasmic, amino-terminal domain but not the carboxyl-terminal domain. Immunoblotting of smaller fusion proteins demonstrated that these autoantibodies recognized a conformational epitope(s) contained within the stretch of amino acids from 1 to 60. These results, combined with those of previous studies, show that autoepitopes of nuclear membrane proteins are located within their nucleocytoplasmic domains and that autoantibodies from patients with PBC predominantly react with one domain of a protein antigen. This work also provides further characterization of anti-LBR antibodies that have found utility as reagents in cell biology research.

  13. Two-color SERS microscopy for protein co-localization in prostate tissue with primary antibody-protein A/G-gold nanocluster conjugates

    NASA Astrophysics Data System (ADS)

    Salehi, Mohammad; Schneider, Lilli; Ströbel, Philipp; Marx, Alexander; Packeisen, Jens; Schlücker, Sebastian

    2014-01-01

    SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates.SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates

  14. Rosuvastatin, inflammation, C-reactive protein, JUPITER, and primary prevention of cardiovascular disease--a perspective.

    PubMed

    Kones, Richard

    2010-12-09

    The major public health concern worldwide is coronary heart disease, with dyslipidemia as a major risk factor. Statin drugs are recommended by several guidelines for both primary and secondary prevention. Rosuvastatin has been widely accepted because of its efficacy, potency, and superior safety profile. Inflammation is involved in all phases of atherosclerosis, with the process beginning in early youth and advancing relentlessly for decades throughout life. C-reactive protein (CRP) is a well-studied, nonspecific marker of inflammation which may reflect general health risk. Considerable evidence suggests CRP is an independent predictor of future cardiovascular events, but direct involvement in atherosclerosis remains controversial. Rosuvastatin is a synthetic, hydrophilic statin with unique stereochemistry. A large proportion of patients achieve evidence-based lipid targets while using the drug, and it slows progression and induces regression of atherosclerotic coronary lesions. Rosuvastatin lowers CRP levels significantly. The Justification for Use of statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial was designed after the observation that when both low density lipoprotein and CRP were reduced, patients fared better than when only LDL was lowered. Advocates and critics alike acknowledge that the benefits of rosuvastatin in JUPITER were real. After a review, the US Food and Drug Administration extended the indications for rosuvastatin to include asymptomatic JUPITER-eligible individuals with one additional risk factor. The American Heart Association and Centers of Disease Control and Prevention had previously recognized the use of CRP in persons with "intermediate risk" as defined by global risk scores. The Canadian Cardiovascular Society guidelines went further and recommended use of statins in persons with low LDL and high CRP levels at intermediate risk. The JUPITER study focused attention on ostensibly healthy individuals with

  15. Rosuvastatin, inflammation, C-reactive protein, JUPITER, and primary prevention of cardiovascular disease – a perspective

    PubMed Central

    Kones, Richard

    2010-01-01

    The major public health concern worldwide is coronary heart disease, with dyslipidemia as a major risk factor. Statin drugs are recommended by several guidelines for both primary and secondary prevention. Rosuvastatin has been widely accepted because of its efficacy, potency, and superior safety profile. Inflammation is involved in all phases of atherosclerosis, with the process beginning in early youth and advancing relentlessly for decades throughout life. C-reactive protein (CRP) is a well-studied, nonspecific marker of inflammation which may reflect general health risk. Considerable evidence suggests CRP is an independent predictor of future cardiovascular events, but direct involvement in atherosclerosis remains controversial. Rosuvastatin is a synthetic, hydrophilic statin with unique stereochemistry. A large proportion of patients achieve evidence-based lipid targets while using the drug, and it slows progression and induces regression of atherosclerotic coronary lesions. Rosuvastatin lowers CRP levels significantly. The Justification for Use of statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial was designed after the observation that when both low density lipoprotein and CRP were reduced, patients fared better than when only LDL was lowered. Advocates and critics alike acknowledge that the benefits of rosuvastatin in JUPITER were real. After a review, the US Food and Drug Administration extended the indications for rosuvastatin to include asymptomatic JUPITER-eligible individuals with one additional risk factor. The American Heart Association and Centers of Disease Control and Prevention had previously recognized the use of CRP in persons with “intermediate risk” as defined by global risk scores. The Canadian Cardiovascular Society guidelines went further and recommended use of statins in persons with low LDL and high CRP levels at intermediate risk. The JUPITER study focused attention on ostensibly healthy individuals

  16. Engineering a horseradish peroxidase C stable to radical attacks by mutating multiple radical coupling sites.

    PubMed

    Kim, Su Jin; Joo, Jeong Chan; Song, Bong Keun; Yoo, Young Je; Kim, Yong Hwan

    2015-04-01

    Peroxidases have great potential as industrial biocatalysts. In particular, the oxidative polymerization of phenolic compounds catalyzed by peroxidases has been extensively examined because of the advantage of this method over other conventional chemical methods. However, the industrial application of peroxidases is often limited because of their rapid inactivation by phenoxyl radicals during oxidative polymerization. In this work, we report a novel protein engineering approach to improve the radical stability of horseradish peroxidase isozyme C (HRPC). Phenylalanine residues that are vulnerable to modification by the phenoxyl radicals were identified using mass spectrometry analysis. UV-Vis and CD spectra showed that radical coupling did not change the secondary structure or the active site of HRPC. Four phenylalanine (Phe) residues (F68, F142, F143, and F179) were each mutated to alanine residues to generate single mutants to examine the role of these sites in radical coupling. Despite marginal improvement of radical stability, each single mutant still exhibited rapid radical inactivation. To further reduce inactivation by radical coupling, the four substitution mutations were combined in F68A/F142A/F143A/F179A. This mutant demonstrated dramatic enhancement of radical stability by retaining 41% of its initial activity compared to the wild-type, which was completely inactivated. Structure and sequence alignment revealed that radical-vulnerable Phe residues of HPRC are conserved in homologous peroxidases, which showed the same rapid inactivation tendency as HRPC. Based on our site-directed mutagenesis and biochemical characterization, we have shown that engineering radical-vulnerable residues to eliminate multiple radical coupling can be a good strategy to improve the stability of peroxidases against radical attack. © 2014 Wiley Periodicals, Inc.

  17. Isolation and primary structure of a methionine- and cystine-rich seed protein of Cannabis sativa.

    PubMed

    Odani, S; Odani, S

    1998-04-01

    A 10-kDa protein was isolated from resting seeds of hemp (Cannabis sativa) by buffer extraction, gel filtration, ion-exchange chromatography, and reversed-phase high-pressure liquid chromatography. The protein did not inhibit bovine trypsin. It consisted of subunits composed of 27 and 61 residues and was held together by two disulfide bonds. The complete amino acid sequence was identified by protein analysis, and had 20 mole% of amino acids containing sulfur. The protein was most similar to a methionine-rich protein of Brazil nut (Bertholletia excelsa) and to Mabinlin IV, a sweetness-inducing protein of Capparis masaikai. The high methionine content and the absence of trypsin inhibitory activity suggested that the seed protein can be used to improve the nutritional quality of plant food-stuffs.

  18. The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation.

    PubMed

    Reales, Elena; Bernabé-Rubio, Miguel; Casares-Arias, Javier; Rentero, Carles; Fernández-Barrera, Jaime; Rangel, Laura; Correas, Isabel; Enrich, Carlos; Andrés, Germán; Alonso, Miguel A

    2015-06-15

    The base of the primary cilium contains a zone of condensed membranes whose importance is not known. Here, we have studied the involvement of MAL, a tetraspanning protein that exclusively partitions into condensed membrane fractions, in the condensation of membranes at the ciliary base and investigated the importance of these membranes in primary cilium formation. We show that MAL accumulates at the ciliary base of epithelial MDCK cells. Knockdown of MAL expression resulted in a drastic reduction in the condensation of membranes at the ciliary base, the percentage of ciliated cells and the length of the cilia, but did not affect the docking of the centrosome to the plasma membrane or produce missorting of proteins to the pericentriolar zone or to the membrane of the remaining cilia. Rab8 (for which there are two isoforms, Rab8A and Rab8b), IFT88 and IFT20, which are important components of the machinery of ciliary growth, were recruited normally to the ciliary base of MAL-knockdown cells but were unable to elongate the primary cilium correctly. MAL, therefore, is crucial for the proper condensation of membranes at the ciliary base, which is required for efficient primary cilium extension. © 2015. Published by The Company of Biologists Ltd.

  19. Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila

    PubMed Central

    Zamparini, Andrea L.; Davis, Marie Y.; Malone, Colin D.; Vieira, Eric; Zavadil, Jiri; Sachidanandam, Ravi; Hannon, Gregory J.; Lehmann, Ruth

    2011-01-01

    In Drosophila, Piwi proteins associate with Piwi-interacting RNAs (piRNAs) and protect the germline genome by silencing mobile genetic elements. This defense system acts in germline and gonadal somatic tissue to preserve germline development. Genetic control for these silencing pathways varies greatly between tissues of the gonad. Here, we identified Vreteno (Vret), a novel gonad-specific protein essential for germline development. Vret is required for piRNA-based transposon regulation in both germline and somatic gonadal tissues. We show that Vret, which contains Tudor domains, associates physically with Piwi and Aubergine (Aub), stabilizing these proteins via a gonad-specific mechanism that is absent in other fly tissues. In the absence of vret, Piwi-bound piRNAs are lost without changes in piRNA precursor transcript production, supporting a role for Vret in primary piRNA biogenesis. In the germline, piRNAs can engage in an Aub- and Argonaute 3 (AGO3)-dependent amplification in the absence of Vret, suggesting that Vret function can distinguish between primary piRNAs loaded into Piwi-Aub complexes and piRNAs engaged in the amplification cycle. We propose that Vret plays an essential role in transposon regulation at an early stage of primary piRNA processing. PMID:21831924

  20. Immunocytochemical Detection of Dentin Matrix Proteins in Primary Teeth from Patients with Dentinogenesis Imperfecta Associated with Osteogenesis Imperfecta

    PubMed Central

    Orsini, G.; Majorana, A.; Mazzoni, A.; Putignano, A.; Falconi, M.; Polimeni, A.; Breschi, L.

    2014-01-01

    Dentinogenesis imperfecta determines structural alterations of the collagen structure still not completely elucidated. Immunohisto-chemical analysis was used to assay type I and VI collagen, various non-collagenous proteins distribution in human primary teeth from healthy patients or from patients affected by type I dentinogenesis imperfecta (DGI-I) associated with osteogenesis imperfecta (OI). In sound primary teeth, an organized well-known ordered pattern of the type I collagen fibrils was found, whereas atypical and disorganized fibrillar structures were observed in dentin of DGI-I affected patients. Expression of type I collagen was observed in both normal and affected primary teeth, although normal dentin stained more uniformly than DGI-I affected dentin. Reactivity of type VI collagen was significantly lower in normal teeth than in dentin from DGI-I affected patients (P<0.05). Expressions of dentin matrix protein-1 (DMP1) and osteopontin (OPN) were observed in both normal dentin and dentin from DGI-I affected patients, without significant differences, being DMP1 generally more abundantly expressed. Immuno labeling for chondroitin sulfate (CS) and biglycan (BGN) was weaker in dentin from DGI-I-affected patients compared to normal dentin, this decrease being significant only for CS. This study shows ultra-structural alterations in dentin obtained from patients affected by DGI-I, supported by immunocytochemical assays of different collagenous and non-collagenous proteins. PMID:25578972

  1. The Arabidopsis pyruvate,orthophosphate dikinase regulatory proteins encode a novel, unprecedented Ser/Thr protein kinase primary structure

    USDA-ARS?s Scientific Manuscript database

    Pyruvate,orthophosphate dikinase (PPDK) is a ubiquitous, low abundance metabolic enzyme of undetermined function in C3 plants. Its activity in C3 chloroplasts is light-regulated via reversible phosphorylation of an active-site Thr residue by the PPDK regulatory protein (RP), a most unusual, bifuncti...

  2. Forgotten Radicals in Biology

    PubMed Central

    Luc, Rochette; Vergely, Catherine

    2008-01-01

    Redox reactions play key roles in intra- and inter-cellular signaling, and in adaptative processes of tissues towards stress. Among the major free radicals with essential functions in cells are reactive oxygen species (ROS) including superoxide anion (O2•-), hydroxyl radical (•OH) and reactive nitrogen species (RNS) such as nitric oxide (•NO). In this article, we review the forgotten and new radicals with potential relevance to cardiovascular pathophysiology. Approximately 0.3% of O2•- present in cytosol exists in its protonated form: hydroperoxyl radical (HO2•). Water (H2O) can be split into two free radicals: •OH and hydrogen radical (H•). Several free radicals, including thiyl radicals (RS•) and nitrogen dioxide (NO2•) are known to isomerize double bonds. In the omega-6 series of poly-unsaturated fatty acids (PUFAs), cis-trans isomerization of γ-linolenate and arachidonate catalyzed by RS• has been investigated. Evidence is emerging that hydrogen disulphide (H2S) is a signaling molecule in vivo which can be a source of free radicals. The Cu-Zn superoxide dismutase (SOD) enzyme can oxidize the ionized form of H2S to hydro-sulphide radical: HS•. Recent studies suggest that H2S plays an important function in cardiovascular functions. Carbonate radical, which can be formed when •OH reacts with carbonate or bicarbonate ions, is also involved in the activity of Cu-Zn-SOD. Recently, it has been reported that carbonate anion were potentially relevant oxidants of nucleic acids in physiological environments. In conclusion, there is solid evidence supporting the formation of many free radicals by cells leading which may play an important role in their homeostasis. PMID:23675099

  3. Multiple myeloma cell lines and primary tumors proteoma: protein biosynthesis and immune system as potential therapeutic targets

    PubMed Central

    Mazzotti, Diego Robles; Evangelista, Adriane Feijó; Braga, Walter Moisés Tobias; de Lourdes Chauffaille, Maria; Leme, Adriana Franco Paes; Colleoni, Gisele Wally Braga

    2015-01-01

    Despite great advance in multiple myeloma (MM) treatment since 2000s, it is still an incurable disease and novel therapies are welcome. Therefore, the purpose of this study was to explore MM plasma cells' (MM-PC) proteome, in comparison with their normal counterparts (derived from palatine tonsils of normal donors, ND-PC), in order to find potential therapeutic targets expressed on the surface of these cells. We also aimed to evaluate the proteome of MM cell lines with different genetic alterations, to confirm findings obtained with primary tumor cells. Bone marrow (BM) samples from eight new cases of MM and palatine tonsils from seven unmatched controls were submitted to PC separation and, in addition to two MM cell lines (U266, RPMI-8226), were submitted to protein extraction for mass spectrometry analyses. A total of 81 proteins were differentially expressed between MM-PC and ND-PC - 72 upregulated and nine downregulated; U266 vs. RPMI 8226 cell lines presented 61 differentially expressed proteins - 51 upregulated and 10 downregulated. On primary tumors, bioinformatics analyses highlighted upregulation of protein biosynthesis machinery, as well as downregulation of immune response components, such as MHC class I and II, and complement receptors. We also provided comprehensive information about U266 and RPMI-8226 cell lines' proteome and could confirm some patients' findings. PMID:26807199

  4. Surface membrane proteins of Biomphalaria glabrata embryonic cells bind fucosyl determinants on the tegumental surface of Schistosoma mansoni primary sporocysts.

    PubMed

    Castillo, Maria G; Wu, Xiao-Jun; Dinguirard, Nathalie; Nyame, A Kwame; Cummings, Richard D; Yoshino, Timothy P

    2007-08-01

    Previous observations that in vitro adherence of Biomphalaria glabrata embryonic (Bge) cells to sporocyst larval stages of Schistosoma mansoni was strongly inhibited by fucoidan, a sulfated polymer of L-fucose, suggested a role for lectinlike Bge cell receptors in sporocyst binding interactions. In the present investigation, monoclonal antibodies with specificities to 3 major glycan determinants found on schistosomes, LacdiNAc, fucosylated LacdiNAc (LDNF), and the Lewis X antigen, were used in adhesion blocking studies to further analyze the molecular interactions at the host-parasite interface. Results showed that only the anti-LDNF antibody significantly reduced snail Bge cell adhesion to the surface of sporocysts, suggesting that fucosyl determinants may be important in larval-host cell interactions. Affinity chromatographic separation of fucosyl-reactive Bge cell proteins from fucoidan-bound Sepharose 4B revealed the presence of polypeptides ranging from 6 to 200 kDa after elution with fucoidan-containing buffer. Pre-elution of the Bge protein-bound affinity column with dextran (Dex) and dextran sulfate (DexS) before introduction of the fucoidan buffer served as controls for protein binding based on nonspecific sugar or negative charge interactions. A subset of polypeptides (approximately 35-150 kDa) released by fucoidan elution was identified as Bge surface membrane proteins, representing putative fucosyl-binding proteins. Far-western blot analysis also demonstrated binding reactivity between Bge cell and sporocyst tegumental proteins. The finding that several of these parasite-binding Bge cell proteins were also fucoidan-reactive suggests the possible involvement of these molecules in mediating cellular interactions with sporocyst tegumental carbohydrates. It is concluded that Bge cells have surface protein(s) that may be playing a role in facilitating host cell adhesion to the surface of schistosome primary sporocysts through larval fucosylated

  5. Primary structure and androgen regulation of a 20-kilodalton protein specific to rat ventral prostate.

    PubMed

    Ho, K C; Snoek, R; Quarmby, V; Viskochil, D H; Rennie, P S; Wilson, E M; French, F S; Bruchovsky, N

    1989-07-25

    Nuclear and cytosolic forms of a 20-kdalton rat ventral prostate protein were purified and partially sequenced from their N-termini. Isolated nuclei were treated with micrococcal nuclease and extracted in 0.6 M NaCl, and proteins were separated by affinity chromatography on Matrex gel green A, ammonium sulfate fractionation, and fast protein liquid chromatography on Superose 12. The 43 amino acid N-terminal sequence of the nuclear 20-kdalton protein was identical with the cytosolic protein except it lacked 7 N-terminal amino acids present in the cytosolic form. The DNA sequence of a full-length complementary DNA clone isolated from a ventral prostate gt11 library extended the N-terminal sequence of the cytosolic form by an additional nine amino acids from the predicted initiation methionine. The cDNA included the nucleotide sequence for the 43 amino acid N-terminal sequence of the purified 20-kdalton protein and predicted molecular weights of 16,686, 17,521, and 18,650, respectively, for the nuclear, cytoplasmic, and nonprocessed proteins. Northern blot analyses of reproductive tract tissue RNAs using the 20-kdalton protein cDNA as probe revealed a single mRNA species of 0.92 kb detectable only in extracts of rat ventral prostate. Expression of the 0.92-kb mRNA was androgen dependent since the mRNA was undetectable in extracts obtained 4 days after castration and was restored 16 h after restimulation with androgen.

  6. Electrophoretic pattern and distribution of cytoskeletal proteins in flat-epitheloid and stellate process-bearing astrocytes in primary culture.

    PubMed

    Ciesielski-Treska, J; Ulrich, G; Mensch, C; Aunis, D

    1984-01-01

    One- and two-dimensional electrophoresis patterns and distribution of major cytoskeletal proteins were studied in primary astrocytes with either flat-epitheloid or stellate appearance. No major differences in the electrophoretic patterns of actin, tubulin, glial fibrillary acidic protein (GFAP) and vimentin were detected between flat-epitheloid and stellate process-bearing astrocytes produced by the exposure of cultures to dibutyryl cyclic AMP (dBcAMP). However the morphological changes of astrocytes were accompanied by marked changes in the quantitative distribution of cytoskeletal proteins. The most prominent change was a large and specific decrease in the amount of actin, detected by [(35)S]methionine incorporation, densitometric scanning of one-dimensional gels and DNase inhibition assay. In stellate astrocytes produced by a 4 day treatment with dibutyryl cyclic AMP, the amount of actin decreased by 50%. This decrease was not apparently related to the depolymerization of actin.

  7. Effects of aluminum on the neurotoxicity of primary cultured neurons and on the aggregation of beta-amyloid protein.

    PubMed

    Kawahara, M; Kato, M; Kuroda, Y

    2001-05-15

    Recent epidemiological, neuropathological, and biochemical studies have suggested a possible link between the neurotoxicity of aluminum and the pathogenesis of Alzheimer's disease. However, this relationship remains controversial. To investigate detailed characteristics of neurotoxicity of aluminum, we used primary cultured neurons of rat cerebral cortex as an in vitro model system for the observation of morphological changes induced by chronic exposure to aluminum. Although the exposure to aluminum chloride (10-100 microM) for 1 week did not cause marked neuronal death, degeneration of neuritic processes and accumulation of tau protein and beta-amyloid protein appeared after chronic exposure to 50 microM aluminum chloride for more than 3 weeks. We also investigated the polymerization of beta-amyloid protein in vitro using the immunoblotting technique. We thus found that aluminum induced conformational changes in beta-amyloid protein and enhanced its aggregation in vitro. The aggregated beta-amyloid protein was dissolved by the addition of desferrioxamine, a chelator of aluminum. The aggregated beta-amyloid protein pre-incubated with aluminum formed fibrillar deposits on the surface of cultured neurons.

  8. The family of light-harvesting-related proteins (LHCs, ELIPs, HLIPs): was the harvesting of light their primary function?

    PubMed

    Montané, M H; Kloppstech, K

    2000-11-27

    Light-harvesting complex proteins (LHCs) and early light-induced proteins (ELIPs) are essential pigment-binding components of the thylakoid membrane and are encoded by one of the largest and most complex higher plant gene families. The functional diversification of these proteins corresponded to the transition from extrinsic (phycobilisome-based) to intrinsic (LHC-based) light-harvesting antenna systems during the evolution of chloroplasts from cyanobacteria, yet the functional basis of this diversification has been elusive. Here, we propose that the original function of LHCs and ELIPs was not to collect light and to transfer its energy content to the reaction centers but to disperse the absorbed energy of light in the form of heat or fluorescence. These energy-dispersing proteins are believed to have originated in cyanobacteria as one-helix, highly light-inducible proteins (HLIPs) that later acquired four helices through two successive gene duplication steps. We suggest that the ELIPs arose first in this succession, with a primary function in energy dispersion for protection of photosynthetic pigments from photo-oxidation. We consider the LHC I and II families as more recent and very successful evolutionary additions to this family that ultimately attained a new function, thereby replacing the ancestral extrinsic light-harvesting system. Our model accounts for the non-photochemical quenching role recently shown for higher plant psbS proteins.

  9. The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation.

    PubMed

    Yin, Yili; Bangs, Fiona; Paton, I Robert; Prescott, Alan; James, John; Davey, Megan G; Whitley, Paul; Genikhovich, Grigory; Technau, Ulrich; Burt, David W; Tickle, Cheryll

    2009-02-01

    The chicken talpid(3) mutant, with polydactyly and defects in other embryonic regions that depend on hedgehog (Hh) signalling (e.g. the neural tube), has a mutation in KIAA0568. Similar phenotypes are seen in mice and in human syndromes with mutations in genes that encode centrosomal or intraflagella transport proteins. Such mutations lead to defects in primary cilia, sites where Hh signalling occurs. Here, we show that cells of talpid(3) mutant embryos lack primary cilia and that primary cilia can be rescued with constructs encoding Talpid3. talpid(3) mutant embryos also develop polycystic kidneys, consistent with widespread failure of ciliogenesis. Ultrastructural studies of talpid(3) mutant neural tube show that basal bodies mature but fail to dock with the apical cell membrane, are misorientated and almost completely lack ciliary axonemes. We also detected marked changes in actin organisation in talpid(3) mutant cells, which may explain misorientation of basal bodies. KIAA0586 was identified in the human centrosomal proteome and, using an antibody against chicken Talpid3, we detected Talpid3 in the centrosome of wild-type chicken cells but not in mutant cells. Cloning and bioinformatic analysis of the Talpid3 homolog from the sea anemone Nematostella vectensis identified a highly conserved region in the Talpid3 protein, including a predicted coiled-coil domain. We show that this region is required to rescue primary cilia formation and neural tube patterning in talpid(3) mutant embryos, and is sufficient for centrosomal localisation. Thus, Talpid3 is one of a growing number of centrosomal proteins that affect both ciliogenesis and Hh signalling.

  10. Primary evaluation of methenamine as a NPN compound with probable effects on increasing ruminal escaped protein.

    PubMed

    Mohebbi-Fani, M; Shekarforoosh, S; Maleki, M; Vahedi, N

    2002-06-01

    The effects of methenamine as a non-protein nitrogenous compound on protein and health status of feedlot lambs were studied in three groups of lambs receiving 5, 10, or 15 g of the substance daily in their feed for 100 days. The results were compared with data obtained from a control group receiving a diet low in crude protein without methenamine. Serum total protein, serum urea nitrogen and serum creatinine were measured every 10 days as indicators of protein metabolism and kidney function. Urine samples were also examined on the same days for possible side-effects of methenamine on the urinary tract. Following slaughter, various internal organs, including the brain and various parts of the gastrointestinal and urinary tracts, were examined both grossly and microscopically to detect any lesions. All groups receiving methenamine had serum total protein and serum urea nitrogen levels higher than those in the control group. The serum creatinine level was normal in all the groups throughout the experiment. No gross or microscopic lesion attributed to the toxic effects of methenamine was detected in any of the internal organs. Therefore, it is concluded that methenamine can be used as a non-protein nitrogenous compound without serious side-effects.

  11. The primary structure of papaya mosaic virus coat protein: a revision.

    PubMed

    Verde, C; Malorni, A; Parente, A

    1989-12-01

    The presence of an acetyl blocking group at the N-terminus of the coat protein of papaya mosaic virus has been identified by FAB mass spectrometry. Furthermore, we have found that the N-terminal sequence of the protein is four amino-acid residues (AC-Ser-Lys-Ser-Ser-) longer than that previously reported, while Glu instead of Gln is the C-terminal residue. The present paper shows that PMV-protein is made up of 215 amino acid residues, with a molecular mass of 22,960 Da.

  12. Hydrogen peroxide-mediated degradation of protein: different oxidation modes of copper- and iron-dependent hydroxyl radicals on the degradation of albumin.

    PubMed

    Kocha, T; Yamaguchi, M; Ohtaki, H; Fukuda, T; Aoyagi, T

    1997-02-08

    Cupric ions (Cu2+) added to hydrogen peroxide (H2O2) were found to generate hydroxyl radicals (HO) capable of benzoate hydroxylation. Although ferrous (Fe2+) and ferric (Fe3+) ions, when added to H2O2, resulted in very little production of HO, the addition of EDTA to the reaction mixture markedly increased their catalytic activity. In the absence of albumin, catalase (a H2O2 scavenger) and mannitol (an HO radical scavenger) effectively inhibited the formation of HO in H2O2/Cu2+ and H2O2/Fe2+/EDTA oxidation systems. On analysis using SDS-polyacrylamide gel electrophoresis, catalase was shown to prevent the degradation of albumin by both oxidation systems, whereas mannitol was an effective scavenger of the H2O2/Fe2+/EDTA oxidation system but not of the H2O2/Cu2+ oxidation system. Furthermore, the effect of alteration of benzoate hydroxylation and H2O2 consumption on the H2O2/Cu2+ and H2O2/Fe2+/EDTA oxidation systems resulted in opposite behavior that was dependent upon the presence or absence of albumin. These observations suggest that copper ions bind to albumin and induce site-specific degradation by HO generated at the copper-binding site, whereas the Fe2+/EDTA-catalyzed oxidation system induces non-specific degradation of albumin by HO generated by the Fenton reaction between H2O2 and free Fe2+/EDTA in solution.

  13. Protein kinase Cμ mediates adenosine-stimulated steroidogenesis in primary rat adrenal cells.

    PubMed

    Chen, Yung-Chia; Chen, Ying; Huang, Shih-Horng; Wang, Seu-Mei

    2010-11-05

    Adenosine (Ado), an endogenous nucleoside, can stimulate corticosterone synthesis in adrenal cells via the A(2A)/A(2B) adenosine receptors (ARs). This study evaluated the contribution of protein kinase C (PKC) isoforms in Ado-induced steroidogenesis. The PKC inhibitor calphostin c blocked Ado-induced steroidogenesis, the mitogen-activated protein kinase (MEK)-extracellular signal-related regulated kinase (ERK)-cyclic AMP responsive element-binding protein cascade, and the mRNA expression of steroidogenic acute regulatory protein and CYP11B1. Further analyses revealed that PKCμ was indeed activated by Ado. Moreover, downregulation of PKCμ by small interfering RNA (siRNA) inhibited Ado-stimulated steroidogenesis and ERK phosphorylation. Finally, inhibition of either A(2A)AR or A(2B)AR led to the suppression of PKCμ phosphorylation. Together, these findings suggest that A(2)AR-PKCμ-MEK signaling mediates Ado-stimulated adrenal steroidogenesis.

  14. Purification and primary structure of novel lipid transfer proteins from germinated lentil (Lens culinaris) seeds.

    PubMed

    Finkina, E I; Balandin, S V; Serebryakova, M V; Potapenko, N A; Tagaev, A A; Ovchinnikova, T V

    2007-04-01

    A subfamily of eight novel lipid transfer proteins designated as Lc-LTP1-8 was found in the lentil Lens culinaris. Lc-LTP2, Lc-LTP4, Lc-LTP7, and Lc-LTP8 were purified from germinated lentil seeds, and their molecular masses (9268.7, 9282.7, 9121.5, 9135.5 daltons) and complete amino acid sequences were determined. The purified proteins consist of 92-93 amino acid residues, have four disulfide bonds, and inhibit growth of Agrobacterium tumefaciens. Total RNA was isolated from germinated lentil seeds, RT-PCR and cloning were performed, and the cDNAs of six LTPs were sequenced. Precursor 116-118-residue proteins with 24-25-residue signal peptides were found, and two of them are purified proteins Lc-LTP2 and Lc-LTP4.

  15. Rapid Proteasomal Degradation of Mutant Proteins Is the Primary Mechanism Leading to Tumorigenesis in Patients With Missense AIP Mutations

    PubMed Central

    Hernández-Ramírez, Laura C.; Martucci, Federico; Morgan, Rhodri M. L.; Trivellin, Giampaolo; Tilley, Daniel; Ramos-Guajardo, Nancy; Iacovazzo, Donato; D'Acquisto, Fulvio; Prodromou, Chrisostomos

    2016-01-01

    Context: The pathogenic effect of mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts. Objective: This study sought to analyze the mechanism/speed of protein turnover of wild-type and missense AIP variants, correlating protein half-life with clinical parameters. Design and Setting: Half-life and protein–protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed in a clinical academic research institution. Patients: Data were obtained from our cohort of pituitary adenoma patients and literature-reported cases. Interventions: Protein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. Glutathione-S-transferase pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by coimmunoprecipitation and gene knockdown. Relevant clinical data was collected. Main Outcome Measures: Half-life of wild-type and mutant AIP proteins and its correlation with clinical parameters. Results: Endogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7 h). AIP variants were divided into stable proteins (median, 77.7 h; interquartile range [IQR], 60.7–92.9 h), and those with short (median, 27 h; IQR, 21.6–28.7 h) or very short (median, 7.7 h; IQR, 5.6–10.5 h) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r = 0.411; P = .002). The FBXO3-containing SKP1–CUL1–F-box protein complex was identified as the E3 ubiquitin-ligase recognizing AIP. Conclusions: AIP is a stable protein, driven to ubiquitination by the SKP1–CUL1–F-box protein complex

  16. Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain.

    PubMed

    Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim; Thomsen, Louiza Bohn; Moos, Torben

    2017-03-14

    Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.

  17. Effects of phytoestrogens on protein turnover in rainbow trout primary myocytes

    USDA-ARS?s Scientific Manuscript database

    Soybean-derived ingredients used in aquaculture feeds may contain phytoestrogens, but it is unknown if these compounds can mimic the catabolic effects of estradiol in fish muscle. Six day-old rainbow trout primary myocytes were exposed to increasing concentrations (10 nM – 100 µM) of either geniste...

  18. An Exercise on the Determination of the Primary Structure of Proteins

    ERIC Educational Resources Information Center

    Leader, David P.

    1976-01-01

    Describes an exercise that extends the actual determination of the amino acid sequence of a simple dipeptide to the theoretical determination of the complete primary structure of a larger polypeptide. Includes diagrams of display cards and duplicated sheets used in the theoretical portion of the exercise. (CS)

  19. An Exercise on the Determination of the Primary Structure of Proteins

    ERIC Educational Resources Information Center

    Leader, David P.

    1976-01-01

    Describes an exercise that extends the actual determination of the amino acid sequence of a simple dipeptide to the theoretical determination of the complete primary structure of a larger polypeptide. Includes diagrams of display cards and duplicated sheets used in the theoretical portion of the exercise. (CS)

  20. Prostaglandin E synthase interacts with inducible heat shock protein 70 after heat stress in bovine primary dermal fibroblast cells.

    PubMed

    Richter, Constanze; Viergutz, Torsten; Schwerin, Manfred; Weitzel, Joachim M

    2015-01-01

    Exposure to heat stress in dairy cows leads to undesired side effects that are reflected by complex alterations in endocrine parameters, such as reduced progesterone, estradiol, and thyroid hormone concentrations. These endocrine maladaptation leads to failure to resume cyclicity, a poor uterine environment and inappropriate immune responses in postpartum dairy cows. Prostaglandins (PG's) are lipid mediators, which serve as signal molecules in response to various external stimuli as well as to cell-specific internal signal molecules. A central role in PG synthesis plays prostaglandin E synthase (PGES) that catalyzes the isomerization of PGH2 to PGE2 .The present study was conducted to investigate heat stress associated PGES expression. Expression of PGES and inducible heat shock protein 70 (HSP70), as a putative chaperonic protein, was studied in bovine primary fibroblasts under different heat shock conditions. Bovine primary fibroblasts produce PGE2 at homoiothermical norm temperature (38.5°C in bovine), but reduce PGE2 production rates under extreme heat stress (at 45°C for 6 h). By contrast, PGE2 production rates are maintained after a milder heat stress (at 41.5°C for 6 h). PGE2 synthesis is abolished by application of cyclooxygenase inhibitor indomethacin, indicating de novo synthesis. Heat stress increases HSP70 but not PGES protein concentrations. HSP70 physically interacts with PGES and the PGES-HSP70 complex did not dissociate upon heat stress at 45°C even after returning the cells to 37°C. The PGE2 production negatively correlates with the portion of PGES-HSP70 complex. These results suggest a protein interaction between HSP70 and PGES in dermal fibroblast cells. Blockage of PGES protein by HSP70 seems to interfere with the regulatory processes essential for cellular adaptive protection. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

  1. Synthesis and characterization of a 'fluorous' (fluorinated alkyl) affinity reagent that labels primary amine groups in proteins/peptides.

    PubMed

    Qian, Jiang; Cole, Richard B; Cai, Yang

    2011-01-01

    Strong non-covalent interactions such as biotin-avidin affinity play critical roles in protein/peptide purification. A new type of 'fluorous' (fluorinated alkyl) affinity approach has gained popularity due especially to its low level of non-specific binding to proteins/peptides. We have developed a novel water-soluble fluorous labeling reagent that is reactive (via an active sulfo-N-hydroxylsuccinimidyl ester group) to primary amine groups in proteins/peptides. After fluorous affinity purification, the bulky fluorous tag moiety and the long oligoethylene glycol (OEG) spacer of this labeling reagent can be trimmed via the cleavage of an acid labile linker. Upon collision-induced dissociation, the labeled peptide ion yields a characteristic fragment that can be retrieved from the residual portion of the fluorous affinity tag, and this fragment ion can serve as a marker to indicate that the relevant peptide has been successfully labeled. As a proof of principle, the newly synthesized fluorous labeling reagent was evaluated for peptide/protein labeling ability in phosphate-buffered saline (PBS). Results show that both the aqueous environment protein/peptide labeling and the affinity enrichment/separation process were highly efficient.

  2. Mutations in g protein encoding genes and chromosomal alterations in primary leptomeningeal melanocytic neoplasms.

    PubMed

    Küsters-Vandevelde, Heidi V N; van Engen-van Grunsven, Ilse A C H; Coupland, Sarah E; Lake, Sarah L; Rijntjes, Jos; Pfundt, Rolph; Küsters, Benno; Wesseling, Pieter; Blokx, Willeke A M; Groenen, Patricia J T A

    2015-04-01

    Limited data is available on the genetic features of primary leptomeningeal melanocytic neoplasms (LMNs). Similarities with uveal melanoma were recently suggested as both entities harbor oncogenic mutations in GNAQ and GNA11. Whether primary LMNs share additional genetic alterations with uveal melanoma including copy number variations is unknown. Twenty primary LMNs ranging from benign and intermediate-grade melanocytomas to melanomas were tested by direct sequencing for hotspot mutations in the genes GNA11, GNAQ, BRAF, NRAS and HRAS. Furthermore, the lesions were tested for copy number variations of chromosomes frequently present in uveal melanoma (1p, 3, 6 and 8q) by multiplex ligation-dependent probe amplification (MLPA). Genome-wide analyses of copy number alterations of two leptomeningeal melanocytic neoplasms were performed using the OncoScan SNP-array. GNAQ(Q209) mutations were present in eleven LMNs, while two of 20 cases carried a GNA11(Q209) mutation. No BRAF, HRAS or NRAS hotspot mutations were detected. Monosomy 3 and gain of 8q were present in one leptomeningeal melanoma, and one intermediate-grade melanocytoma harbored a gain of chromosome 6. With MLPA, the melanocytomas did not show any further gross chromosomal variations. Our data shows that primary LMNs, like uveal melanoma, harbor oncogenic mutations in GNAQ and GNA11 but lack mutations in BRAF, NRAS and HRAS. This finding may help in the differential diagnosis between a primary LMN and a metastasis from a cutaneous melanoma to the central nervous system. Copy number variations in some aggressive LMNs resemble those present in uveal melanoma but their prognostic significance is unclear.

  3. Primary structure of a human arginine-rich nuclear protein that colocalizes with spliceosome components

    SciTech Connect

    Chaudhary, N.; McMahon, C.; Blobel, G. )

    1991-09-15

    The cDNA for a 54-kDa nuclear protein (p54) has been cloned from a human hepatoma expression library. Contained within p54 is an arginine/serine-rich region similar to segments of several proteins that participate in pre-mRNA splicing including the 70-kDa component of U1 small nuclear ribonucleoprotein particle (snRNP) and the Drosophila transformer and suppressor-of-white-apricot proteins. The arginine/serine-rich region is dominated by a series of 8-amino acid imperfect repetitive motifs (consensus sequence, Arg-Arg-Ser-Arg-Ser-Arg-Ser-Arg). Antibodies raised against synthetic peptides of p54 react with an {approximately}70-kDa protein on immunoblots of HeLa cell and rat liver nuclear proteins. This apparent discrepancy in mass is also observed when p54 mRNA is translated in vitro. Indirect immunofluorescence studies in HeLa cells show that p54 is distributed throughout the nucleus in a speckled pattern, with an additional diffuse labeling of the nucleus excluding the nucleoli. Double immunofluorescence experiments indicate that these punctate regions are coincident with the speckles seen in cells stained with antibodies against several constituents of the pre-mRNA splicing machinery. Sedimentation analysis of HeLa cell extracts on sucrose gradients showed that p54 migrates at 4-6 S, indicating that the protein is not a tightly associated component of snRNPs. Although the function of p54 is not yet known, the structure and immunolocalization data suggest that this protein may have a role in pre-mRNA processing.

  4. Primary structure of streptococcal Pep M5 protein: Absence of extensive sequence repeats

    PubMed Central

    Manjula, Belur N.; Mische, Sheenah M.; Fischetti, Vincent A.

    1983-01-01

    Extensive sequence repeats have been observed in a biologically active fragment of type 24 streptococcal M protein, namely Pep M24 [Beachey, E. H., Sayer, J. M. & Kang, A. H. (1978) Proc. Natl. Acad. Sci. USA 75, 3163-3167]. To determine whether such extensive repetition in sequence is a common characteristic of the antiphagocytic streptococcal M proteins, we have determined the sequences of the clostripain peptides of Pep M5, a biologically active fragment of the type 5 M protein that is analogous to Pep M24. These sequences, together with the amino-terminal sequence of the whole molecule, accounted for nearly two thirds of the Pep M5 molecule. However, extensive identical repeats of the kind observed in Pep M24 were not present in Pep M5. Preliminary study of the amino acid sequence analysis of the M protein from type 6 Streptococcus has also indicated the absence of sequence repeats within the regions of this molecule examined so far. These results suggest that extensive sequence repeats may not be a common characteristic of M-protein molecules. On the other hand, the seven-residue periodicity of the nonpolar residues, a characteristic of α-helical coiled-coil structures, appeared to extend over most of the Pep M5 molecule. This feature has been observed previously for the partial sequences of three M protein serotypes. Thus, the important element of the M-protein structure appears to be the seven-residue periodicity necessary for the maintenance of the coiled-coil structure rather than extensive identical amino acid sequence repeats. PMID:16593365

  5. In recurrent primary biliary cirrhosis after liver transplantation, biliary epithelial cells show increased expression of mitochondrial proteins.

    PubMed

    Sasaki, Motoko; Hsu, Maylee; Yeh, Matthew M; Nakanuma, Yasuni

    2015-10-01

    In biliary epithelial lesions in primary biliary cirrhosis (PBC), mitochondrial proteins associated with deregulated autophagy are abnormally expressed. We examined whether this could be used as a diagnostic marker for end-stage PBC and recurrent PBC after liver transplantation. We examined the expression of the mitochondrial protein pyruvate dehydrogenase complex-E2 component and cytochrome c oxidase, subunit I (CCO), the autophagy-related marker microtubule-associated protein-light chain 3 (LC3), and p62/sequestosome-1 and the senescence markers p16(Ink4a) and p21(WAF1/Cip1) in small bile ducts and bile ductules in explanted livers from patients with PBC (n = 20) in comparison with liver tissue from control patients (n = 21) and post-transplant samples including recurrent PBC and cellular rejection (n = 28). Intense granular expression of mitochondrial proteins was significantly more frequent in small bile ducts in explanted livers with PBC than in control livers (p < 0.05). Post-transplant samples comprised of three groups: group A (positive for mitochondrial proteins, n = 7), group B (positive for either autophagy-related or senescence markers but negative for mitochondrial proteins, n = 7), and group C (all negative, n = 14). All but one case of group A were clinically and histologically diagnosed as recurrent PBC. In contrast, all cases of group B were diagnosed as cellular rejection. This study suggests that the expression of mitochondrial proteins in small bile ducts may be a useful diagnostic marker for end-stage PBC and recurrent PBC after liver transplantation.

  6. Primary Role of the Chromophore Bond Length Alternation in Reversible Photoconversion of Red Fluorescence Proteins

    PubMed Central

    Drobizhev, Mikhail; Hughes, Thomas E.; Stepanenko, Yuriy; Wnuk, Pawel; O'Donnell, Kieran; Scott, J. Nathan; Callis, Patrik R.; Mikhaylov, Alexander; Dokken, Leslie; Rebane, Aleksander

    2012-01-01

    Rapid photobleaching of fluorescent proteins can limit their use in imaging applications. The underlying kinetics is multi-exponential and strongly depends on the local chromophore environment. The first, reversible, step may be attributed to a rotation around one of the two exocyclic C-C bonds bridging phenol and imidazolinone groups in the chromophore. However it is not clear how the protein environment controls this motion - either by steric hindrances or by modulating the electronic structure of the chromophore through electrostatic interactions. Here we study the first step of the photobleaching kinetics in 13 red fluorescent proteins (RFPs) with different chromophore environment and show that the associated rate strongly correlates with the bond length alternation (BLA) of the two bridge bonds. The sign of the BLA appears to determine which rotation is activated. Our results present experimental evidence for the dominance of electronic effects in the conformational dynamics of the RFP chromophore. PMID:23008753

  7. FAST EVENTS IN PROTEIN FOLDING: The Time Evolution of Primary Processes

    NASA Astrophysics Data System (ADS)

    Callender, Robert H.; Dyer, R. Brian; Gilmanshin, Rudolf; Woodruff, William H.

    1998-10-01

    Most experimental studies on the dynamics of protein folding have been confined to timescales of 1 ms and longer. Yet it is obvious that many phenomena that are obligatory elements of the folding process occur on much faster timescales. For example, it is also now clear that the formation of secondary and tertiary structures can occur on nanosecond and microsecond times, respectively. Although fast events are essential to, and sometimes dominate, the overall folding process, with a few exceptions their experimental study has become possible only recently with the development of appropriate techniques. This review discusses new approaches that are capable of initiating and monitoring the fast events in protein folding with temporal resolution down to picoseconds. The first important results from those techniques, which have been obtained for the folding of some globular proteins and polypeptide models, are also discussed.

  8. Expression of cell polarity protein scribble differently affects prognosis in primary tumor and lymph node metastasis of breast cancer patients.

    PubMed

    Sakakibara, Junta; Sakakibara, Masahiro; Shiina, Nobumitsu; Fujimori, Toshihiko; Okubo, Yoshiyuki; Fujisaki, Kaoru; Nagashima, Takeshi; Sangai, Takafumi; Nakatani, Yukio; Miyazaki, Masaru

    2017-05-01

    We evaluated the relationship between the immunohistochemically determined expression of the cell polarity protein scribble to prognosis in different environments of estrogen receptor (ER) expression and epithelial-to-mesenchymal transition (EMT). We immunohistochemically evaluated the expression level of scribble in primary tumors and lymph node metastases of 225 node-positive breast cancer patients who had received chemotherapy. We then evaluated metastasis-free survival (MFS) in the absence or presence of ER and the EMT-related protein vimentin. Among patients with ER-positive tumors, patients with low scribble expression in the primary tumor had a significantly shorter MFS than patients with high scribble expression (p = 0.0225). Furthermore, among patients with vimentin-negative tumors, patients with low expression of scribble in the primary tumor had significantly shorter MFS than patients with high expression of scribble (p = 0.0463). In contrast, among patients with vimentin-positive tumors, patients with high expression of scribble in the primary tumor had significantly shorter MFS than patients with low expression of scribble (p = 0.0343). Moreover, among patients with ER-negative tumors, patients with high expression of scribble in lymph node metastases showed significantly higher expression of E-cadherin in metastases (p = 0.0407) and had significantly shorter MFS than patients with low expression of scribble (p = 0.0064). The prognostic significance of cell polarity depended on the ER expression and EMT. Furthermore, the preservation of cell polarity in metastases was associated with mesenchymal-to-epithelial transition and worse prognosis. Cell polarity promotes the diversity of metastasis in combination with malignancy grade in breast cancer patients.

  9. Subpicosecond protein backbone changes detected during the green-absorbing proteorhodopsin primary photoreaction.

    PubMed

    Amsden, Jason J; Kralj, Joel M; Chieffo, Logan R; Wang, Xihua; Erramilli, Shyamsunder; Spudich, Elena N; Spudich, John L; Ziegler, Lawrence D; Rothschild, Kenneth J

    2007-10-11

    Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with approximately 200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm(-1)) and ethylenic stretching (1500-1570 cm(-1)) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm(-1) with a time constant of 500-700 fs, which on the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm(-1) concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport.

  10. An Ensemble Method for Predicting Subnuclear Localizations from Primary Protein Structures

    PubMed Central

    Han, Guo Sheng; Yu, Zu Guo; Anh, Vo; Krishnajith, Anaththa P. D.; Tian, Yu-Chu

    2013-01-01

    Background Predicting protein subnuclear localization is a challenging problem. Some previous works based on non-sequence information including Gene Ontology annotations and kernel fusion have respective limitations. The aim of this work is twofold: one is to propose a novel individual feature extraction method; another is to develop an ensemble method to improve prediction performance using comprehensive information represented in the form of high dimensional feature vector obtained by 11 feature extraction methods. Methodology/Principal Findings A novel two-stage multiclass support vector machine is proposed to predict protein subnuclear localizations. It only considers those feature extraction methods based on amino acid classifications and physicochemical properties. In order to speed up our system, an automatic search method for the kernel parameter is used. The prediction performance of our method is evaluated on four datasets: Lei dataset, multi-localization dataset, SNL9 dataset and a new independent dataset. The overall accuracy of prediction for 6 localizations on Lei dataset is 75.2% and that for 9 localizations on SNL9 dataset is 72.1% in the leave-one-out cross validation, 71.7% for the multi-localization dataset and 69.8% for the new independent dataset, respectively. Comparisons with those existing methods show that our method performs better for both single-localization and multi-localization proteins and achieves more balanced sensitivities and specificities on large-size and small-size subcellular localizations. The overall accuracy improvements are 4.0% and 4.7% for single-localization proteins and 6.5% for multi-localization proteins. The reliability and stability of our classification model are further confirmed by permutation analysis. Conclusions It can be concluded that our method is effective and valuable for predicting protein subnuclear localizations. A web server has been designed to implement the proposed method. It is freely available

  11. Chemical magnetoreception in birds: The radical pair mechanism

    PubMed Central

    Rodgers, Christopher T.; Hore, P. J.

    2009-01-01

    Migratory birds travel vast distances each year, finding their way by various means, including a remarkable ability to perceive the Earth's magnetic field. Although it has been known for 40 years that birds possess a magnetic compass, avian magnetoreception is poorly understood at all levels from the primary biophysical detection events, signal transduction pathways and neurophysiology, to the processing of information in the brain. It has been proposed that the primary detector is a specialized ocular photoreceptor that plays host to magnetically sensitive photochemical reactions having radical pairs as fleeting intermediates. Here, we present a physical chemist's perspective on the “radical pair mechanism” of compass magnetoreception in birds. We outline the essential chemical requirements for detecting the direction of an Earth-strength ≈50 μT magnetic field and comment on the likelihood that these might be satisfied in a biologically plausible receptor. Our survey concludes with a discussion of cryptochrome, the photoactive protein that has been put forward as the magnetoreceptor molecule. PMID:19129499

  12. Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids.

    PubMed

    DiMarco, Rebecca L; Dewi, Ruby E; Bernal, Gabriela; Kuo, Calvin; Heilshorn, Sarah C

    2015-10-15

    Though in vitro culture of primary intestinal organoids has gained significant momentum in recent years, little has been done to investigate the impact of microenvironmental cues provided by the encapsulating matrix on the growth and development of these fragile cultures. In this work, the impact of various in vitro culture parameters on primary adult murine organoid formation and growth are analyzed with a focus on matrix properties and geometric culture configuration. The air-liquid interface culture configuration was found to result in enhanced organoid formation relative to a traditional submerged configuration. Additionally, through use of a recombinantly engineered extracellular matrix (eECM), the effects of biochemical and biomechanical cues were independently studied. Decreasing mechanical stiffness and increasing cell adhesivity were found to increase organoid yield. Tuning of eECM properties was used to obtain organoid formation efficiency values identical to those observed in naturally harvested collagen I matrices but within a stiffer construct with improved ease of physical manipulation. Increased ability to remodel the surrounding matrix through mechanical or enzymatic means was also shown to enhance organoid formation. As the engineering and tunability of recombinant matrices is essentially limitless, continued property optimization may result in further improved matrix performance and may help to identify additional microenvironmental cues that directly impact organoid formation, development, differentiation, and functional behavior. Continued culture of primary organoids in recombinant matrices could therefore prove to be largely advantageous in the field of intestinal tissue engineering for applications in regenerative medicine and in vitro tissue mimics.

  13. Glycyl radical activating enzymes: structure, mechanism, and substrate interactions.

    PubMed

    Shisler, Krista A; Broderick, Joan B

    2014-03-15

    The glycyl radical enzyme activating enzymes (GRE-AEs) are a group of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily and utilize a [4Fe-4S] cluster and SAM to catalyze H-atom abstraction from their substrate proteins. GRE-AEs activate homodimeric proteins known as glycyl radical enzymes (GREs) through the production of a glycyl radical. After activation, these GREs catalyze diverse reactions through the production of their own substrate radicals. The GRE-AE pyruvate formate lyase activating enzyme (PFL-AE) is extensively characterized and has provided insights into the active site structure of radical SAM enzymes including GRE-AEs, illustrating the nature of the interactions with their corresponding substrate GREs and external electron donors. This review will highlight research on PFL-AE and will also discuss a few GREs and their respective activating enzymes.

  14. Hydroxyl radical detection in vivo

    SciTech Connect

    Chevion, M.; Floyd, R.A.

    1986-05-01

    Hydroxyl radicals have been implicated as the actual species responsible for the deleterious effects of active oxygen in biology. However, in most cases, its presence has only been inferred by circumstantial evidence. Using electrochemical detection coupled to HPLC separation technique the authors can identify and quantitate (at sub-picomole level) the hydroxylated products of 3 aromatic compounds (phenol, salicylate, and 2-deoxy-guanosine) as a direct measure of hydroxyl radical formation. Firstly, the authors showed that mixing ascorbate with copper ions (in the absence of presence of a protein) yields catechols, dihydroxybenzoic acids and 8-OH-deoxy-guanosine (8-OHdG). This approach has been used to study the formation of OH in vivo. Human granulocytes stimulated with TPA showed that 8-OHdG was formed in the cellular DNA at high levels (one 8-OHdG/800 DNA bases). Unstimulated granulocytes contained 8-OHdG below detection level. Formation of 8-OHdG in the TPA-stimulated granulocytes DNA was decreased by the addition of SOD and catalase. Using salicylate as an in vivo scavenger of hydroxyl radicals the authors showed that the level of trapped-dihydroxybenzoic acids is increased approx.8 and approx.3 fold in the lungs and liver of paraquat-poisoned mice, respectively, as compared to normal animals. Similarly, the detected level of dihydroxybenzoic acids in the hearts of adriamycin-treated rats was increased over 100-fold as compared to the hearts of control animals.

  15. Leucine and isoleucine reduce protein degradation in rainbow trout (Oncorhynchus mykiss) primary myoblast cultures

    USDA-ARS?s Scientific Manuscript database

    Myogenic precursor cells were isolated from rainbow trout skeletal muscle and incubated in media containing 10% fetal bovine serum for 7 days, thereby differentiating into myoblasts. Rates of protein degradation were determined in response to minimal essential media (MEM) of various amino acid (AA)...

  16. The Primary Role of Fibrinogen-Related Proteins in Invertebrates Is Defense, Not Coagulation

    PubMed Central

    Hanington, Patrick C.; Zhang, Si-Ming

    2010-01-01

    In vertebrates, the conversion of fibrinogen into fibrin is an essential process that underlies the establishment of the supporting protein framework required for coagulation. In invertebrates, fibrinogen-domain-containing proteins play a role in the defense response generated against pathogens; however, they do not function in coagulation, suggesting that this role has been recently acquired. Molecules containing fibrinogen motifs have been identified in numerous invertebrate organisms, and most of these molecules known to date have been linked to defense. Moreover, recent genome projects of invertebrate animals have revealed surprisingly high numbers of fibrinogen-like loci in their genomes, suggesting important and perhaps diverse functions of fibrinogen-like proteins in invertebrates. The ancestral role of molecules containing fibrinogen-related domains (FReDs) with immunity is the focus of this review, with emphasis on specific FReDs called fibrinogen-related proteins (FREPs) identified from the schistosome-transmitting mollusc Biomphalaria glabrata. Herein, we outline the range of invertebrate organisms FREPs can be found in, and detail the roles these molecules play in defense and protection against infection. PMID:21063081

  17. G-protein-coupled receptors and localized signaling in the primary cilium during ventral neural tube patterning.

    PubMed

    Hwang, Sun-Hee; Mukhopadhyay, Saikat

    2015-01-01

    The primary cilium is critical in sonic hedgehog (Shh)-dependent ventral patterning of the vertebrate neural tube. Most mutants that cause disruption of the cilium result in decreased Shh signaling in the neural tube. In contrast, mutations in the intraflagellar complex A (IFT-A) and the tubby family protein, Tulp3, result in increased Shh signaling in the neural tube. Proteomic analysis of Tulp3-binding proteins first pointed to the role of the IFT-A complex in trafficking Tulp3 into the cilia. Tulp3 directs trafficking of rhodopsin family G-protein-coupled receptors (GPCRs) to the cilia, suggesting the role of a GPCR in mediating the paradoxical effects of the Tulp3/IFT-A complex in causing increased Shh signaling. Gpr161 has recently been identified as a Tulp3/IFT-A-regulated GPCR that localizes to the primary cilium. A null knock-out mouse model of Gpr161 phenocopies Tulp3 and IFT-A mutants, and causes increased Shh signaling throughout the neural tube. In the absence of Shh, the bifunctional Gli transcription factors are proteolytically processed into repressor forms in a protein kinase A (PKA) -dependent and cilium-dependent manner. Gpr161 activity results in increased cAMP levels in a Gαs -coupled manner, and determines processing of Gli3. Shh signaling also results in removal of Gpr161 from the cilia, suggesting that Gpr161 functions in a positive feedback loop in the Shh pathway. As PKA-null and Gαs mutant embryos also exhibit increased Shh signaling in the neural tube, Gpr161 is a strong candidate for a GPCR that regulates ciliary cAMP levels, and activates PKA in close proximity to the cilia.

  18. Students' understanding of primary and secondary protein structure: drawing secondary protein structure reveals student understanding better than simple recognition of structures.

    PubMed

    Harle, Marissa; Towns, Marcy H

    2013-01-01

    The interdisciplinary nature of biochemistry courses requires students to use both chemistry and biology knowledge to understand biochemical concepts. Research that has focused on external representations in biochemistry has uncovered student difficulties in comprehending and interpreting external representations in addition to a fragmented understanding of fundamental biochemistry concepts. This project focuses on students' understanding of primary and secondary protein structure and drawings (representations) of hydrogen-bonding in alpha helices and beta sheets. Analysis demonstrated that students can recognize and identify primary protein structure concepts when given a polypeptide. However, when asked to draw alpha helices and beta sheets and explain the role of hydrogen bonding their drawings students exhibited a fragmented understanding that lacked coherence. Faculty are encouraged to have students draw molecular level representations to make their mental models more explicit, complete, and coherent. This is in contrast to recognition and identification tasks, which do not adequately probe mental models and molecular level understanding. © 2013 by The International Union of Biochemistry and Molecular Biology.

  19. Hydroxyl radical scavengers inhibit lymphocyte mitogenesis.

    PubMed Central

    Novogrodsky, A; Ravid, A; Rubin, A L; Stenzel, K H

    1982-01-01

    Agents that are known to be scavengers of hydroxyl radicals inhibit lymphocyte mitogenesis induced by phorbol myristate acetate (PMA) to a greater extent than they inhibit mitogenesis induced by concanavalin A or phytohemagglutinin. These agents include dimethyl sulfoxide, benzoate, thiourea, dimethylurea, tetramethylurea, L-tryptophan, mannitol, and several other alcohols. Their inhibitory effect is not associated with cytotoxicity. The hydroxyl radical scavengers do not inhibit PMA-dependent amino acid transport in T cells or PMA-induced superoxide production by monocytes. Thus, they do not inhibit the primary interaction of PMA with responding cells. Treatment of peripheral blood mononuclear cells with PMA increased cellular guanylate cyclase in most experiments, and dimethyl sulfoxide tended to inhibit this increase. In addition to inhibition of PMA-induced mitogenesis, hydroxyl radical scavengers markedly inhibited the activity of lymphocyte activating factor (interleukin 1). The differential inhibition of lymphocyte mitogenesis induced by different mitogens appears to be related to the differential macrophage requirements of the mitogens. The data suggest that hydroxyl radicals may be involved in mediating the triggering signal for lymphocyte activation. Some of the hydroxyl radical scavengers are inducers of cellular differentiation,. nd it is possible that their differentiating activity is related to their ability to scavenge free radicals. PMID:6122209

  20. Radical chemistry of artemisinin

    NASA Astrophysics Data System (ADS)

    Denisov, Evgenii T.; Solodova, S. L.; Denisova, Taisa G.

    2010-12-01

    The review summarizes physicochemical characteristics of the natural sesquiterpene peroxide artemisinin. The kinetic schemes of transformations of artemisinin radicals under anaerobic conditions are presented and analyzed. The sequence of radical reactions of artemisinin in the presence of oxygen is considered in detail. Special emphasis is given to the intramolecular chain oxidation resulting in the transformation of artemisinin into polyatomic hydroperoxide. The kinetic characteristics of elementary reaction steps involving alkyl, alkoxyl, and peroxyl radicals generated from artemisinin are discussed. The results of testing of artemisinin and its derivatives for the antimalarial activity and the scheme of the biochemical synthesis of artemisinin in nature are considered.

  1. Radical aminomethylation of imines.

    PubMed

    Fujii, Shintaro; Konishi, Takehito; Matsumoto, Yusuke; Yamaoka, Yousuke; Takasu, Kiyosei; Yamada, Ken-Ichi

    2014-09-05

    Taking advantage of the high level of performance of N-alkoxycarbonyl-imines, we achieved the first example of addition of the aminomethyl radical to imine. The reaction efficiency depended on the structure of the radical precursor, whether it is an iodide or a xanthate, and an electron-withdrawing group on the nitrogen atom of the radical. This reaction allows direct introduction of an N-substituted aminomethyl group onto imine to provide 1,2-diamine as well as the short-step synthesis of ICI-199,441.

  2. Reversed cellular polarity in primary cutaneous mucinous carcinoma: A study on tight junction protein expression in sweat gland tumors.

    PubMed

    Nagasawa, Yusuke; Ishida-Yamamoto, Akemi

    2017-04-01

    Primary cutaneous mucinous carcinoma (PCMC) is a rare sweat gland tumor characterized by the presence of abundant mucin around the tumor islands, but the molecular mechanisms for this structure are not well elucidated. Because mucin is epithelial in nature, it is likely to be produced by epithelial tumor cells, not by surrounding stromal cells. We hypothesized that the abundant mucin is a result of reversed cellular polarity of the tumor. To test this hypothesis, we conducted an immunohistological study to investigate expression of tight junction (TJ) proteins occludin and ZO-1 in PCMC, as well as in normal sweat glands and other sweat gland tumors. Dot-like or linear expression of TJ proteins was observed at ductal structures of sweat glands, and ductal or cystic structures of related tumors. In PCMC, however, TJ protein expression was clearly visible at the edges of tumor cell islands. This study provides evidence to show that the characteristic histological structure of PCMC is caused by inverse polarization of the tumor cells, and that TJ proteins are useful markers of ductal differentiation in sweat gland tumors.

  3. The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium

    PubMed Central

    Lier, Johanna Maria; Burmühl, Stephan; Struchtrup, Andreas; Deutschmann, Kathleen; Vetter, Maik; Leu, Tristan; Reeg, Sandra; Grune, Tilman; Rüther, Ulrich

    2015-01-01

    Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l−/− mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2. PMID:26150391

  4. Increased Mitochondrial Pro-oxidant Activity Mediates Up-regulation of Complex I S-glutathionylation via Protein Thiyl Radical in the Murine Heart of eNOS−/−

    PubMed Central

    Kang, Patrick T.; Chen, Chwen-Lih; Chen, Yeong-Renn

    2014-01-01

    In response to oxidative stress, mitochondrial Complex I is reversibly S-glutathionylated. We hypothesized that protein S-glutathionylation (PrSSG) of Complex I is mediated by a kinetic mechanism involving reactive protein thiyl radical (PrS•) and GSH in vivo. Previous studies have shown that in vitro S-glutathionylation of isolated Complex I at the 51 kDa and 75 kDa subunits was detected under the conditions of •O2− production, and mass spectrometry confirmed that formation of Complex I PrS• mediates PrSSG. Exposure of myocytes to menadione resulted in enhanced Complex I PrSSG and PrS• (Kang et al Free Radical Biol. Med. 2012; 52: 962–73). In this investigation, we tested our hypothesis in the murine heart of eNOS−/−. The eNOS−/− mouse is known to be hypertensive and develops the pathological phenotype of progressive cardiac hypertrophy. The mitochondria isolated from the eNOS−/− myocardium exhibited a marked dysfunction with impaired state 3 respiration, a declining respiratory control index, and decreasing enzymatic activities of ETC components. Further biochemical analysis and EPR measurement indicated defective aconitase activity, a marked increase in •O2− generation activity, and a more oxidized physiological setting. These results suggest increasing prooxidant activity and subsequent oxidative stress in the mitochondria of the eNOS−/− murine heart. When Complex I from the mitochondria of the eNOS−/− murine heart was analyzed by immuno-spin trapping and probed with anti-GSH antibody, both PrS• and PrSSG of Complex I were significantly enhanced. Overexpression of SOD2 in the murine heart dramatically diminished the detected PrS•, supporting the conclusion that mediation of Complex I PrSSG by oxidative stress-induced PrS• is a unique pathway for the redox regulation of mitochondrial function in vivo. PMID:25445401

  5. StAR-related lipid transfer domain protein 5 binds primary bile acids[S

    PubMed Central

    Létourneau, Danny; Lorin, Aurélien; Lefebvre, Andrée; Frappier, Vincent; Gaudreault, Francis; Najmanovich, Rafael; Lavigne, Pierre; LeHoux, Jean-Guy

    2012-01-01

    Steroidogenic acute regulatory-related lipid transfer (START) domain proteins are involved in the nonvesicular intracellular transport of lipids and sterols. The STARD1 (STARD1 and STARD3) and STARD4 subfamilies (STARD4–6) have an internal cavity large enough to accommodate sterols. To provide a deeper understanding on the structural biology of this domain, the binding of sterols to STARD5, a member of the STARD4 subfamily, was monitored. The SAR by NMR [1H-15N heteronuclear single-quantum coherence (HSQC)] approach, complemented by circular dichroism (CD) and isothermal titration calorimetry (ITC), was used. Titration of STARD5 with cholic (CA) and chenodeoxycholic acid (CDCA), ligands of the farnesoid X receptor (FXR), leads to drastic perturbation of the 1H-15N HSQC spectra and the identification of the residues in contact with those ligands. The most perturbed residues in presence of ligands are lining the internal cavity of the protein. Ka values of 1.8·10−4 M−1 and 6.3·104 M−1 were measured for CA and CDCA, respectively. This is the first report of a START domain protein in complex with a sterol ligand. Our original findings indicate that STARD5 may be involved in the transport of bile acids rather than cholesterol. PMID:23018617

  6. Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

    SciTech Connect

    Sudhakaran, P.R.; Stamatoglou, S.C.; Hughes, R.C.

    1986-12-01

    Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and section of ..cap alpha..-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as ..cap alpha..-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured.

  7. Partial primary structure of human pregnancy zone protein: extensive sequence homology with human alpha 2-macroglobulin.

    PubMed Central

    Sottrup-Jensen, L; Folkersen, J; Kristensen, T; Tack, B F

    1984-01-01

    Human pregnancy zone protein (PZP) is a major pregnancy-associated protein. Its quaternary structure (two covalently bound 180-kDa subunits, which are further non-covalently assembled into a tetramer of 720 kDa) is similar to that of human alpha 2-macroglobulin (alpha 2M). Here we show, from the results of complete or partial sequence determination of a random selection of 38 tryptic peptides covering 685 residues of the subunit of PZP, that PZP and alpha 2M indeed are extensively homologous. In the stretches of PZP sequenced so far, the degree of identically placed residues in the two proteins is 68%, indicating a close evolutionary relationship between PZP and alpha 2M. Although the function of PZP in pregnancy is largely unknown, its close structural relationship to alpha 2M suggests analogous proteinase binding properties and a potential for being taken up in cells by receptor-mediated endocytosis. In this regard our studies indicate a bait region in PZP significantly different from that present in alpha 2M. PZP could be the human equivalent of the acute-phase alpha-macroglobulins (e.g., rat alpha 2M and rabbit alpha 1M) described earlier. PMID:6209714

  8. Protein fucosylation regulates synapsin Ia/Ib expression and neuronal morphology in primary hippocampal neurons

    PubMed Central

    Murrey, Heather E.; Gama, Cristal I.; Kalovidouris, Stacey A.; Luo, Wen.-I.; Driggers, Edward M.; Porton, Barbara; Hsieh-Wilson, Linda C.

    2006-01-01

    Although fucose-α(1-2)-galactose [Fucα(1-2)Gal] carbohydrates have been implicated in cognitive processes such as long-term memory, the molecular mechanisms by which these sugars influence neuronal communication are not well understood. Here, we present molecular insights into the functions of Fucα(1-2)Gal sugars, demonstrating that they play a role in the regulation of synaptic proteins and neuronal morphology. We show that synapsins Ia and Ib, synapse-specific proteins involved in neurotransmitter release and synaptogenesis, are the major Fucα(1-2)Gal glycoproteins in mature cultured neurons and the adult rat hippocampus. Fucosylation has profound effects on the expression and turnover of synapsin in cells and protects synapsin from degradation by the calcium-activated protease calpain. Our studies suggest that defucosylation of synapsin has critical consequences for neuronal growth and morphology, leading to stunted neurite outgrowth and delayed synapse formation. We also demonstrate that Fucα(1-2)Gal carbohydrates are not limited to synapsin but are found on additional glycoproteins involved in modulating neuronal architecture. Together, our studies identify important roles for Fucα(1-2)Gal sugars in the regulation of neuronal proteins and morphological changes that may underlie synaptic plasticity. PMID:16373512

  9. Morphology and primary crystal structure of a silk-like protein polymer synthesized by genetically engineered Escherichia coli bacteria.

    PubMed

    Anderson, J P; Cappello, J; Martin, D C

    1994-08-01

    The morphology and primary crystal structure of SLPF, a protein polymer produced by genetically engineered Escherichia coli bacteria, were characterized. SLPF is a segmented copolymer consisting of amino acid sequence blocks modeled on the crystalline segments of silk fibroin and the cell attachment domain of human fibronectin. Wide angle x-ray scattering (WAXS), transmission electron microscopy (TEM), selected area electron diffraction (SAED), and molecular simulations were used to analyze the primary crystal structure of SLPF. TEM experiments conducted on SLPF droplets cast from formic acid on amorphous carbon film demonstrated that these protein films have a microstructure formed of woven sheaves. The sheaves are composed of well-defined whisker crystallites. The width of the whiskers, 11.8 +/- 2.2 nm, may be correlated to the length of the silk-like segment in SLPF as predicted by molecular simulations. WAXS data, TEM images, SAED, patterns, molecular simulations, and theoretical diffraction patterns all were consistent with the crankshaft model proposed for Silk I by Lotz and Keith.

  10. Primary structure and functional analysis of the soluble transducer protein HtrXI in the archaeon Halobacterium salinarium.

    PubMed Central

    Brooun, A; Zhang, W; Alam, M

    1997-01-01

    Signal transduction in the archaeon Halobacterium salinarium is mediated by a family of 13 soluble and membrane-bound transducers. Here, we report the primary structure and functional analysis of one of the smallest halobacterial putative transducers, HtrXI. Hydropathy plot analysis of the primary structure predicts no membrane-spanning segments in HtrXI. The fractionation of the H. salinarium proteins confirmed that HtrXI is a soluble protein. Capillary assay with an HtrXI deletion mutant and a complemented strain revealed that this soluble transducer is involved in Asp and Glu taxis. In vivo analysis of the methylesterase activity of the htrXI-1 deletion mutant suggests that HtrXI plays an important role in the adaptation of the chemotactic responses to His, Asp, and Glu, which are attractants for halobacteria. Stimulation by Asp and Glu causes demethylation of HtrXI and of another putative transducer, HtrVII. But addition of His to halobacterial cells increases HtrXI methylation together with that of other putative transducers. In the absence of HtrXI, stimulation by either Glu or His does not decrease or increase the methylation of any putative transducers. Therefire, the HtrXI transducer appears to have a complex role in chemotaxis signal transduction. PMID:9139915

  11. Online Radicalization: Bangladesh Perspective

    DTIC Science & Technology

    2017-06-09

    will work on developing a sustainable social awareness against radicalization. It will be responsible for coordinating and integrating all government...PAGE Form Approved OMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response , including...coordination among the counter-terrorism agencies, and lack of positive initiatives to grow enduring social resilience against radicalization. Bangladesh

  12. Evaluation of DNA binding, DNA cleavage, protein binding, radical scavenging and in vitro cytotoxic activities of ruthenium(II) complexes containing 2,4-dihydroxy benzylidene ligands.

    PubMed

    Mohanraj, Maruthachalam; Ayyannan, Ganesan; Raja, Gunasekaran; Jayabalakrishnan, Chinnasamy

    2016-12-01

    The new ruthenium(II) complexes with hydrazone ligands, 4-Methyl-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(1)), 4-Methoxy-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(2)), 4-Bromo-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(3)), were synthesized and characterized by various spectro analytical techniques. The molecular structures of the ligands were confirmed by single crystal X-ray diffraction technique. The DNA binding studies of the ligands and complexes were examined by absorption, fluorescence, viscosity and cyclic voltammetry methods. The results indicated that the ligands and complexes could interact with calf thymus DNA (CT-DNA) through intercalation. The DNA cleavage activity of the complexes was evaluated by gel electrophoresis assay, which revealed that the complexes are good DNA cleaving agents. The binding interaction of the ligands and complexes with bovine serum albumin (BSA) was investigated using fluorescence spectroscopic method. Antioxidant studies showed that the complexes have a strong radical scavenging properties. Further, the cytotoxic effect of the complexes examined on cancerous cell lines showed that the complexes exhibit significant anticancer activity.

  13. Symptomatology and growth in infants with cow's milk protein intolerance using two different whey-protein hydrolysate based formulas in a Primary Health Care setting.

    PubMed

    Verwimp, J J; Bindels, J G; Barents, M; Heymans, H S

    1995-09-01

    Both growth and the course of allergic symptoms were evaluated in 79 infants with cow's milk protein intolerance, aged three months or younger, diagnosed by standard elimination/provocation and treated with a whey-hydrolysate based infant formula: Nutrilon Pept or Pepti Junior. The efficacy of both products, in terms of symptomatology and growth, was compared with each other. The products differ in fat source (Pepti Junior 50% of its fat as MCT, Nutrilon Pepti normal LCT fat blend) and the presence of lactose (Pepti Junior: lactose free; Nutrilon Pepti: 40% of its carbohydrate as lactose). The study was part of a large project that aimed at standardising the approach towards cow's milk protein intolerance in Baby Health Clinics. Nearly 50 Baby Health Clinics participated in this project. In this study, growth and symptomatology (skin, respiratory tract, gastrointestinal tract) were monitored during an intervention period of at least 10 weeks. Infants in both feeding groups showed normal growth, and in at least 80% of the infants an improvement of the overall symptomatology could be seen during the intervention period. Most profound were the decreases in prevalence and severity of eczema and infantile colic. No differences in efficacy were found in this study between the two infant formulas. It was concluded that the exclusive use of either whey hydrolysate based infant formula resulted in an improvement of allergic symptoms and in normal growth in infants diagnosed by elimination/provocation for cow's milk protein intolerance in a Primary Health Care setting.

  14. Kinetics of phycocyanine bilin groups destruction by peroxyl radicals.

    PubMed

    Lissi, E A; Pizarro, M; Aspee, A; Romay, C

    2000-04-01

    Bilin groups in c-phycocyanine are readily bleached by peroxyl radicals produced in the thermolysis of 2, 2'-azobis(2-amidinopropane). From an evaluation of the bilin groups destroyed per radical that interacts with the protein, it is concluded that the bilin moiety is the main target of the radicals. Kinetic expressions are derived that allows an estimation of the substrate reactivity from the analysis of the rate of bilin group modification as a function of the protein concentration. From this analysis it is concluded that micromolar concentrations of c-phycocyanine are able to reduce the steady state concentration of the peroxyl radicals by one half, indicating a high antioxidant activity for this compound. This conclusion is confirmed by measuring the capacity of the protein to protect 1-naphthol from modification by peroxyl radicals. The results obtained show that the bilin groups have, on a molar basis, an antioxidant activity similar to that of potent antioxidants such as catechin.

  15. Evolutionary history exposes radical diversification among classes of interaction partners of the MLLE domain of plant poly(A)-binding proteins.

    PubMed

    Jiménez-López, Domingo; Bravo, Jaime; Guzmán, Plinio

    2015-09-16

    Poly(A)-binding proteins (PABPs) are evolutionarily conserved proteins that have important functions in the regulation of translation and the control of mRNA stability in eukaryotes. Most PABPs encode a C-terminal domain known as the MLLE domain (previously PABC or CTC), which can mediate protein interactions. In earlier work we identified and predicted that four classes of MLLE-interacting proteins were present in Arabidopsis thaliana, which we named CID A, B, C, and D. These proteins encode transcription-activating domains (CID A), the Lsm and LsmAD domains of ataxin-2 (CID B), the CUE and small MutS-related domains (CID C), and two RNA recognition domains (CID D). We recently found that a novel class that lacks the LsmAD domain is present in CID B proteins. We extended our analysis to other classes of CIDs present in the viridiplantae. We found that novel variants also evolved in classes CID A and CID C. A specific transcription factor domain is present in a distinct lineage in class A, and a variant that lacks at least two distinct domains was also identified in a divergent lineage in class C. We did not detect any variants in Class D CIDs. This class often consists of four to six highly conserved RNA-binding proteins, which suggests that major redundancy is present in this class. CIDs are likely to operate as components of posttranscriptional regulatory assemblies. The evident diversification of CIDs may be neutral or may be important for plant adaptation to the environment and for acquisition of specific traits during evolution. The fact that CIDs subclasses are maintained in early lineages suggest that a presumed interference between duplicates was resolved, and a defined function for each subclass was achieved.

  16. Predicting whole genome protein interaction networks from primary sequence data in model and non-model organisms using ENTS

    PubMed Central

    2013-01-01

    Background The large-scale identification of physical protein-protein interactions (PPIs) is an important step toward understanding how biological networks evolve and generate emergent phenotypes. However, experimental identification of PPIs is a laborious and error-prone process, and current methods of PPI prediction tend to be highly conservative or require large amounts of functional data that may not be available for newly-sequenced organisms. Results In this study we demonstrate a random-forest based technique, ENTS, for the computational prediction of protein-protein interactions based only on primary sequence data. Our approach is able to efficiently predict interactions on a whole-genome scale for any eukaryotic organism, using pairwise combinations of conserved domains and predicted subcellular localization of proteins as input features. We present the first predicted interactome for the forest tree Populus trichocarpa in addition to the predicted interactomes for Saccharomyces cerevisiae, Homo sapiens, Mus musculus, and Arabidopsis thaliana. Comparing our approach to other PPI predictors, we find that ENTS performs comparably to or better than a number of existing approaches, including several that utilize a variety of functional information for their predictions. We also find that the predicted interactions are biologically meaningful, as indicated by similarity in functional annotations and enrichment of co-expressed genes in public microarray datasets. Furthermore, we demonstrate some of the biological insights that can be gained from these predicted interaction networks. We show that the predicted interactions yield informative groupings of P. trichocarpa metabolic pathways, literature-supported associations among human disease states, and theory-supported insight into the evolutionary dynamics of duplicated genes in paleopolyploid plants. Conclusion We conclude that the ENTS classifier will be a valuable tool for the de novo annotation of genome

  17. Linking mutated primary structure of adrenoleukodystrophy protein with X-linked adrenoleukodystrophy.

    PubMed

    Yan, Shaomin; Wu, Guang

    2010-06-01

    The phenotype expression in X-linked adrenoleukodystrophy is one of the most intriguing issues of the disease, because there is no general correlation between the type of ABCD1 gene mutation and the clinical phenotype. In this study, we use the cross-impact analysis to build a descriptively quantitative relationship between mutant adrenoleukodystrophy protein and classification of adrenoleukodystrophy with the amino-acid distribution probability, which is a quantitative measure sensitive to mutation. Then we determine the probability that the adrenoleukodystrophy can be classified under mutations with the help of a Bayesian equation.

  18. PqqD Is a Novel Peptide Chaperone That Forms a Ternary Complex with the Radical S-Adenosylmethionine Protein PqqE in the Pyrroloquinoline Quinone Biosynthetic Pathway*

    PubMed Central

    Latham, John A.; Iavarone, Anthony T.; Barr, Ian; Juthani, Prerak V.; Klinman, Judith P.

    2015-01-01

    Pyrroloquinoline quinone (PQQ) is a product of a ribosomally synthesized and post-translationally modified pathway consisting of five conserved genes, pqqA-E. PqqE is a radical S-adenosylmethionine (RS) protein with a C-terminal SPASM domain, and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the peptide substrate PqqA. PqqD is a 10-kDa protein with an unknown function, but is essential for PQQ production. Recently, in Klebsiella pneumoniae (Kp), PqqD and PqqE were shown to interact; however, the stoichiometry and KD were not obtained. Here, we show that the PqqE and PqqD interaction transcends species, also occurring in Methylobacterium extorquens AM1 (Me). The stoichiometry of the MePqqD and MePqqE interaction is 1:1 and the KD, determined by surface plasmon resonance spectroscopy (SPR), was found to be ∼12 μm. Moreover, using SPR and isothermal calorimetry techniques, we establish for the first time that MePqqD binds MePqqA tightly (KD ∼200 nm). The formation of a ternary MePqqA-D-E complex was captured by native mass spectrometry and the KD for the MePqqAD-MePqqE interaction was found to be ∼5 μm. Finally, using a bioinformatic analysis, we found that PqqD orthologues are associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins. In conclusion, we propose that PqqD is a novel peptide chaperone and that PqqD orthologues may play a similar role in peptide modification pathways that use an RS-SPASM protein. PMID:25817994

  19. Effects of insulin-like growth factor-I, insulin, and leucine on protein turnover and pathways that regulate ubiquitin ligase expression in rainbow trout primary myocytes

    USDA-ARS?s Scientific Manuscript database

    The effects of insulin-like growth factor-I (IGF-I), insulin, and leucine on protein turnover and pathways that regulate proteolytic gene expression and protein polyubiquitination were investigated in primary cultures of four day old rainbow trout myocytes. Supplementing media with 100 nM IGF-I inc...

  20. Loss of p53 protein during radiation transformation of primary human mammary epithelial cells.

    PubMed Central

    Wazer, D E; Chu, Q; Liu, X L; Gao, Q; Safaii, H; Band, V

    1994-01-01

    The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated gamma-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletion within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G1 arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells. Images PMID:7511207

  1. Clathrin assembly protein AP180: primary structure, domain organization and identification of a clathrin binding site.

    PubMed Central

    Morris, S A; Schröder, S; Plessmann, U; Weber, K; Ungewickell, E

    1993-01-01

    Binding of AP180 to clathrin triskelia induces their assembly into 60-70 nm coats. The largest rat brain cDNA clone isolated predicts a molecular weight of 91,430 for AP180. Two cDNA clones have an additional small 57 bp insert. The deduced molecular weight agrees with gel filtration results provided the more chaotropic denaturant 6 M guanidinium thiocyanate is substituted for the weaker guanidinium chloride. The sequence and the proteolytic cleavage pattern suggest a three domain structure. The N-terminal 300 residues (pI 8.7) harbour a clathrin binding site. An acidic middle domain (pI 3.6, 450 residues), interrupted by an uncharged alanine rich segment of 59 residues, appears to be responsible for the anomalous physical properties of AP180. The C-terminal domain (166 residues) has a pI of 10.4. AP180 mRNA is restricted to neuronal sources. AP180 shows no significant homology to known clathrin binding proteins, but is nearly identical to a mouse phosphoprote