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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.

  2. Free radicals in primary photobiological processes.

    PubMed

    Vladimirov, Y A

    1998-01-01

    Formation of a semiquinone free radical derived from chlorophyll in the reaction of photoreduction has been discovered by A. A. Krasnovsky, Sr. in 1953. This review consider the results obtained in the author's laboratory, concerning the participation of free radicals in photochemical reactions under UV-irradiation of aromatic amino acids, proteins, and lipids, as well as in the reactions of chemiluminescence (CL) in the protein and chlorophyll-containing systems. Free radicals are the very first products of photochemical reactions in all systems studied. The back reactions of radicals are accompanied with photon emission. From the point of view of the molecular energetics, the radiativeless electronic transition in molecules is the most probable event, the transition triplet level is less probable, and the transition to the singlet excited level is virtually impossible. This may explain the low quantum yield of CL, similarity of CL and phosphorescence (rather than fluorescence) spectrum of the reaction products, low quantum yield of CL, and its high temperature coefficient.

  3. 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.

  4. Protein hydroperoxides can give rise to reactive free radicals.

    PubMed Central

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

    1995-01-01

    Proteins damaged by free-radical-generating systems in the presence of oxygen yield relatively long-lived protein hydroperoxides. These hydroperoxides have been shown by e.p.r. spectroscopy to be readily degraded to reactive free radicals on reaction with iron(II) complexes. Comparison of the observed spectra with those obtained with free amino acid hydroperoxides had allowed identification of some of the protein-derived radical species (including a number of carbon-centred radicals, alkoxyl radicals and a species believed to be the CO2 radical anion) and the elucidation of novel fragmentation and rearrangement processes involving amino acid side chains. In particular, degradation of hydroperoxide functions on the side chain of glutamic acid is shown to result in decarboxylation at the side-chain carboxy group via the formation of the CO2 radical anion; the generation of an identical radical from hydroperoxide groups on proteins suggests that a similar process occurs with these molecules. In a number of cases these fragmentation and rearrangement reactions give rise to further reactive free radicals (R., O2-./HO2., CO2-.) which may act as chain-carrying species in protein oxidations. These studies suggest that protein hydroperoxides are capable of initiating further radical chain reactions both intra- and inter-molecularly, and provide information on some of the fundamental mechanisms of protein alteration and side-chain fragmentation. PMID:7832784

  5. 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

  6. Absence of an effect of vitamin E on protein and lipid radical formation during lipoperoxidation of LDL by lipoxygenase

    PubMed Central

    Ganini, Douglas; Mason, Ronald P.

    2014-01-01

    LDL oxidation is the primary event in atherosclerosis, where LDL lipoperoxidation leads to modifications in the apolipoprotein B-100 (apo B-100) and lipids. Intermediate species of lipoperoxidation are known to be able to generate amino acid-centered radicals. Thus, we hypothesized that lipoperoxidation intermediates induce protein-derived free radical formation during LDL oxidation. Using DMPO and immuno spin-trapping, we detected the formation of protein free radicals on LDL incubated with Cu2+ or the soybean lipoxidase (LPOx)/phospholipase A2 (PLA2). With low concentrations of DMPO (1 mM), Cu2+ dose-dependently induced oxidation of LDL and easily detected apo B-100 radicals. Protein radical formation in LDL incubated with Cu2+ showed maximum yields after 30 minutes. In contrast, the yields of apo B-100-radicals formed by LPOx/PLA2 followed a typical enzyme-catalyzed kinetics that was unaffected by DMPO concentrations of up to 50 mM. Furthermore, when we analyzed the effect of antioxidants on protein radical formation during LDL oxidation, we found that ascorbate, urate and Trolox dose-dependently reduced apo B-100-free radical formation in LDL exposed to Cu2+. In contrast, Trolox was the only antioxidant that even partially protected LDL from LPOx/PLA2. We also examined the kinetics of lipid radical formation and protein radical formation induced by Cu2+ or LPOx/PLA2 for LDL supplemented with α-tocopherol. In contrast to the potent antioxidant effect of α-tocopherol on the delay of LDL oxidation induced by Cu2+, when we used the oxidizing system LPOx/PLA2, no significant protection was detected. The lack of protection of α-tocopherol on the apo B-100 and lipid free radical formation by LPOx may explain the failure of vitamin E as a cardiovascular protective agent for humans. PMID:25091900

  7. [Reasons of non-radical surgery for patients with primary skin melanoma].

    PubMed

    Gerasimova, A A; Gafmon, G I; Anisimov, V V; Semiletova, Iu V

    2014-01-01

    It was found that up to now a significant number of patients with primary skin melanoma continued to have non-radical surgery. Based on the analysis of clinical and morphological data on 288 of these patients it was revealed that most non-radical treatment was performed for patients who had had primary skin melanoma of linear dimensions of 1 cm and a pink color. It was proved that patients with tumors of the skin should first be examined by the oncologist. A lack of knowledge of semiotics of primary skin melanoma was revealed among doctors. Widely used diagnostic biopsy of the primary tumor with subsequent cytology is recommended. PMID:24919268

  8. [Reasons of non-radical surgery for patients with primary skin melanoma].

    PubMed

    Gerasimova, A A; Gafmon, G I; Anisimov, V V; Semiletova, Iu V

    2014-01-01

    It was found that up to now a significant number of patients with primary skin melanoma continued to have non-radical surgery. Based on the analysis of clinical and morphological data on 288 of these patients it was revealed that most non-radical treatment was performed for patients who had had primary skin melanoma of linear dimensions of 1 cm and a pink color. It was proved that patients with tumors of the skin should first be examined by the oncologist. A lack of knowledge of semiotics of primary skin melanoma was revealed among doctors. Widely used diagnostic biopsy of the primary tumor with subsequent cytology is recommended.

  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. Primary Reactions in Retinal Proteins

    NASA Astrophysics Data System (ADS)

    Diller, R.

    Conversion of sunlight into energy or information and their storage on a chemical level is essential for life on earth. An important family of chromoproteins performing these tasks is that of retinal binding proteins. Prominent examples are rhodopsin (Rh) [1,2] as the visual pigment in vertebrate and invertebrate animals, the archaeal rhodopsins bacteriorhodopsin (BR) [3] as a light driven proton pump, halorhodopsin (HR) [4,5] as a light driven chloride pump, sensory rhodopsin I and II (SRI, SRII) [6] as photoreceptors, and proteorhodopsin (PR) [7] as another bacterial proton pump.

  11. Free-radical-mediated protein inactivation and recovery during protein photoencapsulation.

    PubMed

    Lin, Chien-Chi; Sawicki, Suzanne M; Metters, Andrew T

    2008-01-01

    Photoencapsulation of protein therapeutics is very attractive for preparing biomolecule-loaded hydrogels for a variety of biomedical applications. However, detrimental effects of highly active radical species generated during photoencapsulation must be carefully evaluated to maintain efficient hydrogel cross-linking while preserving the structure and bioactivity of encapsulated biomolecules. Here, we examine the free-radical-mediated inactivation and incomplete release of proteins from photocurable hydrogels utilizing lysozyme as a conservative model system. Various protein photoencapsulation conditions were tested to determine the factors affecting lysozyme structural integrity and bioactivity. It was found that a portion of the lysozyme becomes conjugated to polymer chains at high photoinitiator concentrations and long polymerization times. We also found that the more hydrophilic photoinitiator Irgacure-2959 (I-2959, 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone) causes more damage to lysozyme compared to the hydrophobic photoinitiator Irgacure-651 (I-651, 2,2-dimethoxy-2-phenylacetophenone), even though I-2959 has been previously shown to be more cytocompatible. Furthermore, while nonacrylated PEG provides only limited protection from the denaturing free radicals that are present during hydrogel curing, acrylated PEG macromers effectively preserve lysozyme structural integrity and bioactivity in the presence of either photoinitiator. Overall, these findings indicate how photopolymerization conditions (e.g., photoinitiator type and concentration, UV exposure time, etc.) must be optimized to obtain a functional hydrogel device that can preserve protein bioactivity and provide maximal protein release. PMID:18088094

  12. Hearing outcomes following primary malleostapedial rotation ossiculoplasty in patients undergoing modified radical mastoidectomy

    PubMed Central

    Kanegaonkar, RG; Najuko-Mafemera, A

    2014-01-01

    Introduction Treatment of cholesteatoma consists of either excision or exteriorisation of disease. Approaches have traditionally included a radical or modified radical mastoidectomy and combined approach tympanoplasty. Hearing thresholds following a modified radical mastoidectomy alone have been reported as poor. We assessed hearing outcomes in patients undergoing a primary malleostapedial reconstruction combined with their open cavity surgery. Methods All patients undergoing open cavity mastoidectomy with primary malleostapedial rotation ossiculoplasty between 2009 and 2013 were identified. Case notes were reviewed, and demographic data, recurrence rate and audiometry were recorded. Results Twenty-one patients were identified. The age range was 10–65 years. There was no evidence of recurrence of cholesteatoma. The mean postoperative air-bone gap was 20dBHL, 23dBHL, 10dBHL and 27dBHL at 0.5kHz, 1kHz, 2kHz and 4kHz respectively. Excluding cases consistent with a postoperative ossicular discontinuity (n=3), the mean postoperative air-bone gap was 15dBHL, 19dBHL, 8dBHL and 26dBHL at 0.5kHz, 1kHz, 2kHz and 4kHz respectively. Conclusions The improvement in hearing thresholds demonstrated in this cohort of patients supports the use of this form of ossiculoplasty in those undergoing open cavity procedures. This would also suggest that the subsequent use of hearing aids in these patients would require less amplification and therefore provide superior hearing outcomes. As hearing loss remains a significant concern following modified radical mastoidectomy, we suggest an open cavity with primary malleostapedial rotation ossiculoplasty as a viable alternative to modified radical mastoidectomy alone, in selected cases. PMID:25198979

  13. 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)

  14. Photolysis of nitrous acid as a primary source of OH radicals indoors

    NASA Astrophysics Data System (ADS)

    Gomez Alvarez, E.; Amedro, D.; Afif, C.; Gligorovski, S.; Schoemacker, C.; Fittschen, C. M.; Doussin, J.; Wortham, H.

    2013-12-01

    reactions, potentially more hazardous than the primary pollutants in the indoor air. This calculation indicates that, since the levels of concentration of OH radicals found in this study are one order of magnitude higher than predicted before, reactivity with OH becomes relevant as these reactions take place during a smaller time scale than typical air exchange rates indoors. Gómez Alvarez, E.; Amedro, D.; Afif, C. ; Gligorovski, S.; Schoemacker , C.; Fittschen, C. ; Doussin, J. F.; Wortham, H. (2013) Unexpectedly high indoor hydroxyl radical concentrations associated with nitrous acid. Proc. Natl. Acad. Sci. USA Accepted.

  15. 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

  16. 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

  17. Synthetic use of the primary kinetic isotope effect in hydrogen atom transfer: generation of α-aminoalkyl radicals.

    PubMed

    Wood, Mark E; Bissiriou, Sabine; Lowe, Christopher; Norrish, Andrew M; Sénéchal, Katell; Windeatt, Kim M; Coles, Simon J; Hursthouse, Michael B

    2010-10-21

    The extent to which deuterium can act as a protecting group to prevent unwanted 1,5-hydrogen atom transfer to aryl and vinyl radical intermediates was examined in the context of the generation of α-aminoalkyl radicals in a pyrrolidine ring. Intra- and intermolecular radical trapping following hydrogen atom transfer provides an illustration of the use of the primary kinetic isotope effect in directing the outcome of synthetic C-C bond-forming processes.

  18. Different reactivity of primary fibroblasts and endothelial cells towards crystalline silica: A surface radical matter.

    PubMed

    Pozzolini, Marina; Vergani, Laura; Ragazzoni, Milena; Delpiano, Livia; Grasselli, Elena; Voci, Adriana; Giovine, Marco; Scarfì, Sonia

    2016-06-15

    Quartz is a well-known occupational fibrogenic agent able to cause fibrosis and other severe pulmonary diseases such as silicosis and lung cancer. The silicotic pathology owes its severity to the structural and chemo-physical properties of the particles such as shape, size and abundance of surface radicals. In earlier studies, we reported that significant amounts of surface radicals can be generated on crystalline silica by chemical aggression with ascorbic acid (AA), a vitamin naturally abundant in the lung surfactant, and this reaction led to enhanced cytotoxicity and production of inflammatory mediators in a macrophage cell line. However in the lung, other cells acting in the development of silicosis, like fibroblasts and endothelial cells, can come to direct contact with inhaled quartz. We investigated the cytotoxic/pro-inflammatory effects of AA-treated quartz microcrystals (QA) in human primary fibroblasts and endothelial cells as compared to unmodified microcrystals (Q). Our results show that, in fibroblasts, the abundance of surface radicals on quartz microcrystals (Q vs QA) significantly enhanced cell proliferation (with or without co-culture with macrophages), reactive oxygen species (ROS) production, NF-κB nuclear translocation, smooth muscle actin, fibronectin, Bcl-2 and tissue inhibitor of metalloproteinase-1 expression and collagen production. Contrariwise, endothelial cells reacted to the presence of quartz microcrystals independently from the abundance of surface radicals showing similar levels of cytotoxicity, ROS production, cell migration, MCP-1, ICAM-I and fibronectin gene expression when challenged with Q or QA. In conclusion, our in vitro experimental model demonstrates an important and quite unexplored direct contribute of silica surface radicals to fibroblast proliferation and fibrogenic responses. PMID:27381660

  19. OH Radical Aging of Model Primary Organic Aerosols In Smog Chamber Studies

    NASA Astrophysics Data System (ADS)

    Lambe, A. T.; Zhang, J.; Sage, A. M.; Donahue, N. M.

    2006-12-01

    Most of the fine primary organic carbon (OC) in the atmosphere is saturated. Thus, by OC mass balance alone, OH radicals should be important in organic aerosol processing because saturated OC is unreactive to ozone and vulnerable to NO3 oxidation only at night. However, the importance of OH-induced heterogeneous oxidation remains uncertain because laboratory and ambient analysis is a challenge. Most laboratory aging studies have been conducted in flow tube reactors with extremely high radical concentrations and short exposure times. While these pioneering experiments have revealed efficient OH uptake, a confounding issue remains that both oxidant and reagent losses are rate-limited by diffusion of the oxidant to the phase interface. Examining these systems over much longer timescales in smog chamber experiments is therefore desirable, but established methods of OH radical production in smog chamber studies typically require some combination of UV illumination, high NOx, or radical cycling. Thus, employing these methods results in experimental conditions that either do not represent atmospheric conditions or cannot produce a sufficiently high flux of OH radicals - these shortcomings are acceptable for some applications, but are significant hindrances when heterogeneous aging is the experimental objective. Our approach has been to use alkene ozonolysis as a continuous, low-NOx, dark source of OH. Specifically, we use 2,3-dimethyl-2-butene (tetramethylethylene, TME) because ozonolysis results in an OH yield near unity. Furthermore, the organic TME ozonolysis products have high vapor pressures, so they do not participate directly in condensed-phase chemistry. To maintain a constant radical production rate, fresh reagents are delivered to the chamber to replenish TME and O3 as they react. TME is delivered continuously via capillary liquid flow with constant evaporation from a droplet at the capillary tip into a carrier stream. Ozone is held at a constant, high value by

  20. Reaction of haem containing proteins and enzymes with hydroperoxides: the radical view.

    PubMed

    Svistunenko, Dimitri A

    2005-02-25

    The reaction between hydroperoxides and the haem group of proteins and enzymes is important for the function of many enzymes but has also been implicated in a number of pathological conditions where oxygen binding proteins interact with hydrogen peroxide or other peroxides. The haem group in the oxidized Fe3+ (ferric) state reacts with hydroperoxides with a formation of the Fe4+=O (oxoferryl) haem state and a free radical primarily located on the pi-system of the haem. The radical is then transferred to an amino acid residue of the protein and undergoes further transfer and transformation processes. The free radicals formed in this reaction are reviewed for a number of proteins and enzymes. Their previously published EPR spectra are analysed in a comparative way. The radicals directly detected in most systems are tyrosyl radicals and the peroxyl radicals formed on tryptophan and possibly cysteine. The locations of the radicals in the proteins have been reported as follows: Tyr133 in soybean leghaemoglobin; alphaTyr42, alphaTrp14, betaTrp15, betaCys93, (alphaTyr24-alphaHis20), all in the alpha- and beta-subunits of human haemoglobin; Tyr103, Tyr151 and Trp14 in sperm whale myoglobin; Tyr103, Tyr146 and Trp14 in horse myoglobin; Trp14, Tyr103 and Cys110 in human Mb. The sequence of events leading to radical formation, transformation and transfer, both intra- and intermolecularly, is considered. The free radicals induced by peroxides in the enzymes are reviewed. Those include: lignin peroxidase, cytochrome c peroxidase, cytochrome c oxidase, turnip isoperoxidase 7, bovine catalase, two isoforms of prostaglandin H synthase, Mycobacterium tuberculosis and Synechocystis PCC6803 catalase-peroxidases. PMID:15721611

  1. 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.

  2. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. Primary signet ring cell carcinoma of the prostate treated by radical cystoprostatectomy and chemoradiotherapy

    PubMed Central

    Kim, Sun Wook; Kim, Woohyun; Cho, Yong-Hyun; Kim, Tae-Jung; Woo, Insuk; Sohn, Dong Wan

    2016-01-01

    Primary signet ring cell carcinoma (SRCC) of the prostate is very rare. Although SRCC is primarily found in the stomach and colon, it can also be found in the pancreas, breast, thyroid, bladder, and prostate. We recently diagnosed and treated a case of primary SRCC of the prostate. A 56-year-old Korean man was referred to our institution for evaluation of a one-month history of hematuria and recently identified bladder mass. Transurethral resection of the bladder tumour was performed and histological and immunohistochemical evaluation revealed a diagnosis of SRCC with tumour invading into the outer half of the deep muscularis propria. After three weeks, the patient had radical cystoprostatectomy with ileal conduit. Tumour involved both prostate and bladder, but the centre of the tumour was located in the prostate. Duodenoscopy and colon fibroscopy both indicated no evidence of tumour origin in the gastrointestinal (GI) tract. Overall, this tumour was regarded as primary SRCC of the prostate. Concurrent chemoradiotherapy (CCRT) using leucovorin and fluorouracil was initiated two months later. The patient eventually developed bone and liver metastases and died of hepatopathy.

  5. 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.

  6. Effects of simultaneous exposure of surfactant to serum proteins and free radicals.

    PubMed

    Marzan, Yolanda; Mora, Rene; Butler, Aaron; Butler, Matthew; Ingenito, Edward P

    2002-03-01

    Free radicals (FRs) and serum proteins have both been implicated in the pathophysiology of surfactant dysfunction during acute lung injury (ALI). This study examines how these 2 distinct mechanisms interact to contribute to altered surfactant function in this setting. Calf lung surfactant (2 mg/mL) was incubated with no additives (C = control), and with low = (LD = 125 microM FeCl2; 250 microM H2O2) and high-dose (HD = 250 microM FeCl2, 500 microM H2O2) Fenton reaction reagents to generate hydroxyl radical. Each condition was studied with (1) no protein (N); and with 25%, 200%, and 800% (weight protein/weight phospholipid) protein added as (2) bovine albumin, (3) bovine fibrinogen, (4) hemoglobin, or (5) calf serum. Lipid (LFR) and protein (PFR) free-radical products, and modifications in the tertiary structure of Surfactant Protein A (SPA) on Western blot, were observed in N LD and N HD samples. Added proteins reduced LFR and PFR changes as well as SPA structural changes. Protection was greatest for fibrinogen, hemoglobin, and serum, and least for albumin. Minimal to no dysfunction, assayed by pulsating surfactometry, was observed in all samples. These findings indicate that addition of serum proteins to surfactant at 2 mg/mL protects against, rather than promotes, FR-mediated chemical changes in surfactant lipid and protein constituents.

  7. 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

  8. Kinetic study of coniferyl alcohol radical binding to the (+)-pinoresinol forming dirigent protein.

    PubMed

    Halls, Steven C; Davin, Laurence B; Kramer, David M; Lewis, Norman G

    2004-03-01

    An essential step in lignan and lignin formation in planta is one electron oxidation of (E)-coniferyl alcohol (CA) to generate the radical intermediate (CA(*)), which can then undergo directed radical-radical couplings in vivo. For lignan formation in vitro and in vivo, stereoselective coupling of CA(*) only occurs to afford (+)-pinoresinol in the additional presence of (+)-pinoresinol forming dirigent protein (DP). Presented herein is a kinetic and thermodynamic study which reveals the central mechanistic details of the coupling process involved in DP-mediated coupling. DP activity was maximal between pH 4.25 and pH 6.0, with activity being maintained at temperatures below 33 degrees C. Equilibrium binding assays revealed that coniferyl alcohol was only weakly bound to the DP, with a K(D) of 370 +/- 65 microM. On the other hand, the enantiomeric excess of (+)-pinoresinol formed was dependent on both DP concentration and rate of CA oxidation and, thus, on apparent steady-state [CA(*)]. The data obtained could best be explained using a kinetic model where radical-radical coupling via DP competes with that occurring in open solution. Using this model, an apparent K(M) of about 10 nM was estimated from the saturation behavior of (+)-pinoresinol formation with respect to apparent steady-state [CA(*)]. These data strongly suggest that CA(*), rather than CA, is the substrate for DP, in agreement with earlier predictions. A mechanism of directed radical-radical coupling, where two coniferyl alcohol radical substrates are bound per protein dimer, is proposed.

  9. 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.

  10. Chromophore-assisted laser inactivation of proteins is mediated by the photogeneration of free radicals.

    PubMed Central

    Liao, J C; Roider, J; Jay, D G

    1994-01-01

    Chromophore-assisted laser inactivation (CALI) is a technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions and an understanding of its mechanism is required for its judicious application. We report here that CALI is not mediated by photoinduced thermal denaturation but by photogenerated free radicals. Thermal diffusion calculations suggest that the temperature changes resulting from CALI are too small to cause thermal denaturation, and Arrhenius plots of CALI are inconsistent with a photothermal mechanism. CALI shows an energy dose reciprocity above a threshold and can be inhibited by free-radical quenchers, thus demonstrating a photochemical mechanism of protein inactivation. The type of quenchers that are effective in inhibiting CALI indicates that the active species is a hydrogen abstractor which is not derived from molecular oxygen. We suggest that the active free-radical species is the hydroxyl radical and its very short lifetime explains the spatial specificity of CALI such that half-maximal damage is effected within 15 A from the dye moiety and no significant damage occurs at 34 A. The data are consistent with free-radical formation resulting from a sequential two-photon process. Images PMID:8146171

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

    PubMed Central

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

    2009-01-01

    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 in order to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues. PMID:19278868

  12. 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. PMID:25941968

  13. 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.

  14. 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

  15. A chaperonin as protein nanoreactor for atom-transfer radical polymerization.

    PubMed

    Renggli, Kasper; Nussbaumer, Martin G; Urbani, Raphael; Pfohl, Thomas; Bruns, Nico

    2014-01-27

    The group II chaperonin thermosome (THS) from the archaea Thermoplasma acidophilum is reported as nanoreactor for atom-transfer radical polymerization (ATRP). A copper catalyst was entrapped into the THS to confine the polymerization into this protein cage. THS possesses pores that are wide enough to release polymers into solution. The nanoreactor favorably influenced the polymerization of N-isopropyl acrylamide and poly(ethylene glycol)methylether acrylate. Narrowly dispersed polymers with polydispersity indices (PDIs) down to 1.06 were obtained in the protein nanoreactor, while control reactions with a globular protein-catalyst conjugate only yielded polymers with PDIs above 1.84. PMID:24459061

  16. 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. PMID:26873390

  17. 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

  18. 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

  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. PMID:24406430

  20. 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.

  1. 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

  2. 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.

  3. 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. PMID:25752613

  4. 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.

  5. 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

  6. 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.

  7. Protective activity of green tea against free radical- and glucose-mediated protein damage.

    PubMed

    Nakagawa, Takako; Yokozawa, Takako; Terasawa, Katsutoshi; Shu, Seiji; Juneja, Lekh Raj

    2002-04-10

    Protein oxidation and glycation are posttranslational modifications that are implicated in the pathological development of many age-related disease processes. This study investigated the effects of green tea extract, and a green tea tannin mixture and its components, on protein damage induced by 2,2'-azobis(2-amidinopropane) dihydrochloride (a free radical generator) and glucose in in vitro assay systems. We found that green tea extract can effectively protect against protein damage, and showed that its action is mainly due to tannin. In addition, it was shown that the chemical structures of tannin components are also involved in this activity, suggesting that the presence of the gallate group at the 3 position plays the most important role in the protective activity against protein oxidation and glycation, and that there is also a contribution by the hydroxyl group at the 5' position in the B ring and the sterical structure. These findings demonstrate the mechanisms of the usefulness of green tea in protein oxidation- and glycation-associated diseases.

  8. 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. PMID:23331209

  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. Predicting protein-protein interactions in unbalanced data using the primary structure of proteins

    PubMed Central

    2010-01-01

    Background Elucidating protein-protein interactions (PPIs) is essential to constructing protein interaction networks and facilitating our understanding of the general principles of biological systems. Previous studies have revealed that interacting protein pairs can be predicted by their primary structure. Most of these approaches have achieved satisfactory performance on datasets comprising equal number of interacting and non-interacting protein pairs. However, this ratio is highly unbalanced in nature, and these techniques have not been comprehensively evaluated with respect to the effect of the large number of non-interacting pairs in realistic datasets. Moreover, since highly unbalanced distributions usually lead to large datasets, more efficient predictors are desired when handling such challenging tasks. Results This study presents a method for PPI prediction based only on sequence information, which contributes in three aspects. First, we propose a probability-based mechanism for transforming protein sequences into feature vectors. Second, the proposed predictor is designed with an efficient classification algorithm, where the efficiency is essential for handling highly unbalanced datasets. Third, the proposed PPI predictor is assessed with several unbalanced datasets with different positive-to-negative ratios (from 1:1 to 1:15). This analysis provides solid evidence that the degree of dataset imbalance is important to PPI predictors. Conclusions Dealing with data imbalance is a key issue in PPI prediction since there are far fewer interacting protein pairs than non-interacting ones. This article provides a comprehensive study on this issue and develops a practical tool that achieves both good prediction performance and efficiency using only protein sequence information. PMID:20361868

  11. Redox-sensitive protein phosphatase activity regulates the phosphorylation state of p38 protein kinase in primary astrocyte culture.

    PubMed

    Robinson, K A; Stewart, C A; Pye, Q N; Nguyen, X; Kenney, L; Salzman, S; Floyd, R A; Hensley, K

    1999-03-15

    Reactive oxygen species (ROS) have been implicated as second messengers that activate protein kinase cascades, although the means by which ROS regulate signal transduction remains unclear. In the present study, we show that interleukin 1beta (IL1beta), H2O2, and sorbitol-induced hyperosmolarity mediate a 5- to 10-fold increase in phosphorylation (activation) of the p38 protein kinase in rat primary glial cells as measured by analyses of Western blots using an antibody directed against the dually phosphorylated (active) p38. Additionally, IL1beta was found to elicit H2O2 synthesis in these cells. Concurrent with p38 phosphorylation, all three stimulation paradigms caused an inhibition of protein phosphatase activity. Phenyl-tert-butyl nitrone (PBN), a nitrone-based free radical trap and N-acetyl-cysteine (NAC), a thiol reducing agent, were examined for their effects on the phosphorylation of p38 as well as phosphatase activity. Pretreatment of cells with either PBN or NAC at 1.0 mM suppressed IL1beta H2O2, and sorbitol-mediated activation of p38 and significantly increased phosphatase activity. These data suggest that ROS, particularly H2O2, are used as second messenger substances that activate p38 in part via the transient inactivation of regulatory protein phosphatases.

  12. 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...

  13. Free radical-induced protein modification and inhibition of Ca2+-ATPase of cardiac sarcoplasmic reticulum.

    PubMed

    Kaplan, Peter; Babusikova, Eva; Lehotsky, Jan; Dobrota, Dusan

    2003-06-01

    The effect of oxidative stress on the Ca2+-ATPase activity, lipid peroxidation and protein modification of cardiac sarcoplasmic reticulum (SR) membranes was investigated. Isolated SR vesicles were exposed to FeSO4/EDTA (0.2 micromol Fe2+ per mg of protein) at 37 degrees C for 1 h in the presence or absence of antioxidants. FeSO4/EDTA decreased the maximum velocity of Ca2+-ATPase reaction without a change of affinity for Ca2+ or Hill coefficient. Treatment with radical-generating system led also to conjugated diene formation, loss of sulfhydryl groups, changes in tryptophan and bityrosine fluorescences and to production of lysine conjugates with lipid peroxidation end-products. Lipid antioxidants butylated hydroxytoluene (BHT) and stobadine partially prevented inhibition of Ca2+-ATPase and decrease in tryptophan fluorescence, while the loss of -SH groups and formation of bityrosines or lysine conjugates were completely prevented. Glutathione also partially protected Ca2+-ATPase activity and decreased formation of bityrosine, but it was not able to prevent oxidative modification of tryptophan and lysine. These findings suggest that combination of amino acid modifications, rather than oxidation of amino acids of one kind, is responsible for inhibition of SR Ca2+-ATPase activity. PMID:12870653

  14. Controlled Radical Polymerization as an Enabling Approach for the Next Generation of Protein-Polymer Conjugates.

    PubMed

    Pelegri-O'Day, Emma M; Maynard, Heather D

    2016-09-20

    Protein-polymer conjugates are unique constructs that combine the chemical properties of a synthetic polymer chain with the biological properties of a biomacromolecule. This often leads to improved stabilities, solubilities, and in vivo half-lives of the resulting conjugates, and expands the range of applications for the proteins. However, early chemical methods for protein-polymer conjugation often required multiple polymer modifications, which were tedious and low yielding. To solve these issues, work in our laboratory has focused on the development of controlled radical polymerization (CRP) techniques to improve synthesis of protein-polymer conjugates. Initial efforts focused on the one-step syntheses of protein-reactive polymers through the use of functionalized initiators and chain transfer agents. A variety of functional groups such as maleimide and pyridyl disulfide could be installed with high end-group retention, which could then react with protein functional groups through mild and biocompatible chemistries. While this grafting to method represented a significant advance in conjugation technique, purification and steric hindrance between large biomacromolecules and polymer chains often led to low conjugation yields. Therefore, a grafting from approach was developed, wherein a polymer chain is grown from an initiating site on a functionalized protein. These conjugates have demonstrated improved homogeneity, characterization, and easier purification, while maintaining protein activity. Much of this early work utilizing CRP techniques focused on polymers made up of biocompatible but nonfunctional monomer units, often containing oligoethylene glycol meth(acrylate) or N-isopropylacrylamide. These branched polymers have significant advantages compared to the historically used linear poly(ethylene glycols) including decreased viscosities and thermally responsive behavior, respectively. Recently, we were motivated to use CRP techniques to develop polymers with

  15. Controlled Radical Polymerization as an Enabling Approach for the Next Generation of Protein-Polymer Conjugates.

    PubMed

    Pelegri-O'Day, Emma M; Maynard, Heather D

    2016-09-20

    Protein-polymer conjugates are unique constructs that combine the chemical properties of a synthetic polymer chain with the biological properties of a biomacromolecule. This often leads to improved stabilities, solubilities, and in vivo half-lives of the resulting conjugates, and expands the range of applications for the proteins. However, early chemical methods for protein-polymer conjugation often required multiple polymer modifications, which were tedious and low yielding. To solve these issues, work in our laboratory has focused on the development of controlled radical polymerization (CRP) techniques to improve synthesis of protein-polymer conjugates. Initial efforts focused on the one-step syntheses of protein-reactive polymers through the use of functionalized initiators and chain transfer agents. A variety of functional groups such as maleimide and pyridyl disulfide could be installed with high end-group retention, which could then react with protein functional groups through mild and biocompatible chemistries. While this grafting to method represented a significant advance in conjugation technique, purification and steric hindrance between large biomacromolecules and polymer chains often led to low conjugation yields. Therefore, a grafting from approach was developed, wherein a polymer chain is grown from an initiating site on a functionalized protein. These conjugates have demonstrated improved homogeneity, characterization, and easier purification, while maintaining protein activity. Much of this early work utilizing CRP techniques focused on polymers made up of biocompatible but nonfunctional monomer units, often containing oligoethylene glycol meth(acrylate) or N-isopropylacrylamide. These branched polymers have significant advantages compared to the historically used linear poly(ethylene glycols) including decreased viscosities and thermally responsive behavior, respectively. Recently, we were motivated to use CRP techniques to develop polymers with

  16. Non-local photo-polymerization kinetics including multiple termination mechanisms and dark reactions: Part III. Primary radical generation and inhibition

    SciTech Connect

    Gleeson, Michael R.; Liu Shui; Guo Jinxin; Sheridan, John T.

    2010-09-15

    Photopolymers are playing an ever more important role in diverse areas of research such as holographic data storage, hybrid photonic circuits, and solitary waves. In each of these applications, the production of primary radicals is the driving force of the polymerization processes. Therefore an understanding of the production, removal, and scavenging processes of free radicals in a photopolymer system is crucial in determining a material's response to a given exposure. One such scavenging process is inhibition. In this paper the non-local photo-polymerization driven diffusion model is extended to more accurately model the effects of (i) time varying primary radical production, (ii) the rate of removal of photosensitizer, and (iii) inhibition. The model is presented to specifically analyze the effects of inhibition, which occur most predominantly at the start of grating growth, and comparisons between theory and experiment are performed which quantify these effects.

  17. 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.

  18. 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.

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

    PubMed

    Vahidi, Siavash; Konermann, Lars

    2016-07-01

    Hydroxyl radical (⋅OH) labeling with mass spectrometry detection reports on protein conformations and interactions. Fast photochemical oxidation of proteins (FPOP) involves ⋅OH 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 ⋅OH 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. Graphical Abstract ᅟ. PMID:27067899

  20. Advanced oxidation protein products are generated by bovine neutrophils and inhibit free radical production in vitro.

    PubMed

    Bordignon, Milena; Da Dalt, Laura; Marinelli, Lieta; Gabai, Gianfranco

    2014-01-01

    Despite the recognised importance of oxidative stress in the health and immune function of dairy cows, protein oxidation markers have been poorly studied in this species. The current study aimed to characterise markers of protein oxidation generated by activated bovine neutrophils and investigate the biological effects of advanced oxidation protein products (AOPP) on bovine neutrophils. Markers of protein oxidation (AOPP, dityrosines and carbonyls) were measured in culture medium containing bovine serum albumin (BSA) exposed to neutrophils. The effect of AOPP-BSA on generation of reactive oxygen species (ROS) was assessed by chemiluminescence. Activation of caspases-3, -8 and -9 and the presence of DNA laddering were used as apoptosis markers. Greater amounts of AOPP were generated by phorbol myristate acetate (PMA)-activated than non-activated neutrophils (1.46 ± 0.13 vs. 0.75 ± 0.13 nmol/mg protein, respectively; P<0.05). Activated neutrophils and hypochlorous acid generated slightly different patterns of oxidized protein markers. Exposure to AOPP-BSA did not stimulate ROS production. Activated neutrophils generated a lesser amount of ROS when incubated with AOPP-BSA (P<0.001). Activation with PMA induced a loss of viable neutrophils after 3h, which was greater with AOPP-BSA incubation (P<0.05). Detectable amounts of active caspases-3, -8 and -9 were found in nearly all samples but differences in caspase activation or DNA laddering were not observed comparing treatment groups. Apoptosis was unlikely to be responsible for the greater loss of PMA-activated neutrophils cultured in AOPP-BSA and it is possible that primary necrosis occurred. The results suggest that accumulation of oxidized proteins at an inflammatory site might result in a progressive reduction of neutrophil viability.

  1. Antihypertensive and free radical scavenging properties of enzymatic rapeseed protein hydrolysates.

    PubMed

    He, Rong; Alashi, Adeola; Malomo, Sunday A; Girgih, Abraham T; Chao, Dongfang; Ju, Xingrong; Aluko, Rotimi E

    2013-11-01

    In this study, rapeseed protein isolate (RPI) was digested with various proteases to produce rapeseed protein hydrolysates (RPHs), which were then separated into different peptide fractions (<1, 1-3, 3-5, and 5-10kDa) by membrane ultrafiltration. Membrane fractionation showed that peptides with sizes <3 kDa had significantly (p<0.05) reduced surface hydrophobicity when compared to the RPHs and peptide fractions with sizes >3 kDa. In contrast, the <3 kDa peptides showed significantly (p<0.05) higher oxygen radical scavenging ability when compared to the >3 kDa peptides and RPHs. In vitro inhibition of angiotensin I-converting enzyme (ACE) was significantly (p<0.05) higher for the Thermolysin, Proteinase K and Alcalase RPHs when compared to the pepsin+pancreatin (PP) and Flavourzyme RPHs. The Alcalase RPH had significantly (p<0.05) higher renin inhibition among the RPHs, while with the exception of Thermolysin, the 5-10 kDa peptide fraction had the least renin-inhibitory ability when compared to the <5 kDa peptide fractions. Oral administration (100mg/kg body weight) of the RPHs and RPI to spontaneously hypertensive rats (SHR) showed the Alcalase RPH to be the most effective in blood pressure (BP) reduction (∼24 mm Hg) while Proteinase K RPH was the least effective (∼5 mm Hg) after 8h. However, the PP RPH had the most prolonged effect with BP reduction of ∼20 mm Hg after 24h of oral administration. We conclude that the strong BP-lowering ability of Alcalase and PP RPHs could be due to high resistance of the peptides to structural degradation coupled with high absorption rate within the gastrointestinal tract.

  2. Salvage robotic-assisted laparoscopic radical prostatectomy following failed primary high-intensity focussed ultrasound treatment for localised prostate cancer.

    PubMed

    Murphy, Declan G; Pedersen, John; Costello, Anthony J

    2008-09-01

    We report the first case of salvage robotic-assisted laparoscopic radical prostatectomy (RALP) following failed primary high-intensity focussed ultrasound (HIFU) for localised carcinoma of the prostate. A 66-year-old male with a presenting prostate-specific antigen (PSA) of 5 ng/ml was diagnosed with T1c Gleason 3 + 4 prostate cancer. He underwent transurethral resection of the prostate and HIFU. His PSA dropped to 2.0 ng/ml and repeat biopsy revealed upgrading of his prostate cancer to Gleason 4 + 3. He was referred to us for a second opinion and, following discussion of his options, he underwent RALP. The total operative time was 159 min. There were no intra- or postoperative complications. He was discharged on postoperative day two and was fully continent 10 days following removal of his catheter. His PSA remained undetectable 6 months postoperatively. Salvage RALP was feasible in this case with good functional and short-term oncological outcomes for the patient. PMID:27628262

  3. Signaling the Unfolded Protein Response in primary brain cancers.

    PubMed

    Le Reste, Pierre-Jean; Avril, Tony; Quillien, Véronique; Morandi, Xavier; Chevet, Eric

    2016-07-01

    The Unfolded Protein Response (UPR) is an adaptive cellular program used by eukaryotic cells to cope with protein misfolding stress in the Endoplasmic Reticulum (ER). During tumor development, cancer cells are facing intrinsic (oncogene activation) and extrinsic (limiting nutrient or oxygen supply; exposure to chemotherapies) challenges, with which they must cope to survive. Primary brain tumors are relatively rare but deadly and present a significant challenge in the determination of risk factors in the population. These tumors are inherently difficult to cure because of their protected location in the brain. As such surgery, radiation and chemotherapy options carry potentially lasting patient morbidity and incomplete tumor cure. Some of these tumors, such as glioblastoma, were reported to present features of ER stress and to depend on UPR activation to sustain growth, but to date there is no clear general representation of the ER stress status in primary brain tumors. In this review, we describe the key molecular mechanisms controlling the UPR and their implication in cancers. Then we extensively review the literature reporting the status of ER stress in various primary brain tumors and discuss the potential impact of such observation on patient stratification and on the possibility of developing appropriate targeted therapies using the UPR as therapeutic target. PMID:27016056

  4. 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.

  5. 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.

  6. 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.

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

    PubMed

    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 (alphabeta)4 to three-quarter-barrel structures (alphabeta)6 to full-barrel structures (alphabeta)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 (alphabeta)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.

  8. 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

  9. Synthetic use of the primary kinetic isotope effect in hydrogen atom transfer 2: generation of captodatively stabilised radicals.

    PubMed

    Wood, Mark E; Bissiriou, Sabine; Lowe, Christopher; Windeatt, Kim M

    2013-04-28

    Using C-3 di-deuterated morpholin-2-ones bearing N-2-iodobenzyl and N-3-bromobut-3-enyl radical generating groups, only products derived from the more stabilised C-3, rather than the less stabilised C-5 translocated radicals, were formed after intramolecular 1,5-hydrogen atom transfer, suggesting that any kinetic isotope effect present was not sufficient to offset captodative stabilisation.

  10. 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

  11. Prognostic significance of centrosomal protein 55 in stage I pulmonary adenocarcinoma after radical resection

    PubMed Central

    Jiang, Wenpeng; Chen, Gang; Jia, Yang

    2016-01-01

    Abstract Background Pulmonary adenocarcinoma is a predominant pathologic non‐small cell lung cancer (NSCLC) with a high morbidity in China. Even at histological stage I, many patients still experience recurrence after radical surgery; therefore, it is critical to determine useful indicators to stratify patients according to recurrent risk. Centrosomal protein 55 (CEP55) shares certain characteristics with oncogenes and aberrant expression of CEP55 can lead to tumorigenesis. Therefore, we aimed to clarify the clinicopathological significance and prognostic value of CEP55 in stage I pulmonary adenocarcinoma. Methods We enrolled 106 patients with stage I pulmonary adenocarcinoma who had received complete resection in our study. CEP55 expression levels in the pulmonary tissues of all patients were validated by Western blot analyses and immunohistochemistry. SPSS 17.0 software was employed to analyze the correlation between CEP55 expression and clinicopathological characteristics of patients, as well as prognosis. Results CEP55 overexpression was detected in 67 patients (63.2%). Overexpression is associated with tumor differentiation (P = 0.036), T stage (P = 0.000) and visceral pleural invasion (P = 0.009). Patients with CEP55 overexpression had worse survival compared with those with low expression (P = 0.043). Univariate analysis revealed that T stage (P = 0.000), differentiation degree (P = 0.002), visceral pleural invasion (P = 0.000), and tumor size (P = 0.013) were also significant prognostic factors. Conclusion CEP55 is a useful predicator to improve stratification of patients with stage I pulmonary adenocarcinoma. PMID:27148417

  12. Biological Systems Discovery In Silico: Radical S-Adenosylmethionine Protein Families and Their Target Peptides for Posttranslational Modification▿†

    PubMed Central

    Haft, Daniel H.; Basu, Malay Kumar

    2011-01-01

    Data mining methods in bioinformatics and comparative genomics commonly rely on working definitions of protein families from prior computation. Partial phylogenetic profiling (PPP), by contrast, optimizes family sizes during its searches for the cooccurring protein families that serve different roles in the same biological system. In a large-scale investigation of the incredibly diverse radical S-adenosylmethionine (SAM) enzyme superfamily, PPP aided in building a collection of 68 TIGRFAMs hidden Markov models (HMMs) that define nonoverlapping and functionally distinct subfamilies. Many identify radical SAM enzymes as molecular markers for multicomponent biological systems; HMMs defining their partner proteins also were constructed. Newly found systems include five groupings of protein families in which at least one marker is a radical SAM enzyme while another, encoded by an adjacent gene, is a short peptide predicted to be its substrate for posttranslational modification. The most prevalent, in over 125 genomes, featuring a peptide that we designate SCIFF (six cysteines in forty-five residues), is conserved throughout the class Clostridia, a distribution inconsistent with putative bacteriocin activity. A second novel system features a tandem pair of putative peptide-modifying radical SAM enzymes associated with a highly divergent family of peptides in which the only clearly conserved feature is a run of His-Xaa-Ser repeats. A third system pairs a radical SAM domain peptide maturase with selenocysteine-containing targets, suggesting a new biological role for selenium. These and several additional novel maturases that cooccur with predicted target peptides share a C-terminal additional 4Fe4S-binding domain with PqqE, the subtilosin A maturase AlbA, and the predicted mycofactocin and Nif11-class peptide maturases as well as with activators of anaerobic sulfatases and quinohemoprotein amine dehydrogenases. Radical SAM enzymes with this additional domain, as detected

  13. CO sub 2 ter dot minus radical induced cleavage of disulfide bonds in proteins. A gamma-ray and pulse radiolysis mechanistic investigation

    SciTech Connect

    Favaudon, V.; Tourbez, H.; Lhoste, J-M. ); Houee-Levin, C. )

    1990-12-01

    Disulfide bond reduction by the CO{sub 2}{sup {center dot}{minus}} radical was investigated in aponeocarzinostatin, aporiboflavin-binding protein, and bovine immunoglobulin. Protein-bound cysteine free thiols were formed under {gamma}-ray irradiation in the course of a pH-dependent and protein concentration dependent chain reaction. The chain efficiency increased upon acidification of the medium, with an apparent pK{sub a} around 5, and decreased abruptly below pH 3.6. It decreased also at neutral pH as cysteine accumulated. From pulse radiolysis analysis, CO{sub 2}{sup {center dot}{minus}} proved able to induce rapid one-electron oxidation of thiols and of tyrosine phenolic groups in addition to one-electron donation to exposed disulfide bonds. The bulk rate constant of CO{sub 2}{sup {center dot}{minus}} uptake by the native proteins was 5{minus} to 10-fold faster at pH 3 than at pH 8, and the protonated form of the disulfide radical anion, appeared to be the major protein radical species formed under acidic conditions. Formation of the disulfide radical cation, phenoxyl radical Tyr-O{sup {center dot}} disproportionation, and phenoxyl radical induced oxidation of preformed thiol groups should also be taken into consideration to explain the fate of the oxygen-centered phenoxyl radical.

  14. Radicals in flavoproteins.

    PubMed

    Schleicher, Erik; Weber, Stefan

    2012-01-01

    Current technical and methodical advances in electron paramagnetic resonance (EPR) spectroscopy have proven to be very beneficial for studies of stationary and short-lived paramagnetic states in proteins carrying organic cofactors. In particular, the large number of proteins with flavins as prosthetic groups can be examined splendidly by EPR in all its flavors. To understand how a flavin molecule can be fine-tuned for specific catalysis of different reactions, understanding of its electronic structure mediated by subtle protein-cofactor interactions is of utmost importance. The focus of this chapter is the description of recent research progress from our laboratory on EPR of photoactive flavoproteins. These catalyze a wide variety of important photobiological processes ranging from enzymatic DNA repair to plant phototropism and animal magnetoreception. Whereas increasing structural information on the principal architecture of photoactive flavoproteins is available to date, their primary photochemistry is still largely undetermined. Interestingly, although these proteins carry the same light-active flavin chromophore, their light-driven reactions differ significantly: Formations of photoexcited triplet states and short-lived radical pairs starting out from triplet or singlet-state precursors, as well as generation of stationary radicals have been reported recently. EPR spectroscopy is the method of choice to characterize such paramagnetic intermediates, and hence, to assist in unravelling the mechanisms of these inimitable proteins.

  15. 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

  16. Transient free radicals in iron/oxygen reconstitution of mutant protein R2 Y122F. Possible participants in electron transfer chains in ribonucleotide reductase.

    PubMed

    Sahlin, M; Lassmann, G; Pötsch, S; Sjöberg, B M; Gräslund, A

    1995-05-26

    Ferrous iron/oxygen reconstitution of the mutant R2 apoprotein Y122F leads to formation of a diferric center similar to that of the wild-type R2 protein of Escherichia coli ribonucleotide reductase. This reconstitution reaction requires two extra electrons, supplied or transferred by the protein matrix of R2. We observed several transient free radical species using stopped flow and freeze quench EPR and stopped flow UV-visible spectroscopy. Three of the radicals occur in the time window 0.1-2 s, i.e. concomitant with formation of the diferric site. They include a strongly iron-coupled radical (singlet EPR signal) observed only at < or = 77 K, a singlet EPR signal observed only at room temperature, and a radical at Tyr-356 (light absorption at 410 nm), an invariant residue proposed to be part of an electron transfer chain in catalysis. Three additional transient radicals species are observed in the time window 6 s to 20 min. Two of these are conclusively identified, by specific deuteration, as tryptophan radicals. Comparing side chain geometry and distance to the iron center with EPR characteristics of the radicals, we propose certain Trp residues in R2 as likely to harbor these transient radicals.

  17. 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

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

    PubMed

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

    2010-09-01

    This study initially characterizes the primary photodissociation processes of epichlorohydrin, c-(H(2)COCH)CH(2)Cl. The three dominant photoproduct channels analyzed are c-(H(2)COCH)CH(2)+Cl, c-(H(2)COCH)+CH(2)Cl, and C(3)H(4)O+HCl. In the second channel, the c-(H(2)COCH) photofission product is a higher energy intermediate on C(2)H(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(2)CCO. The final primary photodissociation pathway HCl+C(3)H(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(2)COC)=CH(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(3)H(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((3)P)+allyl bimolecular reaction when the O atom adds to an end C atom. We focus on the HCO+C(2)H(4) and H(2)CO+C(2)H(3) product channels from this radical intermediate in this report. Analysis of the velocity distribution of the momentum-matched signals from the HCO+C(2)H(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(2)CO+C(2)H(3) product channel of the O((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 tangential recoil during the dissociation of highly rotationally excited nascent radicals formed photolytically in this

  19. 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.

  20. Predicting the protein-protein interactions using primary structures with predicted protein surface

    PubMed Central

    2010-01-01

    Background Many biological functions involve various protein-protein interactions (PPIs). Elucidating such interactions is crucial for understanding general principles of cellular systems. Previous studies have shown the potential of predicting PPIs based on only sequence information. Compared to approaches that require other auxiliary information, these sequence-based approaches can be applied to a broader range of applications. Results This study presents a novel sequence-based method based on the assumption that protein-protein interactions are more related to amino acids at the surface than those at the core. The present method considers surface information and maintains the advantage of relying on only sequence data by including an accessible surface area (ASA) predictor recently proposed by the authors. This study also reports the experiments conducted to evaluate a) the performance of PPI prediction achieved by including the predicted surface and b) the quality of the predicted surface in comparison with the surface obtained from structures. The experimental results show that surface information helps to predict interacting protein pairs. Furthermore, the prediction performance achieved by using the surface estimated with the ASA predictor is close to that using the surface obtained from protein structures. Conclusion This work presents a sequence-based method that takes into account surface information for predicting PPIs. The proposed procedure of surface identification improves the prediction performance with an F-measure of 5.1%. The extracted surfaces are also valuable in other biomedical applications that require similar information. PMID:20122202

  1. Everolimus for Primary Intestinal Lymphangiectasia With Protein-Losing Enteropathy.

    PubMed

    Ozeki, Michio; Hori, Tomohiro; Kanda, Kaori; Kawamoto, Norio; Ibuka, Takashi; Miyazaki, Tatsuhiko; Fukao, Toshiyuki

    2016-03-01

    Primary intestinal lymphangiectasia (PIL), also known as Waldmann's disease, is an exudative enteropathy resulting from morphologic abnormalities in the intestinal lymphatics. In this article, we describe a 12-year-old boy with PIL that led to protein-losing enteropathy characterized by diarrhea, hypoalbuminemia associated with edema (serum albumin level: 1.0 g/dL), and hypogammaglobulinemia (serum IgG level: 144 mg/dL). Severe hypoalbuminemia, electrolyte abnormalities, and tetany persisted despite a low-fat diet and propranolol. Everolimus (1.6 mg/m(2)/day) was added to his treatment as an antiangiogenic agent. With everolimus treatment, the patient's diarrhea resolved and replacement therapy for hypoproteinemia was less frequent. Hematologic and scintigraphy findings also improved (serum albumin level: 2.5 g/dL). There were no adverse reactions during the 12-month follow-up. To the best of our knowledge, this is the first report of everolimus use in a patient with PIL. PMID:26908672

  2. Peroxidation radical formation and regiospecificity of recombinated Anabaena sp. lipoxygenase and its effect on modifying wheat proteins.

    PubMed

    Wang, Xiaoming; Lu, Fengxia; Zhang, Chong; Lu, Yingjian; Bie, Xiaomei; Ren, Di; Lu, Zhaoxin

    2014-02-19

    Peroxidation radical formation and the regiospecificity of recombinated lipoxygenase from Anabaena sp. PCC7120 (ana-rLOX) were characterized by using ESR and HPLC-MS. It was found that ana-rLOX oxygenated at the C-13 position of the substrate linoleic acid (LA); at C-13 and C-16 of α-linolenic acid (ALA); at C-9, C-12, and C-15 of arachidonic acid (AA); at C-12, C-15, and C-18 of eicosapentaenoic acid (EPA); and at C-14 and C-16 of docosahexaenoic acid (DHA), respectively. A total of 7, 14, 30, 28, and 18 radical adducts for LA, ALA, AA, EPA, and DHA were respectively identified by HPLC-MS. The functional characteristics of wheat protein, such as foaming capacity (FC), foam stability (FS), emulsifying activity index (EAI), emulsifying stability index (ESI), increased with enzymatic reactions. However, the average particle size of wheat proteins decreased with addition of ana-rLOX/LA. The ana-rLOX was also positivele effective in improving dough properties. These results provided clear evidence that ana-rLOX from Anabaena sp. could effectively improve the quality of wheat flour, which suggested that the enzyme could be applied as flour improver.

  3. 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.

  4. 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…

  5. 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.

  6. 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.

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

    PubMed Central

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

    2015-01-01

    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. Here, 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. 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. PMID:25675500

  8. Using hydroxyl radical footprinting to explore the free energy landscape of protein folding

    PubMed Central

    Calabrese, Antonio N.; Ault, James R.; Radford, Sheena E.; Ashcroft, Alison E.

    2015-01-01

    Characterisation of the conformational states adopted during protein folding, including globally unfolded/disordered structures and partially folded intermediate species, is vital to gain fundamental insights into how a protein folds. In this work we employ fast photochemical oxidation of proteins (FPOP) to map the structural changes that occur in the folding of the four-helical bacterial immunity protein, Im7. Oxidative footprinting coupled with mass spectrometry (MS) is used to probe changes in the solvent accessibility of amino acid side-chains concurrent with the folding process, by quantifying the degree of oxidation experienced by the wild-type protein relative to a kinetically trapped, three-helical folding intermediate and an unfolded variant that lacks secondary structure. Analysis of the unfolded variant by FPOP–MS shows oxidative modifications consistent with the species adopting a solution conformation with a high degree of solvent accessibility. The folding intermediate, by contrast, experiences increased levels of oxidation relative to the wild-type, native protein only in regions destabilised by the amino acid substitutions introduced. The results demonstrate the utility of FPOP–MS to characterise protein variants in different conformational states and to provide insights into protein folding mechanisms that are complementary to measurements such as hydrogen/deuterium exchange labelling and Φ-value analysis. PMID:25746386

  9. [Is it possible to expand the indications for primary radical operations for perforated gastric and duodenal ulcer?].

    PubMed

    Vachev, A N; Adyshirin-Zade, E E; Frolova, E V; Dergal', S V; Kozlov, A A

    2010-01-01

    A retrospective analysis of 365 completed patient records received by the duty surgical hospital with perforated gastric ulcer and duodenal ulcer. All the patients were operated on urgently. Diagnosis of purulent peritonitis was set 17% of patients, "serous"--47.4%, "seroplastic"- 35.6%. 75.3%, perform suturing of perforated openings, regardless of the form of peritonitis. The remaining 24.7%--interventions that address as a perforation, and the impact on the pathogenesis of peptic ulcer. When radical surgery, which were performed in selected patients even with purulent peritonitis, deaths were reported. And when suturing the ulcer defect lethality was 14.5%. In this group of patients were comparable in age and severity of general condition. It is concluded that that the operations of suturing perforated ulcers were used unnecessarily broad, and the majority of patients admitted to hospital with a perforated stomach ulcer and duodenum may increase the volume of surgical benefit for radical treatment of complications as well as most of peptic ulcer.

  10. 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.

  11. 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.

  12. 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. PMID:26340742

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8.

    PubMed

    Jagannathan, Indu; Culver, Gloria M

    2003-07-01

    Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.

  18. 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.

  19. 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.

  20. An oxyferrous heme/protein-based radical intermediate is catalytically competent in the catalase reaction of Mycobacterium tuberculosis catalase-peroxidase (KatG).

    PubMed

    Suarez, Javier; Ranguelova, Kalina; Jarzecki, Andrzej A; Manzerova, Julia; Krymov, Vladimir; Zhao, Xiangbo; Yu, Shengwei; Metlitsky, Leonid; Gerfen, Gary J; Magliozzo, Richard S

    2009-03-13

    A mechanism accounting for the robust catalase activity in catalase-peroxidases (KatG) presents a new challenge in heme protein enzymology. In Mycobacterium tuberculosis, KatG is the sole catalase and is also responsible for peroxidative activation of isoniazid, an anti-tuberculosis pro-drug. Here, optical stopped-flow spectrophotometry, rapid freeze-quench EPR spectroscopy both at the X-band and at the D-band, and mutagenesis are used to identify catalase reaction intermediates in M. tuberculosis KatG. In the presence of millimolar H2O2 at neutral pH, oxyferrous heme is formed within milliseconds from ferric (resting) KatG, whereas at pH 8.5, low spin ferric heme is formed. Using rapid freeze-quench EPR at X-band under both of these conditions, a narrow doublet radical signal with an 11 G principal hyperfine splitting was detected within the first milliseconds of turnover. The radical and the unique heme intermediates persist in wild-type KatG only during the time course of turnover of excess H2O2 (1000-fold or more). Mutation of Met255, Tyr229, or Trp107, which have covalently linked side chains in a unique distal side adduct (MYW) in wild-type KatG, abolishes this radical and the catalase activity. The D-band EPR spectrum of the radical exhibits a rhombic g tensor with dual gx values (2.00550 and 2.00606) and unique gy (2.00344) and gz values (2.00186) similar to but not typical of native tyrosyl radicals. Density functional theory calculations based on a model of an MYW adduct radical built from x-ray coordinates predict experimentally observed hyperfine interactions and a shift in g values away from the native tyrosyl radical. A catalytic role for an MYW adduct radical in the catalase mechanism of KatG is proposed.

  1. 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.

  2. 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. PMID:25463508

  3. Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A.

    PubMed Central

    Shoseyov, O; Takagi, M; Goldstein, M A; Doi, R H

    1992-01-01

    The cbpA gene for the Clostridium cellulovorans cellulose binding protein (CbpA), which is part of the multisubunit cellulase complex, has been cloned and sequenced. When cbpA was expressed in Escherichia coli, proteins capable of binding to crystalline cellulose and of interacting with anti-CbpA were observed. The cbpA gene consists of 5544 base pairs and encodes a protein containing 1848 amino acids with a molecular mass of 189,036 Da. The open reading frame is preceded by a Gram-positive-type ribosome binding site. A signal peptide sequence of 28 amino acids is present at its N terminus. The encoded protein is highly hydrophobic with extremely high levels of threonine and valine residues. There are two types of putative cellulose binding domains of approximately 100 amino acids that are slightly hydrophilic and eight conserved, highly hydrophobic beta-sheet regions of approximately 140 amino acids. These latter hydrophobic regions may be the CbpA domains that interact with the different enzymatic subunits of the cellulase complex. Images PMID:1565642

  4. The primary dynamics in protein folding: the earliest kinetic steps.

    NASA Astrophysics Data System (ADS)

    Callender, Robert

    1996-03-01

    A novel laser-induced temperature jump (T-jump) of 20 C or more is used to initiate the unfolding process of peptides and proteins on the picosecond time scale, and amide I time-resolved infrared absorbance transients are used to characterize the resulting kinetics. We have used this method to study the kinetics of folding and unfolding of a small 21 residue alanine based peptide and molten globule and native states of apomyoglobin, models for the helix which is an basic motif found in proteins. An essential result of our study is that the folding kinetics of a short length of peptide can occur within a few tens of nanoseconds which is much shorter than the time scale of the formation of intramolecular tertiary contacts from one point of a polypeptide chain to another. Furthermore, we observed that helices stabilized by tertiary contact formation unfold slower than helices surrounded by solvent by three orders of magnitude. These results bear directly on the protein folding problem, that is how do proteins fold from a large number of heterogeneous unfolded states to find the specific biologically active folded state on biologically relevent time scales, by suggesting that secondary structure forms first followed by tertiary structure. This work is a collaborative effort with R. GILMANSHIN at City College and S. WILLIAMS, R. B. DYER, and W. H. WOODRUFF at CST-4, Los Alamos National Laboratory, Los Alamos, NM 87545.

  5. Autoregulation of free radicals via uncoupling protein control in pancreatic beta-cell mitochondria.

    PubMed

    Heuett, William J; Periwal, Vipul

    2010-01-20

    Pancreatic beta-cells sense the ambient blood-glucose concentration and secrete insulin to signal other tissues to take up glucose. Mitochondria play a key role in this response as they metabolize nutrients to produce ATP and reactive oxygen species (ROS), both of which are involved in insulin secretion signaling. Based on data available in the literature and previously developed mathematical models, we present a model of glucose-stimulated mitochondrial respiration, ATP synthesis, and ROS production and control in beta-cells. The model is consistent with a number of experimental observations reported in the literature. Most notably, it captures the nonlinear rise in the proton leak rate at high membrane potential and the increase in this leak due to uncoupling protein (UCP) activation by ROS. The functional forms used to model ROS production and UCP regulation yield insight into these mechanisms, as many details have not yet been unraveled in the experimental literature. We examine short- and long-term effects of UCP activation inhibition and changes in the mitochondrial density on mitochondrial responses to glucose. Results suggest increasing mitochondrial density while decreasing UCP activity may be an effective way to increase glucose-stimulated insulin secretion while decreasing oxidative stress.

  6. Rate constant for reaction of vitamin C with protein radicals in γ-irradiated aqueous albumin solution at 295 K

    NASA Astrophysics Data System (ADS)

    Miyazaki, Tetsuo; Yoshimura, Toru; Mita, Kazuya; Suzuki, Keiji; Watanabe, Masami

    1995-02-01

    When an aqueous solution of albumin (0.1 kg dm -3) is irradiated by γ-rays at 295 K, albumin radicals with a long lifetime are observed by ESR. The reaction of vitamin C with the albumin radicals has been studied at 295 K in the albumin solution, which is considered as a model of cells. The rate constant for the reaction of vitamin C with the albumin radicals was measured as 0.014 dm 3 mol -1 s -1, which is much smaller than the reported rate constants (10 6-10 10 dm 3 mol -1 s -1) for the reaction of vitamin C with radicals in a dilute aqueous solution. The small rate constant for the reaction of vitamin C is ascribed to the reaction in polymer coils in the albumin solution, since vitamin C and albumin radicals diffuse very slowly in the coils.

  7. 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.

  8. 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.

  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. Variation in cell signaling protein expression may introduce sampling bias in primary epithelial ovarian cancer.

    PubMed

    Mittermeyer, Gabriele; Malinowsky, Katharina; Beese, Christian; Höfler, Heinz; Schmalfeldt, Barbara; Becker, Karl-Friedrich; Avril, Stefanie

    2013-01-01

    Although the expression of cell signaling proteins is used as prognostic and predictive biomarker, variability of protein levels within tumors is not well studied. We assessed intratumoral heterogeneity of protein expression within primary ovarian cancer. Full-length proteins were extracted from 88 formalin-fixed and paraffin-embedded tissue samples of 13 primary high-grade serous ovarian carcinomas with 5-9 samples each. In addition, 14 samples of normal fallopian tube epithelium served as reference. Quantitative reverse phase protein arrays were used to analyze the expression of 36 cell signaling proteins including HER2, EGFR, PI3K/Akt, and angiogenic pathways as well as 15 activated (phosphorylated) proteins. We found considerable intratumoral heterogeneity in the expression of proteins with a mean coefficient of variation of 25% (range 17-53%). The extent of intratumoral heterogeneity differed between proteins (p<0.005). Interestingly, there were no significant differences in the extent of heterogeneity between phosphorylated and non-phosphorylated proteins. In comparison, we assessed the variation of protein levels amongst tumors from different patients, which revealed a similar mean coefficient of variation of 21% (range 12-48%). Based on hierarchical clustering, samples from the same patient clustered more closely together compared to samples from different patients. However, a clear separation of tumor versus normal tissue by clustering was only achieved when mean expression values of all individual samples per tumor were analyzed. While differential expression of some proteins was detected independently of the sampling method used, the majority of proteins only demonstrated differential expression when mean expression values of multiple samples per tumor were analyzed. Our data indicate that assessment of established and novel cell signaling proteins as diagnostic or prognostic markers may require sampling of serous ovarian cancers at several distinct

  11. Variation in Cell Signaling Protein Expression May Introduce Sampling Bias in Primary Epithelial Ovarian Cancer

    PubMed Central

    Beese, Christian; Höfler, Heinz; Schmalfeldt, Barbara; Becker, Karl-Friedrich; Avril, Stefanie

    2013-01-01

    Although the expression of cell signaling proteins is used as prognostic and predictive biomarker, variability of protein levels within tumors is not well studied. We assessed intratumoral heterogeneity of protein expression within primary ovarian cancer. Full-length proteins were extracted from 88 formalin-fixed and paraffin-embedded tissue samples of 13 primary high-grade serous ovarian carcinomas with 5–9 samples each. In addition, 14 samples of normal fallopian tube epithelium served as reference. Quantitative reverse phase protein arrays were used to analyze the expression of 36 cell signaling proteins including HER2, EGFR, PI3K/Akt, and angiogenic pathways as well as 15 activated (phosphorylated) proteins. We found considerable intratumoral heterogeneity in the expression of proteins with a mean coefficient of variation of 25% (range 17–53%). The extent of intratumoral heterogeneity differed between proteins (p<0.005). Interestingly, there were no significant differences in the extent of heterogeneity between phosphorylated and non-phosphorylated proteins. In comparison, we assessed the variation of protein levels amongst tumors from different patients, which revealed a similar mean coefficient of variation of 21% (range 12–48%). Based on hierarchical clustering, samples from the same patient clustered more closely together compared to samples from different patients. However, a clear separation of tumor versus normal tissue by clustering was only achieved when mean expression values of all individual samples per tumor were analyzed. While differential expression of some proteins was detected independently of the sampling method used, the majority of proteins only demonstrated differential expression when mean expression values of multiple samples per tumor were analyzed. Our data indicate that assessment of established and novel cell signaling proteins as diagnostic or prognostic markers may require sampling of serous ovarian cancers at several

  12. 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

  13. Reactive carbonyls and polyunsaturated fatty acids produce a hydroxyl radical-like species: a potential pathway for oxidative damage of retinal proteins in diabetes.

    PubMed

    Pennathur, Subramaniam; Ido, Yasuo; Heller, Jozsef I; Byun, Jaeman; Danda, Ratna; Pergola, Pablo; Williamson, Joseph R; Heinecke, Jay W

    2005-06-17

    The pattern of oxidized amino acids in aortic proteins of nonhuman primates suggests that a species resembling hydroxyl radical damages proteins when blood glucose levels are high. However, recent studies argue strongly against a generalized increase in diabetic oxidative stress, which might instead be confined to the vascular wall. Here, we describe a pathway for glucose-stimulated protein oxidation and provide evidence of its complicity in diabetic microvascular disease. Low density lipoprotein incubated with pathophysiological concentrations of glucose became selectively enriched in ortho-tyrosine and meta-tyrosine, implicating a hydroxyl radical-like species in protein damage. Model system studies demonstrated that the reaction pathway requires both a reactive carbonyl group and a polyunsaturated fatty acid, involves lipid peroxidation, and is blocked by the carbonyl scavenger aminoguanidine. To explore the physiological relevance of the pathway, we used mass spectrometry and high pressure liquid chromatography to quantify oxidation products in control and hyperglycemic rats. Hyperglycemia raised levels of ortho-tyrosine, meta-tyrosine, and oxygenated lipids in the retina, a tissue rich in polyunsaturated fatty acids. Rats that received aminoguanidine did not show this increase in protein and lipid oxidation. In contrast, rats with diet-induced hyperlipidemia in the absence of hyperglycemia failed to exhibit increased protein and lipid oxidation products in the retina. Our observations suggest that generation of a hydroxyl radical-like species by a carbonyl/polyunsaturated fatty acid pathway might promote localized oxidative stress in tissues vulnerable to diabetic damage. This raises the possibility that antioxidant therapies that specifically inhibit the pathway might delay the vascular complications of diabetes.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27102156

  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. The Radical-Pair Mechanism of Magnetoreception.

    PubMed

    Hore, P J; Mouritsen, Henrik

    2016-07-01

    Although it has been known for almost half a century that migratory birds can detect the direction of the Earth's magnetic field, the primary sensory mechanism behind this remarkable feat is still unclear. The leading hypothesis centers on radical pairs-magnetically sensitive chemical intermediates formed by photoexcitation of cryptochrome proteins in the retina. Our primary aim here is to explain the chemical and physical aspects of the radical-pair mechanism to biologists and the biological and chemical aspects to physicists. In doing so, we review the current state of knowledge on magnetoreception mechanisms. We dare to hope that this tutorial will stimulate new interdisciplinary experimental and theoretical work that will shed much-needed additional light on this fascinating problem in sensory biology. PMID:27216936

  17. 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.

  18. 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

  19. Conserved primary sequences of the DNA terminal proteins of five different human adenovirus groups.

    PubMed

    Green, M; Brackmann, K; Wold, W S; Cartas, M; Thornton, H; Elder, J H

    1979-09-01

    The 31 human adenoviruses (Ad) from five groups (A-E) whose DNAs are <20% homologous by molecular hybridization. Ad5 (group C) DNA contains a 55,000-dalton protein probably covalently bound to each 5' terminus. This covalently bound protein may be analogous to polypeptides found in other viral and nonviral systems that are covalently bound to genomic DNAs or RNAs and that are thought to function in DNA or RNA replication. Because of the importance of proteins linked to nucleic acids, we have investigated whether DNAs from all five groups of human adenoviruses have terminal proteins, as well as the peptide relationships among the different terminal proteins. We show here that DNAs from Ad12, 7, 2, 19, and 4, representing Ad groups A-E, respectively, all contain covalently bound proteins of about 55,000 daltons. To investigate the peptide relatedness among the terminal proteins, we prepared microgram quantities of covalently bound protein from Ads in groups A-E and compared their chymotryptic and tryptic (125)I-labeled peptide maps. We find that the covalently bound protein maps of the five Ad groups are highly related and possibly identical. On the other hand, the tryptic and chymotryptic peptide maps of the major virion protein II and the core proteins V and VII of groups B, C, and E Ads show considerable heterology. Assuming that the covalently bound protein is virally coded, the conserved primary sequence of these proteins suggests a major functional role for the protein in Ad replication. Because the genetic origin of the Ad covalently bound proteins is not established, our data are also consistent with the possibility that the protein is coded by a cellular gene.

  20. Combining polymers with the functionality of proteins: new concepts for atom transfer radical polymerization, nanoreactors and damage self-reporting materials.

    PubMed

    Bruns, Nico; Lörcher, Samuel; Makyła, Katarzyna; Pollarda, Jonas; Renggli, Kasper; Spulber, Mariana

    2013-01-01

    Proteins are macromolecules with a great diversity of functions. By combining these biomolecules with polymers, exciting opportunities for new concepts in polymer sciences arise. This highlight exemplifies the aforementioned with current research results of our group. We review our discovery that the proteins horseradish peroxidase and hemoglobin possess ATRPase activity, i.e. they catalyze atom transfer radical polymerizations. Moreover, a permeabilization method for polymersomes is presented, where the photo-reaction of an α-hydroxyalkylphenone with block copolymer vesicles yields enzyme-containing nanoreactors. A further intriguing possibility to obtain functional nanoreactors is to enclose a polymerization catalyst into the thermosome, a protein cage from the family of chaperonins. Last but not least, fluorescent proteins are discussed as mechanoresponsive molecular sensors that report microdamages within fiber-reinforced composite materials.

  1. 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

  2. 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.

  3. 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. PMID:27635128

  4. 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.

  5. 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

  6. Electromagnetic pulse reduces free radical generation in rat liver mitochondria in vitro.

    PubMed

    Wang, C; Zhou, H; Peng, R; Wang, L; Su, Z; Chen, P; Wang, S; Wang, S; Liu, Y; Cong, J; Wu, K; Hu, X; Fan, E

    2013-04-01

    Non-ionizing radiation electromagnetic pulse (EMP) is generally recorded to induce the generation of free radicals in vivo. Though mitochondria are the primary site to produce free radicals, a rare report is designed to directly investigate the EMP effects on free radical generation at mitochondrial level. Thus the present work was designed to study how EMP induces free radical generation in rat liver mitochondria in vitro using electron paramagnetic resonance technique. Surprisingly, our data suggest that EMP prevents free radical generation by activating antioxidant enzyme activity and reducing oxygen consumption and therefore free radical generation. Electron spin resonance measurements clearly demonstrate that disordering of mitochondrial lipid fluidity and membrane proteins mobility are the underlying contributors to this decreased oxygen consumption. Therefore, our results suggest that EMP might hold the potentiality to be developed as a non-invasive means to benefit certain diseases.

  7. 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

    2014-06-01

    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.

  8. 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

  9. 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

  10. Protein composition and movements of membrane swellings associated with primary cilia

    PubMed Central

    Mohieldin, Ashraf M.; Haymour, Hanan S.; Lo, Shao T.; AbouAlaiwi, Wissam A.; Atkinson, Kimberly F.; Ward, Christopher J.; Gao, Min; Wessely, Oliver

    2015-01-01

    Dysfunction of many ciliary proteins has been linked to a list of diseases, from cystic kidney to obesity and from hypertension to mental retardation. We previously proposed that primary cilia are unique communication organelles that function as microsensory compartments that house mechanosensory molecules. Here we report that primary cilia exhibit membrane swellings or ciliary bulbs, which based on their unique ultrastructure and motility, could be mechanically regulated by fluid-shear stress. Together with the ultrastructure analysis of the swelling, which contains monosialodihexosylganglioside (GM3), our results show that ciliary bulb has a distinctive set of functional proteins, including GM3 synthase (GM3S), bicaudal-c1 (Bicc1), and polycystin-2 (PC2). In fact, results from our cilia isolation demonstrated for the first time that GM3S and Bicc1 are members of the primary cilia proteins. Although these proteins are not required for ciliary membrane swelling formation under static condition, fluid-shear stress induced swelling formation is partially modulated by GM3S. We therefore propose that the ciliary bulb exhibits a sensory function within the mechano-ciliary structure. Overall, our studies provided an important step towards understanding the ciliary bulb function and structure. PMID:25650235

  11. 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.

  12. Primary structures of two proteins from the venom of the Mexican red knee tarantula (Brachypelma smithii).

    PubMed

    Kaiser, I I; Griffin, P R; Aird, S D; Hudiburg, S; Shabanowitz, J; Francis, B; John, T R; Hunt, D F; Odell, G V

    1994-09-01

    Venom of the Mexican red knee tarantula (Brachypelma smithii) was fractionated by gel filtration over Sephadex G-50 Fine. Small polypeptides present in the second and third peaks were subfractionated by cation exchange and reversed-phase FPLC. One major, basic protein was isolated and sequenced from each G-50 fraction using a gas-phase protein sequencer. Primary structures were completed and confirmed using tandem mass spectrometry and carboxypeptidase digestions. Protein 1 contains 39 residues, including six cysteine residues in three disulfide bonds. It is identical to one of the isoforms of ESTX from the venom of the tarantula Eurypelma californicum. Brachypelma smithii Protein 5 contains 34 residues, including six cysteine residues in three disulfide bonds. Disulfide bond assignments for both proteins are provided. Protein 5 shows most similarity with toxin Tx2-9 from the Brazilian 'armed' spider, but only displays 41% sequence identity. Similarities with other proteins are lower. Proteins 1 and 5 appear unrelated to each other. PMID:7801344

  13. Radical Hysterectomy

    MedlinePlus

    ... the base of her partner’s penis during intercourse. Orgasm after radical hysterectomy Women who have had a ... the surgery will affect their ability to have orgasms. This has not been studied a great deal, ...

  14. Quantitative Correlation between the protein primary sequences and secondary structures in spider dragline silks.

    PubMed

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

    2010-01-11

    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 beta-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

  15. Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.

    PubMed

    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 g(1)-value of 2.0090 for the tyrosyl radical was extracted. This g(1)-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, ν(7a) = 1500 cm(-1)) was found to be insensitive to deuterium-oxide exchange. Additionally, the (18)O-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 g(1)-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

  16. 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.

  17. Reduced Glutamate Decarboxylase 65 Protein Within Primary Auditory Cortex Inhibitory Boutons in Schizophrenia

    PubMed Central

    Moyer, Caitlin E.; Delevich, Kristen M.; Fish, Kenneth N.; Asafu-Adjei, Josephine K.; Sampson, Allan R.; Dorph-Petersen, Karl-Anton; Lewis, David A.; Sweet, Robert A.

    2012-01-01

    Background Schizophrenia is associated with perceptual and physiological auditory processing impairments that may result from primary auditory cortex excitatory and inhibitory circuit pathology. High-frequency oscillations are important for auditory function and are often reported to be disrupted in schizophrenia. These oscillations may, in part, depend on upregulation of gamma-aminobutyric acid synthesis by glutamate decarboxylase 65 (GAD65) in response to high interneuron firing rates. It is not known whether levels of GAD65 protein or GAD65-expressing boutons are altered in schizophrenia. Methods We studied two cohorts of subjects with schizophrenia and matched control subjects, comprising 27 pairs of subjects. Relative fluorescence intensity, density, volume, and number of GAD65-immunoreactive boutons in primary auditory cortex were measured using quantitative confocal microscopy and stereologic sampling methods. Bouton fluorescence intensities were used to compare the relative expression of GAD65 protein within boutons between diagnostic groups. Additionally, we assessed the correlation between previously measured dendritic spine densities and GAD65-immunoreactive bouton fluorescence intensities. Results GAD65-immunoreactive bouton fluorescence intensity was reduced by 40% in subjects with schizophrenia and was correlated with previously measured reduced spine density. The reduction was greater in subjects who were not living independently at time of death. In contrast, GAD65-immunoreactive bouton density and number were not altered in deep layer 3 of primary auditory cortex of subjects with schizophrenia. Conclusions Decreased expression of GAD65 protein within inhibitory boutons could contribute to auditory impairments in schizophrenia. The correlated reductions in dendritic spines and GAD65 protein suggest a relationship between inhibitory and excitatory synapse pathology in primary auditory cortex. PMID:22624794

  18. Molecular cloning and primary structure of human glial fibrillary acidic protein

    SciTech Connect

    Reeves, S.A.; Helman, L.J.; Allison, A.; Israel, M.A. )

    1989-07-01

    Glial fibrillary acidic protein (GFAP) is an intermediate-filament (IF) protein that is highly specific for cells of astroglial lineage, although its tissue-specific role is speculative. Determination of the primary structure of this protein should be of importance for understanding the functional role it plays in astroglia. Therefore, the authors isolated a cDNA clone encoding this protein and determined its nucleotide sequence. The predicted amino acid sequence indicates that GFAP shares structural similarities-particularly in the central rod domain and to a lesser degree in the carboxyl-terminal domain-with other IF proteins found in nonepithelial cell types. Considerable sequence divergence in the amino-terminal region of GFAP suggests that the tissue-specific functions of this IF protein might be mediated through this region of the molecule. In contrast, conservation of structural characteristics and a moderate degree of sequence conservation in the carboxyl-terminal region suggest functional similarities. Blot hybridization analysis using the GFAP cDNA as a probe failed to detect GFAP mRNA in both normal and neoplastic human tissues in which IF proteins other than GFAP are known to be expressed.

  19. Zinc Regulates a Switch between Primary and Alternative S18 Ribosomal Proteins in Mycobacterium tuberculosis

    PubMed Central

    Prisic, Sladjana; Hwang, Hyonson; Dow, Allexa; Barnaby, Omar; Pan, Tenny S.; Lonzanida, Jaymes A.; Chazin, Walter J.; Steen, Hanno; Husson, Robert N.

    2015-01-01

    SUMMARY The Mycobacterium tuberculosis genome encodes five putative “alternative” ribosomal proteins whose expression is repressed at high Zn2+ concentration. Each alternative protein has a primary homolog that is predicted to bind Zn2+. We hypothesized that zinc triggers a switch between these paired homologous proteins and therefore chose one of these pairs, S18-1/S18-2, to study mechanisms of the predicted competition for their incorporation into ribosomes. As predicted, our data show that Zn2+-depletion causes accumulation of both S18-2 mRNA and protein. In contrast, S18-1 mRNA levels are unchanged to slightly elevated under Zn2+-limited conditions. However the amount of S18-1 protein is markedly decreased. We further demonstrate that both S18 proteins interact with ribosomal protein S6, a committed step in ribosome biogenesis. Zn2+ is absolutely required for the S18-1/S6 interaction, while it is dispensable for S18-2/S6 dimer formation. These data suggest a model in which the S18-1 is the dominant ribosome constituent in high zinc conditions, e.g. inside of phagosomes, but that it can be replaced by S18-2 when zinc is deficient, e.g. in the extracellular milieu. Consequently, Zn2+-depletion may serve as a signal for building alternative ribosomes when M. tuberculosis is released from macrophages, to allow survival in the extracellular environment. PMID:25858183

  20. Management of patients with respiratory infections in primary care: procalcitonin, C-reactive protein or both?

    PubMed

    Meili, Marc; Müller, Beat; Kulkarni, Prasad; Schütz, Philipp

    2015-10-01

    Use of inflammatory biomarkers to guide antibiotic decisions has shown promising results in the risk-adapted management of respiratory tract infections, mainly in the inpatient setting. Several observational and interventional trials have investigated the benefits of procalcitonin (PCT) and C-reactive protein (CRP) testing in primary care. Both markers have shown promising results, although CRP is an inflammatory biomarker while PCT is more specific for bacterial infections. For CRP, point-of-care testing is widely established. Recently, sensitive point-of-care testing for PCT has also become available. A high-quality trial comparing these two markers for the management of patients in primary care is currently lacking. The aim of this paper is to review the existing literature investigating the use of PCT and CRP in primary care. The authors compare their performance for guiding antibiotic stewardship and analyze the cut-off values and endpoints to put these parameters into context in a low-acuity environment.

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. Role of primary constitutive phosphorylation of Sendai virus P and V proteins in viral replication and pathogenesis.

    PubMed

    Hu, C J; Kato, A; Bowman, M C; Kiyotani, K; Yoshida, T; Moyer, S A; Nagai, Y; Gupta, K C

    1999-10-10

    Functional analysis of the primary constitutive phosphorylation of Sendai virus P and V proteins was performed using both in vitro and in vivo systems. Sendai virus minigenome transcription and replication in transfected cells were not significantly affected in the presence of primary phosphorylation deficient P protein (S249A, S249D, P250A) as measured by either the luciferase activity or the Northern blot analysis. Similarly, recombinant Sendai viruses lacking the primary phosphorylation in P grew to titers close to the wild-type virus in cell cultures and in the natural host of Sendai virus, the mouse. Mutant viruses showed no altered pathogenesis in mice lungs. Oligomerization of P by binding WT P or mutant P to GST-P (WT) Sepharose beads revealed that the primary phosphorylation was not crucial for P protein oligomerization. Similar to P protein primary phosphorylation, the V protein primary phosphorylation at serine249 was not essential for minigenome transcription and replication, as both WT and mutant V proteins were found equally inhibitory to the minigenome replication. These results show that the primary phosphorylation of P protein has no essential role in Sendai virus transcription, replication, and pathogenesis.

  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. 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

  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

    2014-06-01

    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. PMID:24908009

  10. The use of cationic nanogels to deliver proteins to myeloma cells and primary T lymphocytes that poorly express heparan sulfate.

    PubMed

    Watanabe, Kozo; Tsuchiya, Yumiko; Kawaguchi, Yoshinori; Sawada, Shin-ichi; Ayame, Hirohito; Akiyoshi, Kazunari; Tsubata, Takeshi

    2011-09-01

    Fusion proteins containing protein transduction domain (PTD) are widely used for intracellular delivery of exogenous proteins. PTD-mediated delivery requires expression of heparan sulfate on the surface of the target cells. However, some of metastatic tumor cells and primary lymphocytes poorly express heparan sulfate. Here we demonstrate that proteins complexed with nanosize hydrogels formed by cationic cholesteryl group-bearing pullulans (cCHP) are efficiently delivered to myeloma cells and primary CD4(+) T lymphocytes probably by induction of macropinocytosis, although these cells are resistant to PTD-mediated protein delivery as a consequence of poor heparan sulfate expression. The anti-apoptotic protein Bcl-xL delivered by cCHP nanogels efficiently blocked apoptosis of these cells, establishing functional regulation of cells by proteins delivered by cCHP nanogels. Thus, cCHP nanogel is a useful tool to deliver proteins for development of new cancer therapy and immune regulation. PMID:21605901

  11. 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.

  12. Antifreeze proteins in the primary urine of larvae of the beetle Dendroides canadensis.

    PubMed

    Nickell, Philip K; Sass, Sandra; Verleye, Dawn; Blumenthal, Edward M; Duman, John G

    2013-05-01

    To avoid freezing while overwintering beneath the bark of fallen trees, Dendroides canadensis (Coleoptera: Pyrochroidae) larvae produce a family of antifreeze proteins (DAFPs) that are transcribed in specific tissues and have specific compartmental fates. DAFPs and associated thermal hysteresis activity (THA) have been shown previously in hemolymph and midgut fluid, but the presence of DAFPs has not been explored in primary urine, a potentially important site that can contain endogenous ice-nucleating compounds that could induce freezing. A maximum mean THA of 2.65±0.33°C was observed in primary urine of winter-collected D. canadensis larvae. THA in primary urine increased significantly through autumn, peaked in the winter and decreased through spring to levels of 0.2-0.3°C in summer, in a pattern similar to that of hemolymph and midgut fluid. THA was also found in hindgut fluid and excreted rectal fluid, suggesting that these larvae not only concentrate AFPs in the hindgut, but also excrete AFPs from the rectal cavity. Based on dafp transcripts isolated from Malpighian tubule epithelia, cDNAs were cloned and sequenced, identifying the presence of transcripts encoding 24 DAFP isoforms. Six of these Malpighian tubule DAFPs were known previously, but 18 are new. We also provide functional evidence that DAFPs can inhibit ice nucleators present in insect primary urine. This is potentially critical because D. canadensis larvae die if frozen, and therefore ice formation in any body fluid, including the urine, would be lethal.

  13. Antiviral, immunomodulatory, and free radical scavenging activities of a protein-enriched fraction from the larvae of the housefly, Musca domestica.

    PubMed

    Ai, Hui; Wang, Furong; Zhang, Na; Zhang, Lingyao; Lei, Chaoliang

    2013-01-01

    In our previous study, protein-enriched fraction (PEF) that was isolated from the larvae of the housefly, Musca domestica L. (Diptera: Muscidae), showed excellent hepatoprotective activity as well as the potential for clinical application in therapy for liver diseases. In this study, antiviral, immunomodulatory, and free radical scavenging activities of PEF were evaluated. The antiviral results demonstrated that PEF inhibited the infection of avian influenza virus H9N2 and had a virucidal effect against the multicapsid nucleopolyhedrovirus of the alfalfa looper, Autographa californica Speyer (Lepidoptera: Noctuidae) in vitro. The mortality of silkworm larve in a PEF treatment group decreased significantly compared with a negative control. PEF showed excellent scavenging activity for 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radicals, which were similar to those of ascorbic acid. The imunomodulatory results suggested that PEF could effectively improve immune function in experimental mice. Our results indicated that PEF could possibly be used for the prophylaxis and treatment of diseases caused by avian influenza virus infection. In addition, PEF with virucidal activity against insect viruses might provide useful for the development of antimicrobial breeding technology for economically important insects. As a natural product from insects, PEF could be a potential source for the discovery of potent antioxidant and immunomodulatory agents.

  14. TP53 gene mutations and protein accumulation in primary vaginal carcinomas.

    PubMed Central

    Skomedal, H.; Kristensen, G.; Helland, A.; Nesland, J. M.; Kooi, S.; Børresen, A. L.; Holm, R.

    1995-01-01

    Primary carcinomas from 46 patients were screened for TP53 alterations. Immunohistochemistry demonstrated nuclear TP53 protein accumulation in 22 (48%) cases using the polyclonal CM1 antiserum, whereas 15 (33%) cases showed positive nuclear staining with the mononuclear antibody PAb 1801. Constant denaturant gel electrophoresis (CDGE) was used to screen 27 of the vaginal carcinomas for mutations in the conserved regions of the TP53 gene (exons 5-8). Six of these tumours (22%) contained mutations: four were found in exon 5 and two in exon 8. A total of 50% of the primary vaginal carcinomas carried a TP53 alteration. These results indicate that TP53 abnormalities may be involved in the development of these tumours. However, there was no significant association between TP53 abnormalities and survival. Images Figure 1 Figure 2 PMID:7599041

  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. Modulation of primary antibody response by protein A in tumor bearing mice.

    PubMed

    Zaidi, S I; Singh, K P; Raisuddin, S; Jafri, A; Saxena, A K; Choudhary, S; Ray, P K

    1995-11-01

    Protein A (PA) is a cell wall glycoprotein of Staphylococcus aureus Cowan I, which possess a number of immunomodulatory and antitumor properties. We have previously shown that PA suppresses the anti-sheep erythrocyte primary antibody response in normal mice. The present investigation evaluates the effect of protein A on the anti-sheep erythrocyte primary antibody response in tumor-bearing mice. The primary antibody response in tumor-bearing mice immunized with sheep red blood cells (SRBC) was suppressed by the intraperitoneal administration of PA in a dose-dependent fashion. The plaque forming cell (PFC) assay was used to assess this response. Maximum suppression of the PFC response was observed at 12 micrograms PA/animal (p < 0.001) and could be observed at doses as low as 1 microgram PA/animal (p < 0.01). The amount of suppression was proportional to the number of PA doses administered. In addition this effect was critically dependent on the timing of PA administration. PA showed no significant effect on PFC when injected after immunization, but it produced pronounced suppression when injected prior to the immunization with SRBC. Maximum suppression of the PFC response was observed when PA was administered one day before the antigen challenge. PA also reduced splenic localization of 51Cr labeled SRBC to 42% (p < 0.01). The altered localization of antigen in spleen may be responsible for reduced PFC response in tumor-bearing mice. Depletion of B-lymphocyte is reported to exhibit tumor inhibition. Therefore, we propose that the suppression of the primary antibody response by PA helps in tumor regression by reducing the soluble immunosuppressive immune complexes. PMID:8537611

  17. Expression of apoptosis regulatory proteins of the Bcl-2 family and p53 in primary resected non-small-cell lung cancer

    PubMed Central

    Borner, M M; Brousset, P; Pfanner-Meyer, B; Bacchi, M; Vonlanthen, S; Hotz, M A; Altermatt, H J; Schlaifer, D; Reed, J C; Betticher, D C

    1999-01-01

    Proteins of the Bcl-2 family as well as p53 are important regulators of apoptosis. Alterations in the expression of these proteins can contribute to the formation of cancer, as well as influence tumour response to chemo- and radiotherapy. We used antibodies specific for the human Bcl-2, Mcl-1, Bax, Bak and p53 proteins to examine the expression of these apoptosis-regulating genes in 49 archival specimens of patients with radically resected non-small-cell lung cancer (NSCLC). Tumour cells containing immunostaining for the antiapoptotic proteins Bcl-2 and Mcl-1 were present in 31% and 58% of the cases evaluated, respectively, whereas immunopositivity for the proapoptotic proteins Bax and Bak was found in 47% and 58% of the samples. p53 immunopositivity was detected in 61% of the samples. The expression of Bcl-2 and p53 and the expression of Mcl-1 and Bax showed a positive association (P= 0.02 and P= 0.06 respectively), whereas the expression of Bax was inversely related to p53 (P= 0.008). The expression of Bcl-2 had a negative influence on relapse-free survival in this population of primary resected NSCLC patients (P= 0.02). The expression of p53 and Bcl-2 was significantly associated with metastasis-free survival (P< 0.01). Only patients with p53-positive tumours developed metastases during the follow-up period. Our results establish the frequent expression of the Bcl-2 family proteins Bcl-2, Mcl-1, Bax and Bak in NSCLC. It can be expected that Bcl-2 family members have no straightforward impact on clinical outcome in this disease because their interactions in the regulation of apoptosis are complex. © 1999 Cancer Research Campaign PMID:10070896

  18. 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.

  19. Developmental changes of neuron-specific enolase and neurofilament proteins in primary neural culture.

    PubMed

    Schilling, K; Scherbaum, C; Pilgrim, C

    1988-01-01

    The expression of neuron-specific enolase (NSE) and neurofilament (NF) proteins in primary dissociated cell cultures derived from 14-day-old fetal rat diencephalon was studied by immunocytochemistry and quantitative western-blot techniques. Both neuronal marker proteins, NSE and NF, can be detected as early as day 2 in vitro. They show pronounced quantitative increases during the time period studied (12 days), the relative change being highest during the first few days in vitro (DIV). The molar ratio of the medium weight NF to the heavy NF polypeptide is 9.1 after 2 DIV and 2.6 after 12 DIV. Phosphorylation of the heavy NF polypeptide increases steadily during cultivation. Comparison of these results to in vivo data reported in the literature suggests that, qualitatively, neuronal development in vitro follows the pattern observed in vivo, but at an accelerated pace.

  20. Refinement of the primary hydration shell model for molecular dynamics simulations of large proteins

    PubMed Central

    Hamaneh, Mehdi Bagheri; Buck, Matthias

    2009-01-01

    A realistic representation of water molecules is important in molecular dynamics simulation of proteins. However, the standard method of solvating biomolecules, i.e. immersing them in a box of water with periodic boundary conditions, is computationally expensive. The primary hydration shell (PHS) method, developed more than a decade ago and implemented in CHARMM, uses only a thin shell of water around the system of interest, and so greatly reduces the computational cost of simulations. Applying the PHS method, especially to larger proteins, revealed that further optimization and a partial reworking was required and here we present several improvements to its performance. The model is applied to systems with different sizes, and both water and protein behaviors are compared with those observed in standard simulations with periodic boundary conditions and, in some cases, with experimental data. The advantages of the modified PHS method over its original implementation are clearly apparent when it is applied to simulating the 82 KD protein Malate Synthase G. PMID:19382175

  1. 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. PMID:26116512

  2. Photochemical Tyrosine Oxidation in the Structurally Well-Defined α3Y Protein: Proton-Coupled Electron Transfer and a Long-Lived Tyrosine Radical

    PubMed Central

    2014-01-01

    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 × 104 M–1 s–1 (pH 5.5), 1.8 × 105 M–1 s–1 (pH 8.5), 5.4 × 103 M–1 s–1 (pD 5.5), and 4.0 × 104 M–1 s–1 (pD 8.5). kH/kD 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. PMID:25121576

  3. The protein environment surrounding tyrosyl radicals D. and Z. in photosystem II: a difference Fourier-transform infrared spectroscopic study.

    PubMed Central

    Kim, S; Barry, B A

    1998-01-01

    Photosystem II contains two redox-active tyrosine residues, termed D and Z, which have different midpoint potentials and oxidation/reduction kinetics. To understand the functional properties of redox-active tyrosines, we report a difference Fourier-transform infrared (FT-IR) spectroscopic study of these species. Vibrational spectra associated with the oxidation of each tyrosine residue are acquired; electron paramagnetic resonance (EPR) and fluorescence experiments demonstrate that there is no detectable contribution of Q(A)- to these spectra. Vibrational lines are assigned to the radicals by isotopic labeling of tyrosine. Global 15N labeling, 2H exchange, and changes in pH identify differences in the reversible interactions of the two redox-active tyrosines with N-containing, titratable amino acid side chains in their environments. To identify the amino acid residue that contributes to the spectrum of D, mutations at His189 in the D2 polypeptide were examined. Mutations at this site result in substantial changes in the spectrum of tyrosine D. Previously, mutations at the analogous histidine, His190 in the D1 polypeptide, were shown to have no significant effect on the FT-IR spectrum of tyrosine Z (Bernard, M. T., et al. 1995. J. Biol. Chem. 270:1589-1594). A disparity in the number of accessible, proton-accepting groups could influence electron transfer rates and energetics and account for functional differences between the two redox-active tyrosines. PMID:9591683

  4. 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.

  5. 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. PMID:27457987

  6. Effect of single mutations on the specificity of Escherichia coli FPG protein for excision of purine lesions from DNA damaged by free radicals.

    PubMed

    Sidorkina, O; Dizdaroglu, M; Laval, J

    2001-09-15

    The formamidopyrimidine N-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that is specific for the removal of purine-derived lesions from DNA damaged by free radicals and other oxidative processes. We investigated the effect of single mutations on the specificity of this enzyme for three purine-derived lesions in DNA damaged by free radicals. These damaging agents generate a multiplicity of base products in DNA, with the yields depending on the damaging agent. Wild type Fpg protein (wt-Fpg) removes 8-hydroxyguanine (8-OH-Gua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from damaged DNA with similar specificities. We generated five mutant forms of this enzyme with mutations involving Lys-57-->Gly (FpgK57G), Lys-57-->Arg (FpgK57R), Lys-155-->Ala (FpgK155A), Pro-2-->Gly (FpgP2G), and Pro-2-->Glu (FpgP2E), and purified them to homogeneity. FpgK57G and FpgK57R were functional for removal of FapyAde and FapyGua with a reduced activity when compared with wt-Fpg. The removal of 8-OH-Gua was different in that the specificity of FpgK57G was significantly lower for its removal from irradiated DNA, whereas wt-Fpg, FpgK57G, and FpgK57R excised 8-OH-Gua from H2O2/Fe(III)-EDTA/ascorbic acid-treated DNA with almost the same specificity. FpgK155A and FpgP2G had very low activity and FpgP2E exhibited no activity at all. Michaelis-Menten kinetics of excision was measured and kinetic constants were obtained. The results indicate an important role of Lys-57 residue in the activity of Fpg protein for 8-OH-Gua, but a lesser significant role for formamidopyrimidines. Mutations involving Lys-155 and Pro-2 had a dramatic effect with Pro-2-->Glu leading to complete loss of activity, indicating a significant role of these residues. The results show that point mutations significantly change the specificity of Fpg protein and suggest that point mutations are also expected to change specificities of other DNA

  7. Autoinhibition and Signaling by the Switch II Motif in the G-protein Chaperone of a Radical B12 Enzyme*

    PubMed Central

    Lofgren, Michael; Koutmos, Markos; Banerjee, Ruma

    2013-01-01

    MeaB is an accessory GTPase protein involved in the assembly, protection, and reactivation of 5′-deoxyadenosyl cobalamin-dependent methylmalonyl-CoA mutase (MCM). Mutations in the human ortholog of MeaB result in methylmalonic aciduria, an inborn error of metabolism. G-proteins typically utilize conserved switch I and II motifs for signaling to effector proteins via conformational changes elicited by nucleotide binding and hydrolysis. Our recent discovery that MeaB utilizes an unusual switch III region for bidirectional signaling with MCM raised questions about the roles of the switch I and II motifs in MeaB. In this study, we addressed the functions of conserved switch II residues by performing alanine-scanning mutagenesis. Our results demonstrate that the GTPase activity of MeaB is autoinhibited by switch II and that this loop is important for coupling nucleotide-sensitive conformational changes in switch III to elicit the multiple chaperone functions of MeaB. Furthermore, we report the structure of MeaB·GDP crystallized in the presence of AlFx− to form the putative transition state analog, GDP·AlF4−. The resulting crystal structure and its comparison with related G-proteins support the conclusion that the catalytic site of MeaB is incomplete in the absence of the GTPase-activating protein MCM and therefore unable to stabilize the transition state analog. Favoring an inactive conformation in the absence of the client MCM protein might represent a strategy for suppressing the intrinsic GTPase activity of MeaB in which the switch II loop plays an important role. PMID:23996001

  8. Folding and function of the myelin proteins from primary sequence data.

    PubMed

    Inouye, H; Kirschner, D A

    1991-01-01

    To explain how the myelin proteins are involved in the organization and function of the myelin sheath requires knowing their molecular structures. Except for P2 basic protein of PNS myelin, however, their structures are not yet known. As an aid to predicting their molecular folding and possible functions, we have developed a FORTRAN program to analyze the primary sequence data for proteins, and have applied this to the myelin proteins in particular. In this program, propensities for the secondary structure conformations as well as physical-chemical parameters are assigned to the amino acids and the pattern of these parameters is examined by calculating their average values, autocorrelation functions and Fourier transforms. To compare two proteins, their sequences are aligned using a unitary scoring matrix, and homologies are searched by plotting a two-dimensional map of the correlation coefficients. Comparison of the corresponding myelin basic proteins (MBP) and P0 glycoproteins (P0) for rodent and shark showed that the conserved residues included most of the amino acids which were predicted to form the alpha or beta conformations, while the altered residues were mainly in the hydrophilic and turn or coil regions. In both rodent and shark the putative extracellular domain of P0 glycoprotein displayed consecutive peaks of beta propensity similar to that for the immunoglobulins, while the cytoplasmic domain showed alpha-beta-alpha folding. To trace the immunoglobulin fold along the P0 sequence, we compared the beta propensity curve of P0 with that of the immunoglobulin M603, whose three-dimensional structure has been determined. We propose that the flat beta-sheets of P0 are orientated parallel to the membrane surface to facilitate their homotypic interaction in the extracellular space. An extra beta-fold in the extracellular domain of shark P0 compared with rodent P0 was found, and this may result in a greater attraction between the apposed extracellular surfaces

  9. 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

  10. Mass Spectrometry-Based Proteomics for Relative Protein Quantification and Biomarker Identification in Primary Human Hepatocytes.

    PubMed

    Dietz, Lisa; Sickmann, Albert

    2015-01-01

    Liquid chromatography-tandem mass spectrometry-based proteomics is a highly sensitive and effective tool to identify and quantify potential biomarkers in repeated dose toxicity studies using primary cell culture systems. In this respect, 8-plex isobaric tag for relative and absolute quantification labeling is the method of choice for relative quantification. After cell lysis and tryptic protein digestion, an individual isobaric tag is added to the amine groups of arginine and lysine. Then, up to eight differentially labeled samples are mixed and analyzed together in a mass spectrometry experiment. During peptide fragmentation in the mass spectrometer, the individual tag intensity of each identified peptide could be detected, reflecting the peptide intensities in the eight samples. The identified peptides are matched to their specific protein using specific search engines and finally to eight individual relative protein quantities. The two-dimensional fractionation of complex peptide mixtures minimizes the possibility of co-fragmentation of peptides from different origin in the mass spectrometer, which leads to a higher number of peptide search matches and therefore to better identification and quantification results.

  11. 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.

  12. Human Primary Keratinocytes as a Tool for the Analysis of Caspase-1-Dependent Unconventional Protein Secretion.

    PubMed

    Strittmatter, Gerhard E; Garstkiewicz, Martha; Sand, Jennifer; Grossi, Serena; Beer, Hans-Dietmar

    2016-01-01

    Inflammasomes comprise a group of protein complexes, which activate the protease caspase-1 upon sensing a variety of stress factors. Active caspase-1 in turn cleaves and thereby activates the pro-inflammatory cytokines prointerleukin (IL)-1β and -18, and induces unconventional protein secretion (UPS) of mature IL-1β, IL-18, as well as of many other proteins involved in and required for induction of inflammation. Human primary keratinocytes (HPKs) represent epithelial cells able to activate caspase-1 in an inflammasome-dependent manner upon irradiation with a physiological dose of ultraviolet B (UVB) light. Here, we describe the isolation of keratinocytes from human skin, their cultivation, and induction of caspase-1-dependent UPS upon UVB irradiation as well as its siRNA- and chemical-mediated inhibition. In contrast to inflammasome activation of professional immune cells, UVB-irradiated HPKs represent a robust and physiological cell culture system for the analysis of UPS induced by active caspase-1. PMID:27665556

  13. Free Radical Scavenging Activity: Antiproliferative and Proteomics Analyses of the Differential Expression of Apoptotic Proteins in MCF-7 Cells Treated with Acetone Leaf Extract of Diospyros lycioides (Ebenaceae).

    PubMed

    Pilane, M C; Bagla, V P; Mokgotho, M P; Mbazima, V; Matsebatlela, T M; Ncube, I; Mampuru, L

    2015-01-01

    Breast cancer is the most common cancer in South Africa. The acetone leaf extract of Diospyros lycioides was evaluated qualitatively and quantitatively for its antioxidant potential using DPPH assay and nitric oxide radical scavenging effect, while the viability of MCF-7 cells was evaluated using the MTT. MCF-7 treated cells were stained with Hoechst 335258 dye and annexin-V-FITC to be evaluated for apoptotic effect of the extract, while mRNA expression levels of apoptotic genes were assessed by quantitative real-time PCR and deferential protein expression levels using 2D gel electrophoresis and mass spectrometry. Results revealed presence of antioxidant constituents in the extract. Extract was shown to be cytotoxic in a concentration- and time-dependent manner. Cytotoxicity was demonstrated to be due to apoptosis, with 70% of the extract-treated cells being annexin-V-positive/PI negative at 48 hours. The extract was also shown to upregulate the expression of p53 gene with concomitant downregulation of the Bcl-2 antiapoptotic gene while differentially expressed proteins were identified as enolase, pyruvate kinase, and glyceraldehyde-3-phosphate. The extract in this study was shown to induce apoptosis at an early stage which makes it an ideal source that can be explored for compounds that may be used in the treatment and management of cancer. PMID:26457109

  14. Free Radical Scavenging Activity: Antiproliferative and Proteomics Analyses of the Differential Expression of Apoptotic Proteins in MCF-7 Cells Treated with Acetone Leaf Extract of Diospyros lycioides (Ebenaceae)

    PubMed Central

    Pilane, M. C.; Bagla, V. P.; Mokgotho, M. P.; Mbazima, V.; Matsebatlela, T. M.; Ncube, I.; Mampuru, L.

    2015-01-01

    Breast cancer is the most common cancer in South Africa. The acetone leaf extract of Diospyros lycioides was evaluated qualitatively and quantitatively for its antioxidant potential using DPPH assay and nitric oxide radical scavenging effect, while the viability of MCF-7 cells was evaluated using the MTT. MCF-7 treated cells were stained with Hoechst 335258 dye and annexin-V-FITC to be evaluated for apoptotic effect of the extract, while mRNA expression levels of apoptotic genes were assessed by quantitative real-time PCR and deferential protein expression levels using 2D gel electrophoresis and mass spectrometry. Results revealed presence of antioxidant constituents in the extract. Extract was shown to be cytotoxic in a concentration- and time-dependent manner. Cytotoxicity was demonstrated to be due to apoptosis, with 70% of the extract-treated cells being annexin-V-positive/PI negative at 48 hours. The extract was also shown to upregulate the expression of p53 gene with concomitant downregulation of the Bcl-2 antiapoptotic gene while differentially expressed proteins were identified as enolase, pyruvate kinase, and glyceraldehyde-3-phosphate. The extract in this study was shown to induce apoptosis at an early stage which makes it an ideal source that can be explored for compounds that may be used in the treatment and management of cancer. PMID:26457109

  15. 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

  16. Primary photoprocesses involved in the sensory protein for the photophobic response of Blepharisma japonicum.

    PubMed

    Brazard, Johanna; Ley, Christian; Lacombat, Fabien; Plaza, Pascal; Martin, Monique M; Checcucci, Giovanni; Lenci, Francesco

    2008-11-27

    We present new femtosecond transient-absorption and picosecond fluorescence experiments performed on OBIP, the oxyblepharismin-binding protein believed to trigger the photophobic response of the ciliate Blepharisma japonicum. The formerly identified heterogeneity of the sample is confirmed and rationalized in terms of two independent populations, called rOBIP and nrOBIP. The rOBIP population undergoes a fast photocycle restoring the initial ground state in less than 500 ps. Intermolecular electron transfer followed by electron recombination is identified as the excited-state decay route. The experimental results support the coexistence of the oxyblepharismin (OxyBP) radical cation signature with a stimulated-emission signal at all times of the evolution of the transient-absorption spectra. This observation is interpreted by an equilibrium being reached between the locally excited state and a charge-transfer state on the ground of a theory developed by Mataga and co-workers to explain the fluorescence quenching of aromatic hydrogen-bonded donor-acceptor pairs in nonpolar solvents. OxyBP is supposed to bind to an as yet unknown electron acceptor by a hydrogen-bond (HB) and the coordinate along which forward and backward electron transfer proceed is assumed to be the shift of the HB proton. The observed kinetic isotope effect supports this interpretation. Protein relaxation is finally proposed to accompany the whole process and give rise to the highly multiexponential observed dynamics. As previously reported, the fast photocycle of rOBIP can be interpreted as an efficient sunscreen mechanism that protects Blepharisma japonicum from continuous irradiation. The nrOBIP population, the transient-absorption of which strongly reminds that of free OxyBP in solution, might be proposed to actually trigger the photophobic response of the organism through excited-state deprotonation of the chromophore occurring in the nanosecond regime. Additional femtosecond transient

  17. 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

  18. Novel Recombinant Hepatitis B Virus Vectors Efficiently Deliver Protein and RNA Encoding Genes into Primary Hepatocytes

    PubMed Central

    Hong, Ran; Bai, Weiya; Zhai, Jianwei; Liu, Wei; Li, Xinyan; Zhang, Jiming; Cui, Xiaoxian; Zhao, Xue; Ye, Xiaoli; Deng, Qiang; Tiollais, Pierre; Wen, Yumei

    2013-01-01

    Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection. PMID:23552416

  19. Activation and function of murine primary microglia in the absence of the prion protein.

    PubMed

    Pinheiro, Lívia P; Linden, Rafael; Mariante, Rafael M

    2015-09-15

    The prion protein (PrP(C)) is predominantly expressed in the nervous and immune systems and is involved in relevant cell signaling. Microglia participate in neuroimmune interactions, and their regulatory mechanisms are critical for both health and disease. Despite recent reports with a microglial cell line, little is known about the relevance of PrP(C) in brain microglia. We investigated the role of PrP(C) in mouse primary microglia, and found no differences between wild type and Prnp-null cells in cell morphology or the expression of a microglial marker. Translocation of NF-κB to the nucleus also did not differ, nor did cytokine production. The levels of iNOS were also similar and, finally, microglia of either genotype showed no differences in either rates of phagocytosis or migration, even following activation. Thus, functional roles of PrP(C) in primary microglial cells are - if present - much more subtle than in transformed microglial cell lines.

  20. 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

  1. 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. PMID:19215732

  2. 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.

  3. Pyroglutamic acid stimulates DNA synthesis in rat primary hepatocytes through the mitogen-activated protein kinase pathway.

    PubMed

    Inoue, Shinjiro; Okita, Yoichi; de Toledo, Andreia; Miyazaki, Hiroyuki; Hirano, Eiichi; Morinaga, Tetsuo

    2015-01-01

    We purified pyroglutamic acid from human placental extract and identified it as a potent stimulator of rat primary hepatocyte DNA synthesis. Pyroglutamic acid dose-dependently stimulated DNA synthesis, and this effect was inhibited by PD98059, a dual specificity mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitor. Therefore, pyroglutamic acid stimulated DNA synthesis in rat primary hepatocytes via MAPK signaling.

  4. High-resolution neutron protein crystallography with radically small crystal volumes: Application of perdeuteration to human aldose reductase

    SciTech Connect

    Hazemann, I.; Dauvergne, M. T.; Blakeley, M. P.; Meilleur, Flora; Haertlein, M.; Van Dorsselaer, A.; Mitschler, A.; Myles, Dean A A; Podjarny, A.

    2005-08-01

    Neutron diffraction data have been collected to 2.2 {angstrom} resolution from a small (0.15 mm{sup 3}) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm{sup 3} are reported. Neutron data were recorded to 2 {angstrom} resolution, with subsequent data analysis using data to 2.2 {angstrom}. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

  5. 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

  6. 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.

  7. Acceleration of lipid peroxidation in alpha-tocopherol transfer protein-knockout mice following the consumption of drinking water containing a radical initiator.

    PubMed

    Yoshida, Yasukazu; Hayakawa, Mieko; Cynshi, Osamu; Jishage, Kou-ichi; Niki, Etsuo

    2008-01-01

    To assess the antioxidative role of vitamin E (VE) in a mouse model of severe VE deficiency by using biomarkers, alpha-tocopherol transfer protein (alpha-TTP(-/-))-knockout mice were maintained on a VE-deficient diet for 28 weeks [KO group, n = 6]. Wild-type C57BL/6 mice were maintained on a diet containing 0.002% alpha-tocopherol [WT group, n = 6]. The animals were housed individually in a metabolic cage from the age of 9 weeks (Week 0) to 27 weeks. Urine was collected every week, and the levels of total hydroxyoctadecadienoic acid (tHODE), 7-hydroxycholesterol (t7-OHCh), and 8-iso-prostaglandin F(2alpha)(t8-isoPGF(2alpha)), which are biomarkers for lipid peroxidation, were measured by gas chromatography (GC)-mass spectrometry. From the age of 21 weeks (Week 12), three mice in each group were provided drinking water containing the water-soluble radical initiator 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) until the end of the study (Week 19). Blood and tissue samples were collected, and the levels of the abovementioned biomarkers therein were assessed. AIPH consumption clearly elevated the plasma and erythrocyte levels of tHODE and t8-isoPGF(2alpha) in both the WT and KO groups except for the erythrocyte level of tHODE in the WT group. Furthermore, this elevation was more prominent in the KO group than in the WT group. Interestingly, AIPH consumption reduced the stereoisomer ratio of HODE (ZE/EE), which is reflective of the efficacy of a compound as an antioxidant in vivo; this suggests that free radical-mediated oxidation reduces the antioxidant capacity in vivo. The urine levels of tHODE, t7-OHCh, and t8-isoPGF(2alpha) tended to increase with AIPH consumption, but these individual levels fluctuated. It was clearly demonstrated by the proposed biomarkers that maintaining alpha-TTP(-/-) mice on a VE-deficient diet results in a severe VE deficiency and promotes lipid peroxidation.

  8. Robot-assisted total intracorporeal low anterior resection with primary anastomosis and radical dissection for treatment of stage IV endometriosis with bowel involvement: morbidity and its outcome.

    PubMed

    Lim, Peter C; Kang, Elizabeth; Park, Do Hwan

    2011-12-01

    Robot-assisted low anterior resection with primary sigmoid rectal anastomosis/ureterolysis/hysterectomy with bilateral salpingo-oophorectomy (RALARH), and exploratory laparotomy low anterior resection with primary sigmoid rectal anastomosis/ureterolysis/hysterectomy with bilateral salpingo-oophorectomy (ELLARH). A total of eighteen patients (8 RALARH and 10 ELLARH) met the inclusion criteria. The mean age of the RALARH group was 47 and that of the ELLARH group was 36.9. There was no difference between body mass index (RALARH = 30.3 ± 6.6, 95% CI (25.7, 56.0) vs. ELLARH = 26.7 ± 7.5). Total operative time for the RALARH group was 238.5 ± 57.8 min, 95% CI: (164.5, 279.9) whereas that for the ELLARH group was 237 ± 117.7 min. There were no statistically significant differences between blood loss (RALARH = 425 ± 462.1 cc, 95% CI: (104.8, 745.2). ELLARH = 630 ± 432.2 cc) or days of hospitalization (RALARH = 5.5 ± 2.4 days, 95% CI: (3.8, 7.2) vs. ELLARH = 6.2 ± 1.6 days) in the RALARH and ELLARH groups. There were fewer complications in the RALARH group than in the ELLARH group (RALARH two complications vs. ELLARH two blood transfusion and two rectovaginal fistula), but the difference was not significant because of the small sample size. Robotic low anterior resection with primary sigmoid rectal anastomosis with ureterolysis at the time of hysterectomy and bilateral salpingo-oophorectomy for treatment of Stage IV endometriosis is a feasible and safe procedure. PMID:27628117

  9. Myeloid differentiation primary response protein 88 couples reverse cholesterol transport to inflammation.

    PubMed

    Smoak, Kathleen A; Aloor, Jim J; Madenspacher, Jennifer; Merrick, B Alex; Collins, Jennifer B; Zhu, Xuewei; Cavigiolio, Giorgio; Oda, Michael N; Parks, John S; Fessler, Michael B

    2010-06-01

    Crosstalk exists in mammalian cells between cholesterol trafficking and innate immune signaling. Apolipoprotein A-I (apoA-I), a serum apolipoprotein that induces antiatherogenic efflux of macrophage cholesterol, is widely described as anti-inflammatory because it neutralizes bacterial lipopolysaccharide. Conversely, lipopolysaccharide-induced inflammation is proatherogenic. However, whether innate immunity plays an endogenous, physiological role in host cholesterol homeostasis in the absence of infection is undetermined. We report that apoA-I signals in the macrophage through Toll-like receptor (TLR)2, TLR4, and CD14, utilizing myeloid differentiation primary response protein 88 (MyD88)-dependent and -independent pathways, to activate nuclear factor-kappaB and induce cytokines. MyD88 plays a critical role in reverse cholesterol transport in vitro and in vivo, in part through promoting ATP-binding cassette A1 transporter upregulation. Taken together, this work identifies apoA-I as an endogenous stimulus of innate immunity that couples cholesterol trafficking to inflammation through MyD88 and identifies innate immunity as a physiologic signal in cholesterol homeostasis.

  10. Evaluation of salivary gland protein 1 antibodies in patients with primary and secondary Sjogren's syndrome.

    PubMed

    Shen, Long; Kapsogeorgou, Efstathia K; Yu, Meixing; Suresh, Lakshmanan; Malyavantham, Kishore; Tzioufas, Anthanasios G; Ambrus, Julian L

    2014-11-01

    Sjogren's syndrome (SS) has been associated with the expression of anti-Ro and anti-La antibodies. Anti-salivary gland protein 1 (SP1) antibodies have recently been identified in patients with SS. The current work involved a cross sectional study to determine whether anti-SP1 antibodies were identified in particular subgroups of patients with SS. The results of this study revealed that anti-SP1 antibodies were present in the sera of 52% of SS patients while anti-Ro/anti-La was present in 63% of patients. 19% of patients had anti-SP1 without anti-Ro/anti-La. Patients with SS and lymphoma expressed anti-Ro, anti-La and anti-SP1 together. In SS associated with RA, 50% had antibodies anti-SP1 while 40% had anti-Ro/anti-La. In conclusion, anti-SP1 antibodies are commonly seen in both primary and secondary SS and rarely in normal controls. Future studies are needed to determine the roles and timing of expression of anti-SP1 antibodies in Sjogren's syndrome.

  11. Heat shock proteins and chronic fatigue in primary Sjögren’s syndrome

    PubMed Central

    Bårdsen, Kjetil; Nilsen, Mari Mæland; Kvaløy, Jan Terje; Norheim, Katrine Brække; Jonsson, Grete

    2016-01-01

    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. PMID:26921255

  12. The location of protein S8 and surrounding elements of 16S rRNA in the 70S ribosome from combined use of directed hydroxyl radical probing and X-ray crystallography.

    PubMed Central

    Lancaster, L; Culver, G M; Yusupova, G Z; Cate, J H; Yusupov, M M; Noller, H F

    2000-01-01

    Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins. To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems. In addition, cleavage near the 5' terminus of 16S rRNA, in the 300 region of its 5' domain, and in the 1070 region of its 3'-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding. These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-A map of the Thermus thermophilus 70S ribosome. The resulting model is in close agreement with the extensive body of data from previous studies using protein-protein and protein-RNA crosslinking, chemical and enzymatic footprinting, and genetics. PMID:10836793

  13. The location of protein S8 and surrounding elements of 16S rRNA in the 70S ribosome from combined use of directed hydroxyl radical probing and X-ray crystallography.

    PubMed

    Lancaster, L; Culver, G M; Yusupova, G Z; Cate, J H; Yusupov, M M; Noller, H F

    2000-05-01

    Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins. To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems. In addition, cleavage near the 5' terminus of 16S rRNA, in the 300 region of its 5' domain, and in the 1070 region of its 3'-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding. These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-A map of the Thermus thermophilus 70S ribosome. The resulting model is in close agreement with the extensive body of data from previous studies using protein-protein and protein-RNA crosslinking, chemical and enzymatic footprinting, and genetics.

  14. 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...

  15. [Primary hyperparathyroidism].

    PubMed

    Maruani, G; Cornière, N; Nicolet, L; Baron, S; Courbebaisse, M; Renaud, S; Houillier, P

    2013-10-01

    For the past 40 years, primary hyperparathyroidism has been recognized as a common endocrine disease which is, most often, "non-symptomatic", without the occurrence of nephrolithiasis or osteitis fibrosa cystica. Our knowledge in the pathophysiology has increased largely and diagnosis of primary hyperparathyroidism is usually easy. The only radical treatment is surgery and the surgical indications have been codified by several consensus conferences. For patients who do not undergo surgery, prolonged medical monitoring is needed.

  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. Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype.

    PubMed Central

    Ray, R B; Lagging, L M; Meyer, K; Ray, R

    1996-01-01

    We have previously demonstrated that hepatitis C virus (HCV) core protein regulates cellular protooncogenes at the transcriptional level; this observation implicates core protein in the alteration of normal hepatocyte growth. In the present study, the transforming potential of the HCV core gene was investigated by using primary rat embryo fibroblast (REF) cells which were transfected with or without cooperative oncogenes. Integration of the HCV core gene resulted in expression of the viral protein in REF stable transformants. REF cells cotransfected with HCV core and H-ras genes became transformed and exhibited rapid proliferation, anchor-independent growth, and tumor formation in athymic nude mice. Results from these studies suggest that the core protein plays an important role in the regulation of HCV-infected cell growth and in the transformation to tumorigenic phenotype. These observations suggest a possible mechanism for this viral protein in the pathogenesis of hepatocellular carcinoma in HCV-infected humans. PMID:8676467

  18. Monosodium urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human primary macrophages.

    PubMed

    Välimäki, Elina; Miettinen, Juho J; Lietzén, Niina; Matikainen, Sampsa; Nyman, Tuula A

    2013-03-01

    Monosodium urate (MSU) is an endogenous danger signal that is crystallized from uric acid released from injured cells. MSU is known to activate inflammatory response in macrophages but the molecular mechanisms involved have remained uncharacterized. Activated macrophages start to secrete proteins to activate immune response and to recruit other immune cells to the site of infection and/or tissue damage. Secretome characterization after activation of innate immune system is essential to unravel the details of early phases of defense responses. Here, we have analyzed the secretome of human primary macrophages stimulated with MSU using quantitative two-dimensional gel electrophoresis based proteomics as well as high-throughput qualitative GeLC-MS/MS approach combining protein separation by SDS-PAGE and protein identification by liquid chromatography-MS/MS. Both methods showed that MSU stimulation induced robust protein secretion from lipopolysaccharide-primed human macrophages. Bioinformatic analysis of the secretome data showed that MSU stimulation strongly activates unconventional, vesicle mediated protein secretion. The unconventionally secreted proteins included pro-inflammatory cytokines like IL-1β and IL-18, interferon-induced proteins, and danger signal proteins. Also active forms of lysosomal proteases cathepsins were secreted on MSU stimulation, and cathepsin activity was essential for MSU-induced unconventional protein secretion. Additionally, proteins associated to phosphorylation events including Src family tyrosine kinases were increased in the secretome of MSU-stimulated cells. Our functional studies demonstrated that Src, Pyk2, and PI3 kinases act upstream of cathepsins to activate the overall protein secretion from macrophages. In conclusion, we provide the first comprehensive characterization of protein secretion pathways activated by MSU in human macrophages, and reveal a novel role for cathepsins and Src, Pyk2, PI3 kinases in the activation of

  19. Diagnostic value of serum Golgi protein 73 for HBV-related primary hepatic carcinoma

    PubMed Central

    Gao, Guosheng; Dong, Feibo; Xu, Xiaozhen; Hu, Airong; Hu, Yaoren

    2015-01-01

    Background: Alpha-fetoprotein (AFP) levels are routinely used for diagnosis and monitoring of hepatic diseases, but it has a limited value. Golgi protein 73 (GP73) has been suggested as a new marker for hepatic diseases. Objective: To explore the clinical value of serum GP73 in different diseases associated with hepatitis B virus (HBV) infection. Method: Between January 2010 and August 2014, serum samples from 88 patients with chronic hepatitis B (CHB), 78 patients with HBV-related liver cirrhosis (LC), and 194 patients with HBV-related primary hepatic cancer (PHC) were collected. Serum samples from 30 healthy volunteers were used as controls. ELISA and microparticle enzyme immunoassay were used to measure serum GP73 and AFP levels. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic value of serum GP73 and AFP for PHC. Results: For the diagnosis of PHC, GP73 showed a sensitivity of 65.5% and specificity of 66.3%, while AFP levels showed sensitivity of 64.4% and specificity of 76.5%. Serial testing (both tests are positive) could increase the specificity (sensitivity of 45.9% and specificity of 85.5%) while parallel testing (any single positive test result) could increase the sensitivity (sensitivity of 84.0% and specificity of 57.2%). Serum GP73 and AFP levels were significantly different between Child-Pugh grades (P<0.001 for GP73 and P=0.044 for AFP). Significant differences in serum GP73 and AFP were found between TNM stages (all P<0.001). Conclusion: Serum GP73 had limited diagnostic value for HBV-related PHC. The combined use of serum GP73 and AFP levels improved the diagnostic efficacy. PMID:26617863

  20. Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain.

    PubMed

    Bühler, R; Hempel, J; Kaiser, R; de Zalenski, C; von Wartburg, J P; Jörnvall, H

    1984-12-17

    The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE. PMID:6391921

  1. Spectroscopic study of the chromophore--protein association and primary photoinduced events in the photoreceptor of Blepharisma japonicum.

    PubMed

    Plaza, Pascal; Mahet, Mathilde; Martin, Monique M; Angelini, Nicola; Malatesta, Manuela; Checcucci, Giovanni; Lenci, Francesco

    2005-09-01

    Blepharisma japonicum is a ciliated protozoan exhibiting a strong step-up photophobic response upon illumination. The photoreceptor chromophores responsible for this response have been identified to be hypericin-like chromophores (blepharismin and oxyblepharismin), complexed to a 200 kDa non-water-soluble protein. The present work opens up new perspectives on the primary phototransduction steps of B. japonicum's light perception through a joined approach by steady-state fluorescence spectroscopy, time-resolved fluorescence anisotropy and sub-picosecond transient absorption spectroscopy. The free chromophore of the light-adapted form of the cell (oxyblepharismin) was studied in various solvents and its spectroscopic properties, as well as its primary excited-state reactivity, compared with those of the corresponding pigment-protein complex, extracted by phosphate-concentration-step chromatography on a hydroxyapatite column. Fluorescence anisotropy together with SDS PAGE electrophoresis results confirm that oxyblepharismin is non-covalently bound to the apoprotein and show that, in the excited state, it is free to rotate in all directions within the binding site where it experiences a large local viscosity. Time-resolved anisotropy measurements on aromatic amino acids confirm that the molecular weight of the protein is of the order of 200 kDa. Although showing very similar steady-state spectra, free oxyblepharismin and its protein complex have noticeably different excited-state behaviours. In particular, the protein complex exhibits a pronounced short-lived absorption feature in the 640--750 nm range, decaying biexponentially in 4 ps and 56 ps. Those decays, also observed in other spectral regions, are not found in the corresponding kinetics of the isolated pigment in solution. This early behaviour of the protein complex might be the signature of the primary phototransduction process, possibly involving an electron transfer from the pigment to a neighbouring protein

  2. 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.

  3. 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

  4. The contribution of genetic and environmental factors to quantitative variability of erythrocyte membrane proteins in primary hypotension.

    PubMed

    Ivanov, V P; Polonikov, A V; Solodilova, M A

    2005-01-01

    Our previous studies have shown that, compared with healthy individuals, patients with primary arterial hypotension (PAH) have significant quantitative changes in erythrocyte membrane proteins. The purpose of the present study was to evaluate the contribution made by genetic and environmental factors to quantitative variation of erythrocyte membrane proteins in PAH. We studied 109 hypotensive patients, 124 normotensive subjects, 222 of their first-degree relatives and 24 twin pairs by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. The decomposition of total phenotypic variance of erythrocyte membrane proteins to genetic and environmental components was performed on the basis of correlations among first-degree relatives by the least squares method. The genetic dominance and shared environmental factors were found to influence the variability of cytoskeletal membrane proteins whose contents were changed in PAH. Furthermore, variations in alpha-spectrin, actin and anion exchanger in hypotensives were substantially influenced by major gene and maternal effects. Ankyrin 2.1 and actin content was under the control of common underlying genes. Variations in membrane-associated glutathione-S-transferase and tropomyosin were predominantly affected by polygenes. These findings suggest that the putative major genes with pleiotropic effects appear to be involved in the control of quantitative disorders of erythrocyte membrane proteins in primary hypotension. PMID:15638825

  5. Application of a proteomic approach to identify proteins associated with primary graft non-function after liver transplantation.

    PubMed

    Kornasiewicz, Oskar; Bojarczuk, Kamil; Bugajski, Marek; Golab, Jakub; Krawczyk, Marek

    2012-10-01

    Primary graft non-function (PNF) is a rare, life-threatening complication of liver transplantation. Increasing use of extended criteria donor pools and high-risk recipients seem to influence the incidence of PNF. Primary failure is associated with high patient morbidity and inferior graft survival. The only available treatment for PNF is emergency hepatic retransplantation, which is also correlated with significant morbidity and mortality. Therefore, researchers are working to identify risk factors of diagnostic value to prevent PNF. The current study attempted to explore liver proteomic patterns in patients with PNF. Using two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry (LC-MS), we compared liver protein homogenates from 3 patients with PNF to those obtained from 6 healthy liver samples to identify potential new biomarkers of PNF. Our comparisons revealed 21 proteins with differential expression (13 upregulated and 8 downregulated). Most of these proteins are involved in energy metabolism, lipid metabolism, peptide cleavage, cell differentiation, and apoptosis. Although none of these proteins appeared more than once in separate analyses, this preliminary study shows that two-dimensional gel electrophoresis and LC-MS may allow identification of characteristic proteins to be used as biomarkers of a life-threatening complication of liver transplantation. Larger-scale analyses could improve patient care by finding suitable prognostic and therapeutic options. These data represent the first global proteomic approach to study PNF.

  6. Improved protocols for protein and RNA isolation from three-dimensional collagen sandwich cultures of primary hepatocytes.

    PubMed

    Heidebrecht, F; Schulz, I; Keller, M; Behrens, S-E; Bader, A

    2009-10-01

    The sandwich culture is the most widely used long-term culture system for functional primary hepatocytes. Despite its advantages, the currently available protocols for protein and RNA extraction are either time-consuming or contain steps that may skewer the results. This paper describes improved protocols for RNA and protein extraction from sandwich cultures that are easy to perform, require short working time, and use no additional enzymatic reactions that could change the expression profile of the cells. The quality of the RNA is excellent, allowing also applications requiring high purity such as microarrays. In general, the protocols are suited for any cells in 3D collagen culture. PMID:19539596

  7. 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

  8. 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

  9. 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.

  10. Radical-Mediated Enzymatic Polymerizations.

    PubMed

    Zavada, Scott R; Battsengel, Tsatsral; Scott, Timothy F

    2016-01-01

    Polymerization reactions are commonly effected by exposing monomer formulations to some initiation stimulus such as elevated temperature, light, or a chemical reactant. Increasingly, these polymerization reactions are mediated by enzymes--catalytic proteins--owing to their reaction efficiency under mild conditions as well as their environmental friendliness. The utilization of enzymes, particularly oxidases and peroxidases, for generating radicals via reduction-oxidation mechanisms is especially common for initiating radical-mediated polymerization reactions, including vinyl chain-growth polymerization, atom transfer radical polymerization, thiol-ene step-growth polymerization, and polymerization via oxidative coupling. While enzyme-mediated polymerization is useful for the production of materials intended for subsequent use, it is especially well-suited for in situ polymerizations, where the polymer is formed in the place where it will be utilized. Such polymerizations are especially useful for biomedical adhesives and for sensing applications. PMID:26848652

  11. The Cholinergic Signaling Responsible for the Expression of a Memory-Related Protein in Primary Rat Cortical Neurons.

    PubMed

    Chen, Tsan-Ju; Chen, Shun-Sheng; Wang, Dean-Chuan; Hung, Hui-Shan

    2016-11-01

    Cholinergic dysfunction in the brain is closely related to cognitive impairment including memory loss. In addition to the degeneration of basal forebrain cholinergic neurons, deficits in the cholinergic receptor signaling may also play an important role. In the present study, to examine the cholinergic signaling pathways responsible for the induction of a memory-related postsynaptic protein, a cholinergic agonist carbachol was used to induce the expression of activity-regulated cytoskeleton associated protein (Arc) in primary rat cortical neurons. After pretreating neurons with various antagonists or inhibitors, the levels of carbachol-induced Arc protein expression were detected by Western blot analysis. The results show that carbachol induces Arc protein expression mainly through activating M1 acetylcholine receptors and the downstream phospholipase C pathway, which may lead to the activation of the MAPK/ERK signaling pathway. Importantly, carbachol-mediated M2 receptor activation exerts negative effects on Arc protein expression and thus counteracts the enhanced effects of M1 activation. Furthermore, it is suggested for the first time that M1-mediated enhancement of N-methyl-D-aspartate receptor (NMDAR) responses, leading to Ca(2+) entry through NMDARs, contributes to carbachol-induced Arc protein expression. These findings reveal a more complete cholinergic signaling that is responsible for carbachol-induced Arc protein expression, and thus provide more information for developing treatments that can modulate cholinergic signaling and consequently alleviate cognitive impairment. J. Cell. Physiol. 231: 2428-2438, 2016. © 2016 Wiley Periodicals, Inc. PMID:26895748

  12. 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.

  13. Immunocytochemical detection of dentin matrix proteins in primary teeth from patients with dentinogenesis imperfecta associated with osteogenesis imperfecta.

    PubMed

    Orsini, G; Majorana, A; Mazzoni, A; Putignano, A; Falconi, M; Polimeni, A; Breschi, L

    2014-12-01

    Dentinogenesis imperfecta determines structural alterations of the collagen structure still not completely elucidated. Immunohistochemical 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 (DMP)-1 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. Immunolabeling 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 ultrastructural alterations in dentin obtained from patients affected by DGI-I, supported by immunocytochemical assays of different collagenous and non-collagenous proteins.

  14. 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.

  15. 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

  16. Real-time probing of radical events with sulfide molecules

    NASA Astrophysics Data System (ADS)

    Gauduel, Yann A.; Glinec, Yannick; Malka, Victor

    2007-02-01

    The physio-pathological roles of sulfide biomolecules in cellular environments involves redox processes and radical reactions that alter or protect the functional properties of enzymatic systems, proteins and nucleic acids repair. We focus on micromolar monitoring of sulfur-centered radical anions produced by direct electron attachment, using sulfide molecules (a thioether and a disulfide biomolecule) and two complementary spectroscopic approaches: low energy radiation femtochemistry (1-8 eV) and high energy radiation femtochemistry (2.5-15 MeV). The early step of a disulfide bond making RS∴SR from thiol molecules involves a very-short lived odd-electron bonded intermediate for which an excess electron is transiently localized by a preexisting two sulfide monomers complex. The reactive center of oxidized glutathione (cystamine), a major cytoplasmic disulfide biomolecule, is also used as sensor for the real-time IR investigation of effective reaction radius r eff in homogenous aqueous environments and interfacial water of biomimetic systems. Femtosecond high-energy electrons beams, typically in the 2.5 - 15 MeV range, may conjecture the picosecond observation of primary radical events in nanometric radiation spurs. The real-time investigation of sulfide and disulfide molecules opens exciting opportunities for sensitisation of confined environments (aqueous groove of DNA, protein pockets, sub-cellular systems) to ionizing radiation. Low and high-energy femtoradical probing foreshadow the development of new applications in radiobiology (low dose effect at the nanometric scale) and anticancer radiotherapy (pro-drogue activation).

  17. The Arabidopsis pyruvate,orthophosphate dikinase regulatory proteins encode a novel, unprecedented Ser/Thr protein kinase primary structure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. 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

  19. Multiple myeloma cell lines and primary tumors proteoma: protein biosynthesis and immune system as potential therapeutic targets.

    PubMed

    Fernando, Rodrigo Carlini; de Carvalho, Fabricio; Mazzotti, Diego Robles; Evangelista, Adriane Feijó; Braga, Walter Moisés Tobias; de Lourdes Chauffaille, Maria; Paes Leme, Adriana Franco; Colleoni, Gisele Wally Braga

    2015-11-01

    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

  20. Low Protein A20 in Minor Salivary Glands is Associated with Lymphoma in Primary Sjögren's Syndrome.

    PubMed

    Johnsen, S J; Gudlaugsson, E; Skaland, I; Janssen, E A M; Jonsson, M V; Helgeland, L; Berget, E; Jonsson, R; Omdal, R

    2016-03-01

    Patients with primary Sjögren's syndrome (pSS) have an increased risk of developing lymphomas, particularly the subtype mucosa-associated lymphoid tissue (MALT) lymphoma. Chronic antigen stimulation and increased activation of nuclear factor-κB (NF-κB) are important factors for the pathogenesis of MALT lymphomas. Protein A20 is an inhibitor of NF-κB. A recent study of pSS-associated MALT lymphomas identified potential functional abnormalities in the TNFAIP3 gene, which encodes protein A20. The present study aimed to assess protein A20 by immunohistochemistry (IHC) in minor salivary glands (MSGs) and lymphoma tissue sections of patients with pSS and investigate a potential association with lymphoma development. Protein A20 staining in lymphocytes was scored in four categories (0 = negative, 1 = weak, 2 = moderate and 3 = strong). For statistical purposes, these scores were simplified into negative (scores 0-1) and positive (scores 2-3). We investigated associations between protein A20-staining, focus scores, germinal centre (GC)-like structures and monoclonal B-cell infiltration in MSGs. MSG protein A20 staining was weaker in pSS patients with lymphomas than in those without lymphomas (P = 0.01). Weak protein A20 staining was also highly associated with a lack of GC formation (P < 0.01). Finally, weaker A20 staining was observed in the majority of pSS-associated MALT lymphoma tissues. In conclusion, we found absent or weak protein A20 immunoreactivity in MSGs of patients with pSS with lymphomas. This finding indicates that protein A20 downregulation in lymphocytes might be a mechanism underlying lymphoma genesis in patients with pSS. PMID:26679293

  1. Free radicals, antioxidants, and nutrition.

    PubMed

    Fang, Yun-Zhong; Yang, Sheng; Wu, Guoyao

    2002-10-01

    Radiation hazards in outer space present an enormous challenge for the biological safety of astronauts. A deleterious effect of radiation is the production of reactive oxygen species, which result in damage to biomolecules (e.g., lipid, protein, amino acids, and DNA). Understanding free radical biology is necessary for designing an optimal nutritional countermeasure against space radiation-induced cytotoxicity. Free radicals (e.g., superoxide, nitric oxide, and hydroxyl radicals) and other reactive species (e.g., hydrogen peroxide, peroxynitrite, and hypochlorous acid) are produced in the body, primarily as a result of aerobic metabolism. Antioxidants (e.g., glutathione, arginine, citrulline, taurine, creatine, selenium, zinc, vitamin E, vitamin C, vitamin A, and tea polyphenols) and antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidases) exert synergistic actions in scavenging free radicals. There has been growing evidence over the past three decades showing that malnutrition (e.g., dietary deficiencies of protein, selenium, and zinc) or excess of certain nutrients (e.g., iron and vitamin C) gives rise to the oxidation of biomolecules and cell injury. A large body of the literature supports the notion that dietary antioxidants are useful radioprotectors and play an important role in preventing many human diseases (e.g., cancer, atherosclerosis, stroke, rheumatoid arthritis, neurodegeneration, and diabetes). The knowledge of enzymatic and non-enzymatic oxidative defense mechanisms will serve as a guiding principle for establishing the most effective nutrition support to ensure the biological safety of manned space missions. PMID:12361782

  2. Flies, worms and the Free Radical Theory of ageing.

    PubMed

    Clancy, David; Birdsall, John

    2013-01-01

    Drosophila and Caenorhabditis elegans have provided the largest body of evidence addressing the Free Radical Theory of ageing, however the evidence has not been unequivocally supportive. Oxidative damage to DNA is probably not a major contributor, damage to lipids is assuming greater importance and damage to proteins probably the source of pathology. On balance the evidence does not support a primary role of oxidative damage in ageing in C. elegans, perhaps because of its particular energy metabolic and stress resistance profile. Evidence is more numerous, varied and consistent and hence more compelling for Drosophila, although not conclusive. However there is good evidence for a role of oxidative damage in later life pathology. Future work should: 1/ make more use of protein oxidative damage measurements; 2/ use inducible transgenic systems or pharmacotherapy to ensure genetic equivalence of controls and avoid confounding effects during development; 3/ to try to delay ageing, target interventions which reduce and/or repair protein oxidative damage. PMID:22504404

  3. Flies, worms and the Free Radical Theory of ageing.

    PubMed

    Clancy, David; Birdsall, John

    2013-01-01

    Drosophila and Caenorhabditis elegans have provided the largest body of evidence addressing the Free Radical Theory of ageing, however the evidence has not been unequivocally supportive. Oxidative damage to DNA is probably not a major contributor, damage to lipids is assuming greater importance and damage to proteins probably the source of pathology. On balance the evidence does not support a primary role of oxidative damage in ageing in C. elegans, perhaps because of its particular energy metabolic and stress resistance profile. Evidence is more numerous, varied and consistent and hence more compelling for Drosophila, although not conclusive. However there is good evidence for a role of oxidative damage in later life pathology. Future work should: 1/ make more use of protein oxidative damage measurements; 2/ use inducible transgenic systems or pharmacotherapy to ensure genetic equivalence of controls and avoid confounding effects during development; 3/ to try to delay ageing, target interventions which reduce and/or repair protein oxidative damage.

  4. Radical prostatectomy - discharge

    MedlinePlus

    ... prostatectomy - discharge; Laparoscopic radical prostatectomy - discharge; LRP - discharge; Robotic-assisted laparoscopic prostatectomy - discharge ; RALP - discharge; Pelvic lymphadenectomy - ...

  5. Expressions and clinical significances of CD133 protein and CD133 mRNA in primary lesion of gastric adenocacinoma

    PubMed Central

    2010-01-01

    Background To study on expressions and clinical significances of CD133 protein and CD133 mRNA in primary lesion of gastric adenocarcinoma (GC). Methods Expressions of CD133 protein by immunostaining (99 cases) and CD133 mRNA by semi-quantitative RT-PCR (31 cases) were detected in primary lesion and in noncancerous gastric mucosa tissue (NCGT). Correlations of CD133 protein expression with clinicopathological parameters and post-operative survival were analyzed. Relations of CD133 mRNA level with Ki-67 labeling index (LI), and lymphatic metastasis were assessed too. Results Brown particles indicating CD133 protein positivity occurred in some parts of tumor cells and epithelium. Expressive percentage of CD133 protein positivity was significantly higher in subgroups with >5 cm diameter (P = 0.041), later TNM stage (P = 0.044), severer lymph node metastasis (P = 0.017), occurrences of lymphatic invasion (P = 0.000) and vascular invasion (P = 0.000) respectively. Severer invasion depth (P = 0.011), lymph node metastasis occurrence (P = 0.043) and later TNM stage (P = 0.049) were the independent risk factors for CD133 protein expression. Average brightness scale value (BSV) of CD133 mRNA was significantly higher in subgroups with >5 cm diameter (P = 0.041), lymph node metastasis occurrence (P = 0.004) and in lower Ki-67 LI (P = 0.02). Relative analysis revealed that BSV of CD133 mRNA related positively to metastatic lymphatic nodes ratio (P = 0.008) and metastatic lymph node number (P = 0.009), but negatively to Ki-67 LI (P = 0.009). Survival of positive subgroup of CD 133 protein was significantly poorer (P = 0.047). Lymph node metastasis occurrence (P = 0.042), later TNM stage (P = 0.046) and CD 133 protein positive expression (P = 0.046) were respectively the independent risk factors to survival. Conclusion Higher expressive level of CD133 mRNA is associated to lower Ki-67 LI and severer lymphatic metastasis. Therefore, the expressive level of CD133 mRNA can play an

  6. 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

  7. Lipid peroxidation product acrolein as a predictive biomarker of prostate carcinoma relapse after radical surgery.

    PubMed

    Custovic, Zajim; Zarkovic, Kamelija; Cindric, Marina; Cipak, Ana; Jurkovic, Ilija; Sonicki, Zdenko; Uchida, Koji; Zarkovic, Neven

    2010-05-01

    Cancer recurrence after radical surgery might happen even in the case of patients with localized prostate carcinoma treated by radical prostatectomy. Therefore, identifying predictive markers of tumour recurrence is very important, so this study evaluated the presence of lipid peroxidation product acrolein in primary prostate carcinomas, assuming that acrolein could be involved in prostate carcinogenesis as was recently shown for colon cancer. Samples obtained by radical prostatectomy of 70 patients were analysed, out of which 27 patients suffered afterwards from tumour recurrence, while 43 patients were disease free. Immunohistochemistry using genuine monoclonal antibodies against acrolein-protein adducts revealed the association of acrolein with progression of carcinoma. The logistic regression combining clinical parameters together with the biochemical markers of disease and acrolein immunohistochemistry has shown that the relapse might be predicted with 90% accuracy if tumour-positive surgical margins, stage of disease and the intensity of acrolein presence in tumour stroma were taken together.

  8. Nuclear Protein of the Testis Midline Carcinoma Masquerading as a Primary Mediastinal Seminoma

    PubMed Central

    Sayapina, Maria S.; Savelov, Nikita A.; Karseladze, Apollon I.; Bulanov, Anatoly A.; Tryakin, Alexey A.; Nosov, Dmitry A.; Garin, Avgust M.; Tjulandin, Sergey A.

    2016-01-01

    Nuclear protein of the testis (NUT) midline carcinomas are rare aggressive carcinomas characterized by chromosomal rearrangements that involve the gene encoding the NUT. This article reviews the clinicopathologic features and the differential diagnosis of these malignancies. PMID:27441078

  9. 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.

  10. A descriptive analysis of populations of three-dimensional structures calculated from primary sequences of proteins by OSIRIS.

    PubMed

    Benhabilès, N; Gallet, X; Thomas-Soumarmon, A; Brasseur, R

    1998-01-01

    Among different ab initio approaches to calculate 3D-structures of proteins out of primary sequences, a few are using restricted dihedral spaces and empirical equations of energy as is OSIRIS. All those approaches were calibrated on a few proteins or fragments of proteins. To optimize the calculation over a larger diversity of structures, we need first to define for each sequence what are good conditions of calculations in order to choose a consensus procedure fitting most 3D-structures best. This requires objective classification of calculated 3D-structures. In this work, populations of avian and bovine pancreatic polypeptides (APP, BPP) and of calcium-binding protein (CaBP) are obtained by varying the rate of the angular dynamics of the second step of OSIRIS. Then, 3D-structures are clustered using a nonhierarchical method, SICLA, using rmsd as a distance parameter. A good clustering was obtained for four subpopulations of APP, BPP and CaBP. Each subpopulation was characterized by its barycenter, relative frequency and dispersion. For the three alpha-helix proteins, after the step 1 of OSIRIS, most secondary structures were correct but molecules have a few atomic contacts. Step 2, i.e., the angular dynamics, resolves those atomic contacts and clustering demonstrates that it generates subpopulations of topological conformers as the barycenter topologies show.

  11. Effects of phytoestrogens on protein turnover in rainbow trout primary myocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. 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.

  13. Prediction of Membrane Transport Proteins and Their Substrate Specificities Using Primary Sequence Information

    PubMed Central

    Mishra, Nitish K.; Chang, Junil; Zhao, Patrick X.

    2014-01-01

    Background Membrane transport proteins (transporters) move hydrophilic substrates across hydrophobic membranes and play vital roles in most cellular functions. Transporters represent a diverse group of proteins that differ in topology, energy coupling mechanism, and substrate specificity as well as sequence similarity. Among the functional annotations of transporters, information about their transporting substrates is especially important. The experimental identification and characterization of transporters is currently costly and time-consuming. The development of robust bioinformatics-based methods for the prediction of membrane transport proteins and their substrate specificities is therefore an important and urgent task. Results Support vector machine (SVM)-based computational models, which comprehensively utilize integrative protein sequence features such as amino acid composition, dipeptide composition, physico-chemical composition, biochemical composition, and position-specific scoring matrices (PSSM), were developed to predict the substrate specificity of seven transporter classes: amino acid, anion, cation, electron, protein/mRNA, sugar, and other transporters. An additional model to differentiate transporters from non-transporters was also developed. Among the developed models, the biochemical composition and PSSM hybrid model outperformed other models and achieved an overall average prediction accuracy of 76.69% with a Mathews correlation coefficient (MCC) of 0.49 and a receiver operating characteristic area under the curve (AUC) of 0.833 on our main dataset. This model also achieved an overall average prediction accuracy of 78.88% and MCC of 0.41 on an independent dataset. Conclusions Our analyses suggest that evolutionary information (i.e., the PSSM) and the AAIndex are key features for the substrate specificity prediction of transport proteins. In comparison, similarity-based methods such as BLAST, PSI-BLAST, and hidden Markov models do not provide

  14. Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes

    PubMed Central

    1975-01-01

    The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented. PMID:166079

  15. [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.

  16. CD46 measles virus receptor polymorphisms influence receptor protein expression and primary measles vaccine responses in naive Australian children.

    PubMed

    Clifford, Holly D; Hayden, Catherine M; Khoo, Siew-Kim; Zhang, Guicheng; Le Souëf, Peter N; Richmond, Peter

    2012-05-01

    Despite the availability of measles vaccines, infants continue to die from measles. Measles vaccine responses vary between individuals, and poor immunogenicity is likely to preclude protection against measles. CD46 is a ubiquitously expressed specific receptor for vaccine strains of measles virus. CD46 polymorphisms have not been functionally investigated but may affect CD46 protein expression, which in turn may mediate primary measles antibody responses in infants. In a cohort of children aged 12 to 14 months from Perth, Australia (n = 137), after their first dose of measles-mumps-rubella (MMR) vaccine, CD46 polymorphisms were genotyped, and postvaccination measles IgG and CD46 protein expression before and after measles lysate stimulation of cells were measured. Three CD46 variants (rs7144, rs11118580, and rs2724384) were significantly associated with measles virus-specific IgG levels (P = 0.008, P = 0.026, and P = 0.018, respectively). There were significant differences between CD46 rs7144 genotypes and CD46 protein expression on T cells, as well as the downregulation of CD46 and T-cell frequency after measles lysate stimulation. We show that CD46 polymorphisms were associated with primary measles antibody responses in naive infants. We also report the first association of a measles virus receptor polymorphism with functional effects on the receptor, suggesting a possible mechanism through which antibody responses are altered. Elucidating all of the interconnecting genetic factors that alter primary measles vaccine responses may be important for identifying children at risk of poor immunogenicity or vaccine failure and for the future design of vaccine strategies to help these children.

  17. 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

  18. Primary Role of the Chromophore Bond Length Alternation in Reversible Photoconversion of Red Fluorescence Proteins

    NASA Astrophysics Data System (ADS)

    Drobizhev, Mikhail; Hughes, Thomas E.; Stepanenko, Yuriy; Wnuk, Pawel; O'Donnell, Kieran; Scott, J. Nathan; Callis, Patrik R.; Mikhaylov, Alexander; Dokken, Leslie; Rebane, Aleksander

    2012-09-01

    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.

  19. 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. PMID:17880126

  20. Primary radiation damage of protein crystals by an intense synchrotron X-ray beam.

    PubMed

    Teng, T Y; Moffat, K

    2000-09-01

    X-ray radiation damage of a lysozyme single crystal by an intense monochromatic beam from a third-generation radiation source at the Advanced Photon Source has been studied. The results show that primary radiation damage is linearly dependent on the X-ray dose even when the crystal is at cryogenic temperatures. The existence of an upper limit for the primary radiation damage was observed. Above the threshold of approximately 1 x 10(7) Gy, excessive damage of the crystal develops which is interpreted as the onset of secondary and/or tertiary radiation damage. This upper limit of X-ray dose is compared with Henderson's limit [Henderson (1990). Proc. R. Soc. London, B241, 6-8], and its implication for the amount of useful X-ray diffraction data that can be obtained for crystals of a given scattering power is also discussed. PMID:16609214

  1. Leucine and isoleucine reduce protein degradation in rainbow trout (Oncorhynchus mykiss) primary myoblast cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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)...

  2. 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

  3. 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.

  4. UL16-binding proteins, novel MHC class I-related proteins, bind to NKG2D and activate multiple signaling pathways in primary NK cells.

    PubMed

    Sutherland, Claire L; Chalupny, N Jan; Schooley, Kenneth; VandenBos, Tim; Kubin, Marek; Cosman, David

    2002-01-15

    The UL16-binding proteins (ULBPs) are a novel family of MHC class I-related molecules that were identified as targets of the human CMV glycoprotein, UL16. We have previously shown that ULBP expression renders a relatively resistant target cell sensitive to NK cytotoxicity, presumably by engaging NKG2D, an activating receptor expressed by NK and other immune effector cells. In this study we show that NKG2D is the ULBP counterstructure on primary NK cells and that its expression is up-regulated by IL-15 stimulation. Soluble forms of ULBPs induce marked protein tyrosine phosphorylation, and activation of the Janus kinase 2, STAT5, extracellular signal-regulated kinase, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signal transduction pathways. ULBP-induced activation of Akt and extracellular signal-regulated kinase and ULBP-induced IFN-gamma production are blocked by inhibitors of PI 3-kinase, consistent with the known binding of PI 3-kinase to DAP10, the membrane-bound signal-transducing subunit of the NKG2D receptor. While all three ULBPs activate the same signaling pathways, ULBP3 was found to bind weakly and to induce the weakest signal. In summary, we have shown that NKG2D is the ULBP counterstructure on primary NK cells and for the first time have identified signaling pathways that are activated by NKG2D ligands. These results increase our understanding of the mechanisms by which NKG2D activates immune effector cells and may have implications for immune surveillance against pathogens and tumors. PMID:11777960

  5. Localization of Trk neurotrophin receptor-like proteins in avian primary lymphoid organs (thymus and bursa of Fabricius).

    PubMed

    Ciriaco, E; Dall'Aglio, C; Hannestad, J; Huerta, J J; Laurà, R; Germanà, G; Vega, J A

    1996-09-01

    The avian thymus and bursa of Fabricius are the specific organs where the maturation and differentiation of T- and B-lymphocytes, respectively, take place. In the mammalian lymphoid organs mRNAs of the neurotrophins and their receptors have been identified but their localization at the protein level remains still unknown. This study was undertaken to analyze the localization of the Trk family of tyrosine kinase receptors in the avian primary lymphoid organs (thymus and bursa of Fabricius) during the posthatching development using immunohistochemistry. These proteins serve as essential constituents of the high affinity receptors for neurotrophins. In the thymus of all groups of age specific immunoreactivity (IR) was observed for all three Trks: TrkA-like IR was found labelling medullary epithelial cells and a subpopulation of cortical epithelial cells; TrkB-like IR was found in the medullar dendritic cells and cortical macrophages; TrkC-like IR labelled the cortical epithelial cells and scattered medullar clusters of epithelial cells (including Hassal's corpuscles). Quantitative analysis revealed age-dependent decrease in the area occupied by TrkA-like IR in the cortex, and age-dependent increase in the medulla; no changes were detected in the area occupied by TrkB-like IR; the TrkC-like immunoreactive cells increase from 7 to 30 days and then decrease. Regarding to the bursa of Fabricius, TrkA-and TrkC-like IR were exclusively found in the epithelial cells of the follicle associated and the interfollicular epithelia, as well as TrkC-like IR in some medullary reticular epithelial cells of adult animals. Nevertheless, TrkB-like IR labelled extrafollicular unidentified cells in 7 days old animals, and the follicular secretory dendritic cells at 30 and 60 post-hatching. The area occupied by the medullary TrkB-like IR cells increased between 30 and 60 days. No immunostaining of lymphocytes was observed for any of the assessed antigens. The blood vessels of both the

  6. Forgotten radicals in biology.

    PubMed

    Luc, Rochette; Vergely, Catherine

    2008-12-01

    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

  7. Gains of REL in primary mediastinal B-cell lymphoma coincide with nuclear accumulation of REL protein.

    PubMed

    Weniger, Marc A; Gesk, Stefan; Ehrlich, Steve; Martin-Subero, José I; Dyer, Martin J S; Siebert, Reiner; Möller, Peter; Barth, Thomas F E

    2007-04-01

    Gains or amplifications of the REL locus are frequently seen in primary mediastinal B-cell lymphoma (PMBL). In classical Hodgkin's lymphoma, genomic overrepresentation of REL correlated with nuclear REL protein accumulation. To investigate the correlation between REL gene copies and its RNA and protein expression in PMBL, we analyzed genomic, transcriptional, and protein levels in 20 PMBLs and the PMBL derived cell lines MedB-1 and Karpas1106P. We found gains/amplifications in 75% of the PMBLs by fluorescence in situ hybridization (FISH) and genomic REL overrepresentation in the PMBL lines. Three of the five PMBLs with amplifications displayed elevated REL transcripts, while only 3/10 PMBLs with gains showed increased REL transcripts by real-time PCR. One PMBL without gains displayed increased REL transcription. REL protein expression exhibited a variable pattern across the PMBLs except for a single case that was completely negative by immunohistochemistry despite having gained REL. Although transcript levels were generally low and nuclear REL staining was weak in the lymphoma cell lines, these nevertheless exhibited high NF-kappaB activation. By fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms, genomic gains/amplifications of REL significantly correlated with nuclear REL expression (P < 0.05). In conclusion, the frequent genomic overrepresentation of REL in PMBL does not necessarily trigger an increased transcription/translation of REL. However, combined genomic and protein analysis revealed a significant association of gained REL and nuclear REL accumulation at the single cell level. PMID:17243160

  8. Expression of genes coding for antioxidant enzymes and heat shock proteins is altered in primary cultures of rat hepatocytes.

    PubMed

    Van Remmen, H; Williams, M D; Heydari, A R; Takahashi, R; Chung, H Y; Yu, B P; Richardson, A

    1996-02-01

    The expression of genes for heat shock proteins in the HSP70 family and genes for antioxidant enzymes was studied in rat hepatocytes cultured in either L-15 or Williams E media on a collagen matrix for up to 48 hours. The mRNA transcripts for the heat shock proteins hsp70, hsc70, and grp78 were induced dramatically when hepatocytes were cultured in L-15, and to a lesser extent when cultured in Williams E. The increase in hsp70 and hsc70 mRNA levels in the cultured hepatocytes was correlated with an increase in the nuclear transcription of these two genes and the binding activity of the heat shock transcription factor to the heat shock element. Culturing rat hepatocytes in either L-15 or Williams E resulted in a decrease in the levels of the mRNA transcripts for catalase and glutathione peroxidase and the activities of these two enzymes. However, the expression of Cu/Zn-superoxide dismutase, i.e., the level of the mRNA transcript or the enzymatic activity, did not change appreciably when hepatocytes were cultured for up to 48 hours. The decline in catalase and glutathione peroxidase expression in the cultured hepatocytes was correlated with a decrease in the GSH/GSSG ratio and an increase in lipid peroxidation. These data show that the expression of several genes involved in cellular protection change when hepatocytes are placed in primary cultures. Therefore, one must be careful in extrapolating from primary cultures to the liver in vivo, especially when studying processes that might be affected by heat shock proteins or antioxidant enzymes.

  9. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Periodicity in DNA primary structure is defined by secondary structure of the coded protein.

    PubMed Central

    Zhurkin, V B

    1981-01-01

    A 10.5-base periodicity found earlier is inherent in both eu- and prokaryotic coding nucleotide sequences. In the case of noncoding eukaryotic sequences no periodicity is found, so the 10.5-base oscillation seemingly does not correlate with the nucleosomal organization of DNA. It is shown that the DNA fragments, coding the alpha-helical protein segments, manifest the pronounced 10.5-base periodicity, while those regions of DNA which code the beta-structure have a 6-base oscillation. The repeating pattern of nucleotide sequences can be used for comparison of the DNA segments with low degree of homology. PMID:7243595

  11. Smoothened determines β-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium.

    PubMed

    Pal, Kasturi; Hwang, Sun-Hee; Somatilaka, Bandarigoda; Badgandi, Hemant; Jackson, Peter K; DeFea, Kathryn; Mukhopadhyay, Saikat

    2016-03-28

    Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein-coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves β-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161-β-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment. PMID:27002170

  12. Distribution of neurons immunoreactive for calcium-binding proteins varies across areas of cat primary somatosensory cortex.

    PubMed

    Schwark, H D; Li, J

    2000-03-15

    The primary somatosensory (SI) cortex in the cat contains four cytoarchitectonic areas that appear to contain separate body representations and have different functions. We tested whether functional differences among these areas are reflected in the densities of neurons containing each of three calcium-binding proteins: parvalbumin (PV), calbindin (CB), and calretinin (CR). Colocalization experiments revealed that CR was localized in a population of neurons distinct from those containing PV or CB. The general laminar distributions of the three calcium-binding proteins were similar to those described in other species and cortical areas, but there were significant density differences in layers II and III across SI. The density of PV-immunoreactive neurons was higher in areas 3b and 1 than in areas 3a and 2. CB-immunoreactive neurons were found in higher densities in anterior SI than in posterior SI, and the pattern of CR-immunoreactive neurons was reciprocal to that of CB, with significantly higher densities in posterior regions of SI. Since the firing characteristics of nonpyramidal neurons appear to be related to their calcium-binding protein content, differences in regional distributions of these neurons in layers II and III may contribute to functional differences between the cytoarchitectonic areas of SI cortex.

  13. HOCO radical chemistry.

    PubMed

    Francisco, Joseph S; Muckerman, James T; Yu, Hua-Gen

    2010-12-21

    Free radicals are important species in atmospheric chemistry, combustion, plasma environments, interstellar clouds, and biochemistry. Therefore, researchers would like to understand the formation mechanism, structure, stability, reactivity, spectroscopy, and dynamics of these chemical species. However, due to the presence of one or more unpaired electrons, radicals are often very reactive and have short lifetimes, which makes it difficult to conduct experiments. The HOCO radical appears in the atmosphere as well as in combustion environments and plays an important role in the conversion of CO to CO(2). Through the interplay between theoretical and experimental investigations, researchers have only recently understood the chemical role of the HOCO radical. In this Account, we systematically describe the current state of knowledge of the HOCO radical based on recent theoretical and experimental studies. This radical's two stable conformers, trans- and cis-HOCO, have been identified by high-level ab initio calculations and experimental spectroscopy. trans-HOCO is more stable by approximately 1.8 kcal/mol. The heat of formation of HOCO (298 K) was determined to be -43.0 ± 0.5 kcal/mol, giving a potential well depth of 30.1 ± 0.5 kcal/mol relative to the asymptote of the reactants OH + CO. The HOCO radical is very reactive. In most reactions between the HOCO radical and atoms, the HOCO radical acts as a hydrogen donor to reaction partners. Generally, the hydrogen is transferred through the formation of an association intermediate, which then proceeds through a molecular elimination step to produce the reaction products. The reaction rates of HOCO with some small radicals fall in the range of 10(-11)-10(-10) cm(3) molecule(-1) s(-1). These results clearly illustrate important features in the reactivity of the HOCO radical with other molecules.

  14. Protein Catalysis of the Retinal Subpicosecond Photoisomerization in the Primary Process of Bacteriorhodopsin Photosynthesis

    NASA Astrophysics Data System (ADS)

    Song, Li; El-Sayed, M. A.; Lanyi, J. K.

    1993-08-01

    The rate of retinal photoisomerization in wild-type bacteriorhodopsin (wt bR) is compared with that in a number of mutants in which a positively charged (Arg82), a negatively charged (Asp85 or Asp212), or neutral hydrogen bonding (Asp115 or Tyr185) amino acid residue known to be functionally important within the retinal cavity is replaced by a neutral, non-hydrogen bonding one. Only the replacements of the charged residues reduced the photoisomerization rate of the 13-cis and all-trans isomers present in these mutants by factors of ~1/4 and ~1/20, respectively. Retinal photo-and thermal isomerization catalysis and selectivity in wt bR by charged residues is discussed in terms of the known protein structure, the valence-bond wave functions of the ground and excited state of the retinal, and the electrostatic stabilization interactions within the retinal cavity.

  15. Cholesteryl ester transfer protein gene expression during differentiation of human preadipocytes to adipocytes in primary culture.

    PubMed

    Gauthier, B; Robb, M; McPherson, R

    1999-02-01

    The expression pattern of the CETP gene in relationship to that of LPL, adipsin, PPARgamma, C/EBPalpha, ADD1/SREBPI and actin was examined by RT-PCR during differentiation of human fibroblastic preadipocytes to adipocytes in primary culture. Preadipocytes were isolated from subcutaneous fat obtained from healthy female subjects undergoing mammary reduction procedures, and induced to differentiate in culture. Morphologically, adipogenesis was confirmed by the accumulation of lipid droplets in cells. We show that the gene encoding CETP is expressed in preadipocytes and is present throughout differentiation as compared to LPL and adipsin which were detected in the majority of samples by day 2 or 3 of adipogenesis. The transcription factors, PPARgamma, ADD1/SREBP1 and C/EBPalpha were expressed by day 2, concomitant with the appearance of LPL and adipsin but subsequent to the appearance of CETP. CETP mRNA was not detectable in human skin fibroblasts. These studies demonstrate that CETP. expression is induced at an early stage of commitment to the adipocyte lineage and may be activated by transcription factor(s), which are not members of the PPAR, ADD1/SREBP1 or C/EBP families. PMID:10030381

  16. Recombinant soluble gp130 protein reduces DEN-induced primary hepatocellular carcinoma in mice

    PubMed Central

    Hong, Jing; Wang, Hang; Shen, Guoying; Lin, Da; Lin, Yanxue; Ye, Nanhui; Guo, Yashan; Li, Qiaoling; Ye, Nanhui; Deng, Chengjun; Meng, Chun

    2016-01-01

    IL-6 (interleukin 6) plays an important role in the development and growth of hepatocellular carcinoma (HCC) via both classic signaling and trans-signaling pathways. Soluble gp130 (sgp130) is known to be a natural inhibitor of the trans-signaling pathway. In the present study, our goal was to investigate whether recombinant sgp130 could suppress the initiation and progression of HCC in mouse models. Our results demonstrate that sgp130 induced an apoptosis of HepG2 cells and inhibited the clonogenicity of HepG2 in vitro. Moreover, the IL-6 trans-signaling pathway is significantly suppressed by sgp130 as reflected by the decrease in the level of STAT3 phosphorylation and other inflammatory factors both in vitro and in vivo. In the DEN-induced HCC mouse model, intravenous injection of sgp130 attenuated hepatic fibrosis at 16 weeks and reduced the initiation and progression of primary HCC at 36 weeks. Furthermore, our results also demonstrate that intravenous administration of sgp130 significantly suppressed the growth and metastasis of xenograft human HCC in NOD/SCID mice. PMID:27080032

  17. Lamin B1 protein is required for dendrite development in primary mouse cortical neurons

    PubMed Central

    Giacomini, Caterina; Mahajani, Sameehan; Ruffilli, Roberta; Marotta, Roberto; Gasparini, Laura

    2016-01-01

    Lamin B1, a key component of the nuclear lamina, plays an important role in brain development and function. A duplication of the human lamin B1 (LMNB1) gene has been linked to adult-onset autosomal dominant leukodystrophy, and mouse and human loss-of-function mutations in lamin B1 are susceptibility factors for neural tube defects. In the mouse, experimental ablation of endogenous lamin B1 (Lmnb1) severely impairs embryonic corticogenesis. Here we report that in primary mouse cortical neurons, LMNB1 overexpression reduces axonal outgrowth, whereas deficiency of endogenous Lmnb1 results in aberrant dendritic development. In the absence of Lmnb1, both the length and complexity of dendrites are reduced, and their growth is unresponsive to KCl stimulation. This defective dendritic outgrowth stems from impaired ERK signaling. In Lmnb1-null neurons, ERK is correctly phosphorylated, but phospho-ERK fails to translocate to the nucleus, possibly due to delocalization of nuclear pore complexes (NPCs) at the nuclear envelope. Taken together, these data highlight a previously unrecognized role of lamin B1 in dendrite development of mouse cortical neurons through regulation of nuclear shuttling of specific signaling molecules and NPC distribution. PMID:26510501

  18. The transmembrane protein of HIV-1 primary isolates modulates cell surface expression of their envelope glycoproteins.

    PubMed

    Lebigot, S; Roingeard, P; Thibault, G; Lemiale, F; Verrier, B; Barin, F; Brand, D

    2001-11-10

    We have recently shown that the level of cell surface expression of envelope glycoproteins derived from various human immunodeficiency virus type 1 (HIV-1) primary isolates (PI) was lower than those of envelope glycoproteins derived from T-cell laboratory-adapted (TCLA) HIV-1 (D. Brand et al., 2000, Virology 271, 350-362). We investigated this phenomenon by comparing the cell surface expression of chimeric envelope glycoproteins constructed by swapping the gp120 surface and gp41 transmembrane glycoproteins of the TCLA HIV-1MN and the PI HIV-1(133), HIV-1G365, or HIV-1EFRA. We found that each chimeric envelope construct had a cell surface-specific pattern of expression similar to that of the parental envelope glycoproteins corresponding to the gp41. Thus, the difference in cell surface expression observed between TCLA viruses and various PI is probably due to a signal located in gp41. Identification of this signal may be important for the design of PI envelope-derived immunogens and may increase our understanding of the mechanisms by which HIV-1 escapes from the immune system.

  19. The biosynthesis of acute-phase proteins in primary cultures of rat hepatocytes.

    PubMed

    Andus, T; Gross, V; Tran-Thi, T A; Schreiber, G; Nagashima, M; Heinrich, P C

    1983-07-01

    The biosynthesis and secretion of alpha 2-macroglobulin, transferrin, alpha 1-acid glycoprotein and alpha 1-proteinase inhibitor were studied in rat hepatocyte primary cultures. After labeling with [35S]methionine, two forms, which can be separated electrophoretically differing by molecular weight, were found for each of the four glycoproteins. The following molecular weights were estimated for the intracellular precursors and the secreted forms: alpha 2-macroglobulin, 176 000 and 182 000; transferrin, 84 000 and 86 000; alpha 1-acid glycoprotein, 39 000 and 43 000-60 000; alpha 1-proteinase inhibitor, 49 000 and 54 000. Carbohydrate moieties could be removed from intracellular forms by treatment with endoglucosaminidase H indicating that their oligosaccharide chains were of the high-mannose type. The extracellular forms were sensitive to sialidase. They incorporated [3H]galactose and [3H]fucose showing that their oligosaccharide chains were of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the high-mannose and the complex type glycoproteins. In the hepatocyte medium newly synthesized albumin was detected after 30 min and newly synthesized glycoproteins after 60 min. Unglycosylated alpha 2-macroglobulin (162 000), transferrin (79 000), alpha 1-acid glycoprotein (23 000), and alpha 1-proteinase inhibitor (41 000) were found in the cells as well as in the medium, when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked reduction of the secretion of alpha 2-macroglobulin, alpha 1-acid glycoprotein and alpha 1-proteinase inhibitor, whereas the secretion of transferrin was less affected. PMID:6602705

  20. Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1

    PubMed Central

    Milenkovic, Ljiljana; Weiss, Lucien E.; Yoon, Joshua; Roth, Theodore L.; Su, YouRong S.; Sahl, Steffen J.; Scott, Matthew P.; Moerner, W. E.

    2015-01-01

    Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation. PMID:26100903

  1. Risk Stratification for Primary Prevention of Coronary Artery Disease: Roles of C-Reactive Protein and Coronary Artery Calcium.

    PubMed

    Qureshi, Waqas T; Rana, Jamal S; Yeboah, Joseph; Bin Nasir, Usama; Al-Mallah, Mouaz H

    2015-12-01

    Risk stratification of individuals at risk for atherosclerotic cardiovascular disease (ASCVD) plays an important role in primary prevention of cardiovascular disease. In addition to risk scores derived from conventional cardiovascular risk factors, high sensitivity C-reactive protein (hs-CRP) and coronary artery calcium (CAC) have emerged as two of the widely accepted non traditional risk factors for atherosclerotic disease that have shown incremental prognostic value in predicting cardiovascular events. This review systematically assesses the role of hs-CRP and CAC in various studies and demonstrates meta-analyses of the incremental prognostic value of hs-CRP and CAC in identifying patients at risk of future CVD events. Compared with this, CAC showed better incremental prognostic value and might be a better indicator of ASCVD risk in asymptomatic adults.

  2. Positive Selection in Bone Morphogenetic Protein 15 Targets a Natural Mutation Associated with Primary Ovarian Insufficiency in Human

    PubMed Central

    Meslin, Camille; Monestier, Olivier; Di Pasquale, Elisa; Pascal, Géraldine; Persani, Luca; Fabre, Stéphane

    2013-01-01

    Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in

  3. Positive selection in bone morphogenetic protein 15 targets a natural mutation associated with primary ovarian insufficiency in human.

    PubMed

    Auclair, Sylvain; Rossetti, Raffaella; Meslin, Camille; Monestier, Olivier; Di Pasquale, Elisa; Pascal, Géraldine; Persani, Luca; Fabre, Stéphane

    2013-01-01

    Bone Morphogenetic Protein 15 (BMP15) is a TGFβ-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFβ family member was performed. A maximum likelihood phylogenetic tree of several TGFβ/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in

  4. Swelling-activated chloride and potassium conductance in primary cultures of mouse proximal tubules. Implication of KCNE1 protein.

    PubMed

    Barrière, H; Rubera, I; Belfodil, R; Tauc, M; Tonnerieux, N; Poujeol, C; Barhanin, J; Poujeol, P

    2003-06-01

    Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 -/- mice. In wild-type PCT cells in primary culture, a Cl- conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I- > Br- > Cl- > > gluconate. This conductance was sensitive to 1 mM 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K+ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 microM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 microM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl- and K+ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures. PMID:12962276

  5. Establishment of Green Fluorescent Protein and Firefly Luciferase Expressing Mouse Primary Macrophages for In Vivo Bioluminescence Imaging

    PubMed Central

    Pajarinen, Jukka; Lin, Tzu-hua; Sato, Taishi; Loi, Florence; Yao, Zhenyu; Konttinen, Yrjö T.; Goodman, Stuart B.

    2015-01-01

    Macrophages play a key role in tissue homeostasis as well as in a range of pathological conditions including atherosclerosis, cancer, and autoimmunity. Many aspects of their in vivo behavior are, however, poorly understood. Bioluminescence imaging (BLI) with green fluorescent protein (GFP) and firefly luciferase (FLUC) labelled autologous reporter macrophages could potentially offer a powerful tool to study macrophage biology, but this approach has been hindered by the relative difficulty of efficient gene transfer into primary macrophages. Here we describe a straightforward method for producing large numbers of GFP/FLUC expressing mouse primary macrophages utilizing lentivirus vector, cyclosporine, and a double infection strategy. Using this method we achieved up to 60% of macrophages to express GFP with correspondingly high FLUC signal. When injected into the circulation using a mouse model of local biomaterial induced inflammation and osteolysis, macrophages were initially detectable within the lungs, followed by systemic homing to the local area of chronic inflammation in the distal femur. In addition, transduced macrophages maintained their ability to assume M1 and M2 phenotypes although the GFP/FLUC expression was altered by the polarizing signals. These reporter macrophages could prove to be valuable tools to study the role of macrophages in health and disease. PMID:26555613

  6. Electron transfer pathways and protein response to charge separation in photosynthetic reaction centers: time-resolved high-field ENDOR of the spin-correlated radical pair P865(+)QA(-).

    PubMed

    Poluektov, Oleg G; Utschig, Lisa M; Dubinskij, Alexander A; Thurnauer, Marion C

    2005-03-23

    Recently we reported the first observation of time-resolved (TR) high-frequency (HF) electron nuclear double resonance (ENDOR) of the transient charge separated state P865(+)Q(-)A in purple photosynthetic bacterial reaction centers (RC) (Poluektov, O. G., et al. J. Am. Chem. Soc. 2004, 126, 1644-1645). The high resolution and orientational selectivity of HF ENDOR allows us to directly probe protein environments by spectrally selecting specific nuclei in isotopically labeled samples. A new phenomenon associated with the spin correlated radical pair (SCRP) nature of P865(+)Q(-)A was observed. The TR-HF ENDOR spectra of protein nuclei (protons) surrounding deuterated QA(-) exhibit a derivative-like, complicated line shape, which differs considerably from the HF ENDOR spectrum of the protein nuclei surrounding thermally equilibrated QA(-). Here, a theoretical analysis of these observations is presented that shows that the positions and amplitudes of ENDOR lines contain information on hyperfine interactions (HFI) of a particular nucleus (a proton of the protein) with both correlated electron spins. Thus, spin density delocalization in the protein environment between the SCRP donor and acceptor molecules can be revealed via HF ENDOR. Novel approaches for acquiring and analyzing SCRP ENDOR that simplify interpretation of the spectra are discussed. Furthermore, we report here that the positions of the ENDOR lines of the SCRP shift with an increase in the time after laser flash, which initiates electron transfer. These shifts provide direct spectroscopic evidence of reorganization of the protein environment to accommodate the donor-acceptor charge-separated state P865(+)QA(-).

  7. 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.

  8. Interdependent TTF1 - ErbB4 interactions are critical for surfactant protein-B homeostasis in primary mouse lung alveolar type II cells.

    PubMed

    Marten, Elger; Nielsen, Heber C; Dammann, Christiane E L

    2015-09-01

    ErbB4 receptor and thyroid transcription factor (TTF)-1 are important modulators of fetal alveolar type II (ATII) cell development and injury. ErbB4 is an upstream regulator of TTF-1, promoting its expression in MLE-12 cells, an ATII cell line. Both proteins are known to promote surfactant protein-B gene (SftpB) and protein (SP-B) expression, but their feedback interactions on each other are not known. We hypothesized that TTF-1 expression has a feedback effect on ErbB4 expression in an in-vitro model of isolated mouse ATII cells. We tested this hypothesis by analyzing the effects of overexpressing HER4 and Nkx2.1, the genes of ErbB4 and TTF-1 on TTF-1 and ErbB4 protein expression, respectively, as well as SP-B protein expression in primary fetal mouse lung ATII cells. Transient ErbB4 protein overexpression upregulated TTF-1 protein expression in primary fetal ATII cells, similarly to results previously shown in MLE-12 cells. Transient TTF-1 protein overexpression down regulated ErbB4 protein expression in both cell types. TTF-1 protein was upregulated in primary transgenic ErbB4-depleted adult ATII cells, however SP-B protein expression in these adult transgenic ATII cells was not affected by the absence of ErbB4. The observation that TTF-1 is upregulated in fetal ATII cells by ErbB4 overexpression and also in ErbB4-deleted adult ATII cells suggests additional factors interact with ErbB4 to regulate TTF-1 levels. We conclude that the interdependency of TTF-1 and ErbB4 is important for surfactant protein levels. The interactive regulation of ErbB4 and TTF-1 needs further elucidation.

  9. RABL6A, a Novel RAB-Like Protein, Controls Centrosome Amplification and Chromosome Instability in Primary Fibroblasts

    PubMed Central

    Zhang, Xuefeng; Hagen, Jussara; Muniz, Viviane P.; Smith, Tarik; Coombs, Gary S.; Eischen, Christine M.; Mackie, Duncan I.; Roman, David L.; Van Rheeden, Richard; Darbro, Benjamin; Tompkins, Van S.; Quelle, Dawn E.

    2013-01-01

    RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the Alternative Reading Frame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53−/− MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis. PMID:24282525

  10. 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

  11. The prognostic significance of expression of the multidrug resistance-associated protein (MRP) in primary breast cancer.

    PubMed Central

    Nooter, K.; Brutel de la Riviere, G.; Look, M. P.; van Wingerden, K. E.; Henzen-Logmans, S. C.; Scheper, R. J.; Flens, M. J.; Klijn, J. G.; Stoter, G.; Foekens, J. A.

    1997-01-01

    In the present study, we determined the frequency and intensity of MRP protein expression by monoclonal antibody immunohistochemistry in a series of 259 resected invasive primary breast carcinomas, and we evaluated MRP immunoreactivity in relation to patient and tumour characteristics, relapse-free (RFS) and overall survival (OS). The immunostaining was graded on a semiquantitative scale that ranged from (-) to ( ). Overall, 34% of the tumours were positive for anti-MRP antibody: 19% showed weak cytoplasmic staining (+), 14% had clear cytoplasmic staining (++) and only 1% of the tumours had a strong cytoplasmic as well as membranous staining ( ). MRP expression was not related to patient's age, menopausal status, tumour size, differentiation grade, oestrogen and progesterone receptor level or lymph node involvement. In an exploratory univariate analysis of all patients, only primary tumour size and number of lymph nodes involved were significantly associated with shortened RFS (P < 0.001 and P < 0.001 respectively) and OS (P = 0.02 and P < 0.001 respectively). In Cox univariate analysis for RFS in subgroups of patients stratified by menopausal status, tumour size, nodal status, adjuvant systemic therapy and oestrogen and progesterone receptor status, MRP expression was associated with increased risk for failure in patients with small tumours (T1), in node-negative patients and in node-positive patients who received adjuvant systemic chemotherapy with cyclophosphamide, methotrexate and 5-fluorouracil (CMF); the relative hazard rate (RHR) for relapse was increased in the presence of MRP, with RHR values with 95% confidence limits (CL) of 2.8 (1.2-6.9), 2.1 (1.0-4.2) and 2.8 (0.8-9.9) respectively. In analysis for OS, expression of MRP was also associated with increased risk for failure in patients with small tumours (T1) [RHR (95% CL) 2.3 (0.9-6.0)] and in node-positive patients who received adjuvant systemic chemotherapy with CMF [RHR (95% CL) 3.7 (0.8-17.1)] but

  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. PMID:27612830

  13. Unusual copper-induced sensitization of the biological damage due to superoxide radicals. [Gamma radiation

    SciTech Connect

    Samuni, A.; Chevion, M.; Czapski, G.

    1981-12-25

    The role of superoxide radicals in biological damage in the presence of copper(II) ions has been investigated. Solutions of purified penicillinase in phosphate buffer, saturated with either air N/sub 2/, N/sub 2/O, or N/sub 2/O/O/sub 3/, were ..gamma..-irradiated in the presence and in the absence of formate. The residual activity of the enzyme in catalyzing the cleavage of the ..beta..-lactam ring of benzylpenicillin, was the monitored. The results indicate that in metal-free systems only the primary water-derived radicals, H, e/sub aq//sup -/, and OH, contribute toward enzyme inactivation, while the secondary species O/sub 2//sup -/ and CO/sub 2//sup -/ do not. The effect of copper(II) ions on the radiation-induced damage depended on which of the active species was predominant. With OH radicals, no effect of copper was detected. The damage originating from e/sub aq//sup -/ and H radicals decreased with the addition of copper, presumably due to their trapping by the copper(II) ions. In contrast, with O/sub 2//sup -/ radicals predominant in the system, copper dramatically enhanced the damage. This copper-induced sensitization was further increased in the presence of H/sub 2/O/sub 2/ and the dose modifying factor of copper exceeded 100. This copper effect could be completely eliminated by EDTA. The present results suggest that the presence of both transition metal ions and H/sub 2/O/sub 2/ and the binding of the metal ions to the target bimolecules are required for the manifestation of the deleterious role of O/sub 2//sup -/. According to this model, the enhancement of the damage by copper results from the attack, of reducing radical species on copper(II) ions bound to the bimolecule. These protein-Cu(II) complexes can be reduced by O/sub 2//sup -/ radicals yielding protein-Cu(I) species, which in turn react with H/sub 2/O/sub 2/ to locally form secondary hydroxyl radicals that react, on that site, with the protein impairing its biological function.

  14. Radical-Mediated Fluoroalkylations.

    PubMed

    Cho, Eun Jin

    2016-02-01

    Recently, the development of eco-friendly radical processes has become of great interest in synthetic chemistry. In particular, visible-light photocatalysis has drawn tremendous attention for its environmental compatibility and versatility in promoting many synthetically important reactions. In addition, inorganic electrides as electron donors have emerged as new eco-friendly tools for radical transformations since they consist of non-toxic and naturally abundant main metals such as calcium. The design of new fluoroalkylation reactions has benefited greatly from recent advances in visible-light photocatalysis and the chemistry of inorganic electrides. Since adding fluoroalkyl groups can dramatically change the physical and chemical properties of organic compounds, using these processes to promote eco-friendly radical fluoroalkylations will have a major impact in areas such as pharmaceuticals, agrochemicals, and material sciences. This Personal Account reviews radical-mediated fluoroalkylations, such as trifluoromethylations and difluoroalkylations, recently developed in our laboratory. PMID:26497950

  15. Discovery and Validation of Predictive Biomarkers of Survival for Non-small Cell Lung Cancer Patients Undergoing Radical Radiotherapy: Two Proteins With Predictive Value.

    PubMed

    Walker, Michael J; Zhou, Cong; Backen, Alison; Pernemalm, Maria; Williamson, Andrew J K; Priest, Lynsey J C; Koh, Pek; Faivre-Finn, Corinne; Blackhall, Fiona H; Dive, Caroline; Whetton, Anthony D

    2015-08-01

    Lung cancer is the most frequent cause of cancer-related death world-wide. Radiotherapy alone or in conjunction with chemotherapy is the standard treatment for locally advanced non-small cell lung cancer (NSCLC). Currently there is no predictive marker with clinical utility to guide treatment decisions in NSCLC patients undergoing radiotherapy. Identification of such markers would allow treatment options to be considered for more effective therapy. To enable the identification of appropriate protein biomarkers, plasma samples were collected from patients with non-small cell lung cancer before and during radiotherapy for longitudinal comparison following a protocol that carries sufficient power for effective discovery proteomics. Plasma samples from patients pre- and during radiotherapy who had survived > 18 mo were compared to the same time points from patients who survived < 14 mo using an 8 channel isobaric tagging tandem mass spectrometry discovery proteomics platform. Over 650 proteins were detected and relatively quantified. Proteins which showed a change during radiotherapy were selected for validation using an orthogonal antibody-based approach. Two of these proteins were verified in a separate patient cohort: values of CRP and LRG1 combined gave a highly significant indication of extended survival post one week of radiotherapy treatment.

  16. Hydroxyl radicals in indoor environments

    NASA Astrophysics Data System (ADS)

    Sarwar, Golam; Corsi, Richard; Kimura, Yosuke; Allen, David; Weschler, Charles J.

    Indoor hydroxyl radical concentrations were estimated using a new indoor air quality model which employs the SAPRC-99 atmospheric chemistry model to simulate indoor homogenous reactions. Model results indicate that typical indoor hydroxyl radical concentrations are lower than typical outdoor summertime urban hydroxyl radical levels of 5-10×10 6 molecules cm -3; however, indoor levels can be similar to or greater than typical nighttime outdoor hydroxyl radical levels of approximately 5×10 4 molecules cm -3. Effects of selected parameters on indoor hydroxyl radical concentrations are presented herein. Indoor hydroxyl radical concentrations are predicted to increase non-linearly with increasing outdoor ozone concentrations, indoor alkene emission rates, and air exchange rates. Indoor hydroxyl radical concentrations decrease with increasing outdoor nitric oxide concentrations. Indoor temperature and indoor light intensity have moderate impacts on indoor hydroxyl radical concentrations. Outdoor hydroxyl radical concentrations, outdoor nitrate (NO 3rad ) radical concentrations, outdoor hydroperoxy radical concentrations, and hydroxyl radical removal by indoor surfaces are predicted to have no appreciable impact on indoor hydroxyl radical concentrations. Production of hydroxyl radicals in indoor environments appears to be controlled primarily by reactions of alkenes with ozone, and nitric oxide with hydroperoxy radical. Estimated indoor hydroxyl radical levels may potentially affect indoor air quality. Two examples are presented in which reactions of d-limonene and α-pinene with indoor hydroxyl radicals produce aldehydes, which may be of greater concern than the original compounds.

  17. Primary endosymbiosis events date to the later Proterozoic with cross-calibrated phylogenetic dating of duplicated ATPase proteins.

    PubMed

    Shih, Patrick M; Matzke, Nicholas J

    2013-07-23

    Chloroplasts and mitochondria descended from bacterial ancestors, but the dating of these primary endosymbiosis events remains very uncertain, despite their importance for our understanding of the evolution of both bacteria and eukaryotes. All phylogenetic dating in the Proterozoic and before is difficult: Significant debates surround potential fossil calibration points based on the interpretation of the Precambrian microbial fossil record, and strict molecular clock methods cannot be expected to yield accurate dates over such vast timescales because of strong heterogeneity in rates. Even with more sophisticated relaxed-clock analyses, nodes that are distant from fossil calibrations will have a very high uncertainty in dating. However, endosymbiosis events and gene duplications provide some additional information that has never been exploited in dating; namely, that certain nodes on a gene tree must represent the same events, and thus must have the same or very similar dates, even if the exact date is uncertain. We devised techniques to exploit this information: cross-calibration, in which node date calibrations are reused across a phylogeny, and cross-bracing, in which node date calibrations are formally linked in a hierarchical Bayesian model. We apply these methods to proteins with ancient duplications that have remained associated and originated from plastid and mitochondrial endosymbionts: the α and β subunits of ATP synthase and its relatives, and the elongation factor thermo unstable. The methods yield reductions in dating uncertainty of 14-26% while only using date calibrations derived from phylogenetically unambiguous Phanerozoic fossils of multicellular plants and animals. Our results suggest that primary plastid endosymbiosis occurred ∼900 Mya and mitochondrial endosymbiosis occurred ∼1,200 Mya.

  18. Primary endosymbiosis events date to the later Proterozoic with cross-calibrated phylogenetic dating of duplicated ATPase proteins.

    PubMed

    Shih, Patrick M; Matzke, Nicholas J

    2013-07-23

    Chloroplasts and mitochondria descended from bacterial ancestors, but the dating of these primary endosymbiosis events remains very uncertain, despite their importance for our understanding of the evolution of both bacteria and eukaryotes. All phylogenetic dating in the Proterozoic and before is difficult: Significant debates surround potential fossil calibration points based on the interpretation of the Precambrian microbial fossil record, and strict molecular clock methods cannot be expected to yield accurate dates over such vast timescales because of strong heterogeneity in rates. Even with more sophisticated relaxed-clock analyses, nodes that are distant from fossil calibrations will have a very high uncertainty in dating. However, endosymbiosis events and gene duplications provide some additional information that has never been exploited in dating; namely, that certain nodes on a gene tree must represent the same events, and thus must have the same or very similar dates, even if the exact date is uncertain. We devised techniques to exploit this information: cross-calibration, in which node date calibrations are reused across a phylogeny, and cross-bracing, in which node date calibrations are formally linked in a hierarchical Bayesian model. We apply these methods to proteins with ancient duplications that have remained associated and originated from plastid and mitochondrial endosymbionts: the α and β subunits of ATP synthase and its relatives, and the elongation factor thermo unstable. The methods yield reductions in dating uncertainty of 14-26% while only using date calibrations derived from phylogenetically unambiguous Phanerozoic fossils of multicellular plants and animals. Our results suggest that primary plastid endosymbiosis occurred ∼900 Mya and mitochondrial endosymbiosis occurred ∼1,200 Mya. PMID:23776247

  19. The interactive association between heat shock factor 1 and heat shock proteins in primary myocardial cells subjected to heat stress.

    PubMed

    Tang, Shu; Chen, Hongbo; Cheng, Yanfen; Nasir, Mohammad Abdel; Kemper, Nicole; Bao, Endong

    2016-01-01

    Heat shock factor 1 (HSF1) is a heat shock transcription factor that rapidly induces heat shock gene transcription following thermal stress. In this study, we subjected primary neonatal rat myocardial cells to heat stress in vitro to create a model system for investigating the trends in expression and association between various heat shock proteins (HSPs) and HSF1 under adverse environmental conditions. After the cells were subjected to heat stress at 42˚C for different periods of time, HSP and HSF1 mRNA and protein levels were detected by qPCR and western blot analysis in the heat-stressed cells. The HSF1 expression levels significantly increased in the cells following 120 min of exposure to heat stess compared to the levels observed at the beginning of heat stress exposure. HSP90 followed a similar trend in expression to HSF1, whereas HSP70 followed an opposite trend. However, no significant changes were observed in the crystallin, alpha B (CRYAB, also known as HSP beta-5) expression levels during the 480‑min period of exposure to heat stress. The interaction between the HSPs and HSF1 was analyzed by STRING 9.1, and it was found that HSF1 interacted with HSP90 and HSP70, and that it did not play a role in regulating CRYAB expression. Based on our findings, HSP70 may suppress HSF1 in rat myocardial cells under conditions of heat stress. Furthermore, our data demonstrate that HSF1 is not the key factor for all HSPs, and this was particularly the case for CRYAB.

  20. Chronic Alcohol Exposure Decreases 53BP1 Protein Levels Leading to a Defective DNA Repair in Cultured Primary Cortical Neurons.

    PubMed

    Romero, Ana M; Palanca, Ana; Ruiz-Soto, Maria; Llorca, Javier; Marín, María P; Renau-Piqueras, Jaime; Berciano, Maria T; Lafarga, Miguel

    2016-01-01

    Chronic alcohol consumption may cause neurodevelopmental and neurodegenerative disorders. Alcohol neurotoxicity is associated with the production of acetaldehyde and reactive oxygen species that induce oxidative DNA damage. However, the molecular mechanisms by which ethanol disturbs the DNA damage response (DDR), resulting in a defective DNA repair, remain unknown. Here, we have used cultured primary cortical neurons exposed to 50 or 100 mM ethanol for 7 days to analyze the ethanol-induced DDR. Ethanol exposure produced a dose-dependent generation of double strand breaks and the formation of DNA damage foci immunoreactive for the histone γH2AX, a DNA damage marker, and for the ubiquitylated H2A, which is involved in chromatin remodeling at DNA damage sites. Importantly, these DNA damage foci failed to recruit the protein 53BP1, a crucial DNA repair factor. This effect was associated with a drop in 53BP1 mRNA and protein levels and with an inhibition of global transcription. Moreover, ethanol-exposed neurons treated with ionizing radiation (2 Gy) also failed to recruit 53BP1 at DNA damage foci and exhibited a greater vulnerability to DNA lesions than irradiated control neurons. Our results support that defective DNA repair, mediated by the deficient expression and recruitment of 53BP1 to DNA damage sites, represents a novel mechanism involved in ethanol neurotoxicity. The design of therapeutic strategies that increase or stabilize 53BP1 levels might potentially promote DNA repair and partially compensate alcohol neurotoxicity.

  1. Photoionization and electron radical recombination dynamics in photoactive yellow protein investigated by ultrafast spectroscopy in the visible and near-infrared spectral region.

    PubMed

    Zhu, Jingyi; Paparelli, Laura; Hospes, Marijke; Arents, Jos; Kennis, John T M; van Stokkum, Ivo H M; Hellingwerf, Klaas J; Groot, Marie Louise

    2013-09-26

    Photoinduced ionization of the chromophore inside photoactive yellow protein (PYP) was investigated by ultrafast spectroscopy in the visible and near-infrared spectral regions. An absorption band that extended from around 550 to 850 nm was observed and ascribed to solvated electrons, ejected from the p-hydroxycinnamic acid anion chromophore upon the absorption of two 400 nm photons. Global kinetic analysis showed that the solvated electron absorption decayed in two stages: a shorter phase of around 10 ps and a longer phase of more than 3 ns. From a simulation based on a diffusion model we conclude that the diffusion rate of the electron is about 0.8 Å(2)/ps in wild type PYP, and that the electron is ejected to a short distance of only several angstroms away from the chromophore. The chromophore-protein pocket appears to provide a water-similar local environment for the electron. Because mutations at different places around the chromophore have different effect on the electron recombination dynamics, we suggest that solvated electrons could provide a new method to investigate the local dielectric environment inside PYP and thus help to understand the role of the protein in the photoisomerization process.

  2. Scavenging of superoxide anion radical by chaparral.

    PubMed

    Zang, L Y; Cosma, G; Gardner, H; Starks, K; Shi, X; Vallyathan, V

    1999-06-01

    Chaparral is considered to act as an antioxidant. However, the inhibitory effects of chaparral on specific radical species are not well understood. Using electron paramagnetic resonance (EPR) spectroscopy in combination with spin trapping techniques, we have found that chaparral scavenges superoxide anion radical (O2*-) in a dose-dependent manner. 5,5-dimethyl-lpyrroline-N-oxide (DMPO) was used as a spin trapping agent and the reaction of xanthine and xanthine oxidase as a source of O2*-. The kinetic parameters, IC50 and Vmax, for chaparral scavenging of O2*- were found to be 0.899 microg/mL and 8.4 ng/mL/sec, respectively. The rate constant for chaparral scavenging O2*- was found to be 1.22 x 10(6) g(-1) s(-1). Our studies suggest that the antioxidant properties of chaparral may involve a direct scavenging effect of the primary oxygen radical, O2*-.

  3. HCMV protein LUNA is required for viral reactivation from latently infected primary CD14⁺ cells.

    PubMed

    Keyes, Lisa R; Hargett, Danna; Soland, Melisa; Bego, Mariana G; Rossetto, Cyprian C; Almeida-Porada, Graca; St Jeor, Stephen

    2012-01-01

    Human cytomegalovirus (HCMV) is a member of the Herpesviridae family that infects individuals throughout the world. Following an initial lytic stage, HCMV can persist in the individual for life in a non-active (or latent) form. During latency, the virus resides within cells of the myeloid lineage. The mechanisms controlling HCMV latency are not completely understood. A latency associated transcript, UL81-82ast, encoding the protein LUNA (Latency Unique Natural Antigen) was identified from latently infected donors in vivo. To address the role of the UL81-82ast protein product LUNA, in the context of the viral genome, we developed a recombinant HCMV bacterial artificial chromosome (BAC) that does not express LUNA. This construct, LUNA knockout FIX virus (FIX-ΔLUNA), was used to evaluate LUNA's role in HCMV latency. The FIX-ΔLUNA virus was able to lytically infect Human Fibroblast (HF) cells, showing that LUNA is not required to establish a lytic infection. Interestingly, we observed significantly higher viral copy numbers in HF cells infected with FIX-ΔLUNA when compared to FIX-WT virus. Furthermore, FIX-WT and FIX-ΔLUNA genomic DNA and transcription of UL81-82ast persisted over time in primary monocytes. In contrast, the levels of UL138 transcript expression in FIX-ΔLUNA infected HF and CD14⁺ cells was 100 and 1000 fold lower (respectively) when compared to the levels observed for FIX-WT infection. Moreover, FIX-ΔLUNA virus failed to reactivate from infected CD14⁺ cells following differentiation. This lack of viral reactivation was accompanied by a lack of lytic gene expression, increase in viral copy numbers, and lack of the production of infectious units following differentiation of the cells. Our study suggests that the LUNA protein is involved in regulating HCMV reactivation, and that in the absence of LUNA, HCMV may not be able to enter a proper latent state and therefore cannot be rescued from the established persistent infection in CD14⁺ cells.

  4. GM-CSF reduces expression of chondroitin sulfate proteoglycan (CSPG) core proteins in TGF-β-treated primary astrocytes.

    PubMed

    Choi, Jung-Kyoung; Park, Sang-Yoon; Kim, Kil Hwan; Park, So Ra; Lee, Seok-Geun; Choi, Byung Hyune

    2014-12-01

    GM-CSF plays a role in the nervous system, particularly in cases of injury. A therapeutic effect of GM-CSF has been reported in rat models of various central nervous system injuries. We previously showed that GM-CSF could enhance long-term recovery in a rat spinal cord injury model, inhibiting glial scar formation and increasing the integrity of axonal structure. Here, we investigated molecular the mechanism(s) by which GM-CSF suppressed glial scar formation in an in vitro system using primary astrocytes treated with TGF-β. GM-CSF repressed the expression of chondroitin sulfate proteoglycan (CSPG) core proteins in astrocytes treated with TGF-β. GM-CSF also inhibited the TGF-β-induced Rho-ROCK pathway, which is important in CSPG expression. Finally, the inhibitory effect of GM-CSF was blocked by a JAK inhibitor. These results may provide the basis for GM-CSF's effects in glial scar inhibition and ultimately for its therapeutic effect on neural cell injuries.

  5. Increased protein kinase A type Iα regulatory subunit expression in parathyroid gland adenomas of patients with primary hyperparathyroidism.

    PubMed

    Hibi, Yatsuka; Kambe, Fukushi; Imai, Tsuneo; Ogawa, Kimio; Shimizu, Yoshimi; Shibata, Masahiro; Kagawa, Chikara; Mizuno, Yutaka; Ito, Asako; Iwase, Katsumi

    2013-01-01

    Protein kinase A (PKA) regulatory subunit type Iα (RIα) is a major regulatory subunit that functions as an inhibitor of PKA kinase activity. We have previously demonstrated that elevated RIα expression is associated with diffuse-to-nodular transformation of hyperplasia in parathyroid glands of renal hyperparathyroidism. The aim of the current study was to determine whether or not RIα expression is increased in adenomas of primary hyperparathyroidism (PHPT), because monoclonal proliferation has been demonstrated in both adenomas and nodular hyperplasia. Surgical specimens comprising 22 adenomas and 11 normal glands, obtained from 22 patients with PHPT, were analyzed. Western blot and immunohistochemical analyses were employed to evaluate RIα expression. PKA activities were determined in several adenomas highly expressing RIα. RIα expression was also separately evaluated in chief and oxyphilic cells using the "Allred score" system. Expression of proliferating cell nuclear antigen (PCNA), a proliferation marker, was also immunohistochemically examined. Western blot analysis revealed that 5 out of 8 adenomas highly expressed RIα, compared with normal glands. PKA activity in adenomas was significantly less than in normal glands. Immunohistochemical analysis further demonstrated high expression of RIα in 20 out of 22 adenomas. In adenomas, the greater RIα expression and more PCNA positive cells were observed in both chief and oxyphilic cells. The present study suggested that high RIα expression could contribute to monoclonal proliferation of parathyroid cells by impairing the cAMP/PKA signaling pathway. PMID:23197043

  6. Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia

    PubMed Central

    Picaud, Sarah; Leonards, Katharina; Lambert, Jean-Philippe; Dovey, Oliver; Wells, Christopher; Fedorov, Oleg; Monteiro, Octovia; Fujisawa, Takao; Wang, Chen-Yi; Lingard, Hannah; Tallant, Cynthia; Nikbin, Nikzad; Guetzoyan, Lucie; Ingham, Richard; Ley, Steven V.; Brennan, Paul; Muller, Susanne; Samsonova, Anastasia; Gingras, Anne-Claude; Schwaller, Juerg; Vassiliou, George; Knapp, Stefan; Filippakopoulos, Panagis

    2016-01-01

    Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET (bromo and extra-terminal) inhibitors and their significant activity in diverse tumor models have rapidly translated into clinical studies and have motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of BRD protein complexes complicates predictions of the consequences of their pharmacological targeting. To address this issue, we developed a promiscuous BRD inhibitor [bromosporine (BSP)] that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle, we studied the effects of BSP on leukemic cell lines known to be sensitive to BET inhibition and found, as expected, strong antiproliferative activity. Comparison of the modulation of transcriptional profiles by BSP after a short exposure to the inhibitor resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, nonselective targeting of BRDs identified BETs, but not other BRDs, as master regulators of context-dependent primary transcription response. PMID:27757418

  7. Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells

    PubMed Central

    1992-01-01

    Chloride-dependent K transport ([K-Cl] cotransport) in dog red cells is activated by cell swelling. Whether the volume signal is generated by a change in cell configuration or by the dilution of some cytosolic constituent is not known. To differentiate between these two alternatives we prepared resealed ghosts that, compared with intact red cells, had the same surface area and similar hemoglobin concentration, but a greatly diminished volume. Swelling-induced [K-Cl] cotransport was activated in the ghosts at a volume (20 fl) well below the activation volume for intact cells (70 fl), but at a similar hemoglobin concentration (30-35 g dry solids per 100 g wet weight). Ghosts made to contain 40% albumin and 60% hemoglobin showed activation of [K-Cl] cotransport at a concentration of cell solids similar to intact cells or ghosts containing only hemoglobin. [K-Cl] cotransport in the resealed ghosts became quiescent at a dry solid concentration close to that at which shrinkage-induced Na/H exchange became activated. These results support the notion that the primary volume sensor in dog red cells is cytosolic protein concentration. We speculate that macromolecular crowding is the mechanism by which cells initiate responses to volume perturbation. PMID:1512553

  8. Chemistry of carotenoid neutral radicals.

    PubMed

    Ligia Focsan, A; Magyar, Adam; Kispert, Lowell D

    2015-04-15

    Proton loss from the carotenoid radical cations (Car(+)) to form neutral radicals (#Car) was investigated by numerous electrochemical, EPR, ENDOR and DFT studies described herein. The radical cation and neutral radicals were formed in solution electrochemically and stabilized on solid silica-alumina and MCM-41 matrices. Carotenoid neutral radicals were recently identified in Arabidopsis thaliana plant and photosystem II samples. Deprotonation at the terminal ends of a zeaxanthin radical cation could provide a secondary photoprotection pathway which involves quenching excited state chlorophyll by the long-lived zeaxanthin neutral radicals formed. PMID:25687648

  9. Radical formation in cytochrome c oxidase☆

    PubMed Central

    Yu, Michelle A.; Egawa, Tsuyoshi; Shinzawa-Itoh, Kyoko; Yoshikawa, Shinya; Yeh, Syun-Ru; Rousseau, Denis L.; Gerfen, Gary J.

    2015-01-01

    The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O2 redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O2 with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77 K at reaction times ranging from 50 µs to 6 ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9 GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130 GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO2•− ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O2 with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H2O2) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50 µs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (R – O – O•) species was formed, presumably by the reaction between O2 and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical

  10. Sequence-regulated vinyl copolymers by metal-catalysed step-growth radical polymerization.

    PubMed

    Satoh, Kotaro; Ozawa, Satoshi; Mizutani, Masato; Nagai, Kanji; Kamigaito, Masami

    2010-04-12

    Proteins and nucleic acids are sequence-regulated macromolecules with various properties originating from their perfectly sequenced primary structures. However, the sequence regulation of synthetic polymers, particularly vinyl polymers, has not been achieved and is one of the ultimate goals in polymer chemistry. In this study, we report a strategy to obtain sequence-regulated vinyl copolymers consisting of styrene, acrylate and vinyl chloride units using metal-catalysed step-growth radical polyaddition of designed monomers prepared from common vinyl monomer building blocks. Unprecedented ABCC-sequence-regulated copolymers with perfect vinyl chloride-styrene-acrylate-acrylate sequences were obtained by copper-catalysed step-growth radical polymerization of designed monomers possessing unconjugated C=C and reactive C-Cl bonds. This strategy may open a new route in the study of sequence-regulated synthetic polymers.

  11. Photolysis of CH{sub 3}CHO at 248 nm: Evidence of triple fragmentation from primary quantum yield of CH{sub 3} and HCO radicals and H atoms

    SciTech Connect

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

    2014-06-07

    Radical quantum yields have been measured following the 248 nm photolysis of acetaldehyde, CH{sub 3}CHO. 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 HO{sub 2} radicals by reaction with O{sub 2}. The CH{sub 3} radical yield has been determined using the same technique following their conversion into CH{sub 3}O{sub 2}. Absolute yields have been deduced for HCO radicals and H atoms through fitting of time resolved HO{sub 2} profiles, obtained under various O{sub 2} concentrations, to a complex model, while the CH{sub 3} yield has been determined relative to the CH{sub 3} yield from 248 nm photolysis of CH{sub 3}I. Time resolved HO{sub 2} profiles under very low O{sub 2} concentrations suggest that another unknown HO{sub 2} forming reaction path exists in this reaction system besides the conversion of HCO radicals and H atoms by reaction with O{sub 2}. HO{sub 2} profiles can be well reproduced under a large range of experimental conditions with the following quantum yields: CH{sub 3}CHO + hν{sub 248nm} → CH{sub 3}CHO{sup *}, CH{sub 3}CHO{sup *} → CH{sub 3} + HCO ϕ{sub 1a} = 0.125 ± 0.03, CH{sub 3}CHO{sup *} → CH{sub 3} + H + CO ϕ{sub 1e} = 0.205 ± 0.04, CH{sub 3}CHO{sup *}→{sup o{sub 2}}CH{sub 3}CO + HO{sub 2} ϕ{sub 1f} = 0.07 ± 0.01. The CH{sub 3}O{sub 2} quantum yield has been determined in separate experiments as ϕ{sub CH{sub 3}} = 0.33 ± 0.03 and is in excellent agreement with the CH{sub 3} yields derived from the HO{sub 2} measurements considering that the triple fragmentation (R1e) is an important reaction path in the 248 nm photolysis of CH{sub 3}CHO. From arithmetic considerations taking into account the HO{sub 2} and CH{sub 3} measurements we deduce a remaining quantum yield for the molecular pathway: CH{sub 3}CHO{sup *} → CH{sub 4} + CO ϕ{sub 1b} = 0.6. All experiments can be

  12. Antinephritis and radical scavenging activity of prenylflavonoids.

    PubMed

    Fukai, Toshio; Satoh, Kazue; Nomura, Taro; Sakagami, Hiroshi

    2003-12-01

    Antinephritis activity of 5 prenylflavonoids similar to glabridin (1-5), isolated from Morus alba, Artocarpus communis, Glycyrrhiza uralensis and G. inflata, was evaluated in mice with glomerular disease (Masugi-nephritis). Oral administrations of artonin E (2) or licochalcone A (4) for 10 days (30 mg kg(-1) day(-1)) reduced the amount of urinary protein excretion compared to nephritic mice. ESR spectroscopy demonstrated that morusin (1) and licorisoflavan A (5) increased the radical intensity of sodium ascorbate by about two times. Morusin, licoricidin (3), licochalcone A and licorisoflavan A showed weak scavenging activity against superoxide anion radical.

  13. Development of immunoblotting techniques for DNA radical detection

    PubMed Central

    Summers, Fiona A.; Mason, Ronald P.; Ehrenshaft, Marilyn

    2012-01-01

    Radical damage to DNA has been implicated in cell death, cellular dysfunction and cancer. A recently developed method for detecting DNA radicals uses the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) to trap radicals. The trapped radicals then decay into stable nitrone adducts detectable with anti-DMPO antibodies and quantifiable by ELISA or dot blot assays. However, the sequences of DNA that are damaged are likely to be as important as the total level of damage. Therefore, we have developed immunoblotting methods for detection of DNA nitrone adducts on electrophoretically separated DNA, comparable to Western blotting for proteins. These new techniques not only allow the assessment of relative radical adduct levels, but can reveal specific DNA fragments, and ultimately nucleotides, as radical targets. Moreover, we have determined that denaturation of samples into single-stranded DNA enhances the detection of DNA-DMPO adducts in our new blotting methods and also ELISA assays. PMID:23142572

  14. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    PubMed

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images. PMID:23085529

  15. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    PubMed

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images.

  16. Catalytically impaired hMYH and NEIL1 mutant proteins identified in patients with primary sclerosing cholangitis and cholangiocarcinoma

    PubMed Central

    Forsbring, Monika; Vik, Erik S.; Dalhus, Bjørn; Karlsen, Tom H.; Bergquist, Annika; Schrumpf, Erik; Bjørås, Magnar; Boberg, Kirsten M.; Alseth, Ingrun

    2009-01-01

    The human hMYH and NEIL1 genes encode DNA glycosylases involved in repair of oxidative base damage and mutations in these genes are associated with certain cancers. Primary sclerosing cholangitis (PSC), a chronic cholestatic liver disease characterized by inflammatory destruction of the biliary tree, is often complicated by the development of cholangiocarcinoma (CCA). Here, we aimed to investigate the influence of genetic variations in the hMYH and NEIL1 genes on risk of CCA in PSC patients. The hMYH and NEIL1 gene loci in addition to the DNA repair genes hOGG1, NTHL1 and NUDT1 were analyzed in 66 PSC patients (37 with CCA and 29 without cancer) by complete genomic sequencing of exons and adjacent intronic regions. Several single-nucleotide polymorphisms and mutations were identified and severe impairment of protein function was observed for three non-synonymous variants. The NEIL1 G83D mutant was dysfunctional for the major oxidation products 7,8-dihydro-8-oxoguanine (8oxoG), thymine glycol and dihydrothymine in duplex DNA, and the ability to perform δ-elimination at abasic sites was significantly reduced. The hMYH R260Q mutant had severe defect in adenine DNA glycosylase activity, whereas hMYH H434D could excise adenines from A:8oxoG pairs but not from A:G mispairs. We found no overall associations between the 18 identified variants and susceptibility to CCA in PSC patients; however, the impaired variants may be of significance for carcinogenesis in general. Our findings demonstrate the importance of complete resequencing of selected candidate genes in order to identify rare genetic variants and their possible contribution to individual susceptibility to cancer development. PMID:19443904

  17. Primary light-induced reaction steps of reversibly photoswitchable fluorescent protein Padron0.9 investigated by femtosecond spectroscopy.

    PubMed

    Walter, Arne; Andresen, Martin; Jakobs, Stefan; Schroeder, Jörg; Schwarzer, Dirk

    2015-04-23

    The reversible photoswitching of the photochromic fluorescent protein Padron0.9 involves a cis-trans isomerization of the chromophore. Both isomers are subjected to a protonation equilibrium between a neutral and a deprotonated form. The observed pH dependent absorption spectra require at least two protonating groups in the chromophore environment modulating its proton affinity. Using femtosecond transient absorption spectroscopy, we elucidate the primary reaction steps of selectively excited chromophore species. Employing kinetic and spectral modeling of the time dependent transients, we identify intermediate states and their spectra. Excitation of the deprotonated trans species is followed by excited state relaxation and internal conversion to a hot ground state on a time scale of 1.1-6.5 ps. As the switching yield is very low (Φtrans→cis = 0.0003 ± 0.0001), direct formation of the cis isomer in the time-resolved experiment is not observed. The reverse switching route involves excitation of the neutral cis chromophore. A strong H/D isotope effect reveals the initial reaction step to be an excited state proton transfer with a rate constant of kH = (1.7 ps)(-1) (kD = (8.6 ps)(-1)) competing with internal conversion (kic = (4.5 ps)(-1)). The deprotonated excited cis intermediate relaxes to the well-known long-lived fluorescent species (kr = (24 ps)(-1)). The switching quantum yield is determined to be low as well, Φcis→trans = 0.02 ± 0.01. Excitation of both the neutral and deprotonated cis chromophores is followed by a ground state proton transfer reaction partially re-establishing the disturbed ground state equilibrium within 1.6 ps (deuterated species: 5.6 ps). The incomplete equilibration reveals an inhomogeneous population of deprotonated cis species which equilibrate on different time scales.

  18. Peroxyl radical reactions with carotenoids in microemulsions: Influence of microemulsion composition and the nature of peroxyl radical precursor.

    PubMed

    El-Agamey, Ali; McGarvey, David J

    2016-01-01

    The reactions of acetylperoxyl radicals with different carotenoids (7,7'-dihydro-β-carotene and ζ-carotene) in SDS and CTAC microemulsions of different compositions were investigated using laser flash photolysis (LFP) coupled with kinetic absorption spectroscopy. The primary objective of this study was to explore the influence of microemulsion composition and the type of surfactant used on the yields and kinetics of various transients formed from the reaction of acetylperoxyl radicals with carotenoids. Also, the influence of the site (hydrocarbon phases or aqueous phase) of generation of the peroxyl radical precursor was examined by using 4-acetyl-4-phenylpiperidine hydrochloride (APPHCl) and 1,1-diphenylacetone (11DPA) as water-soluble and lipid-soluble peroxyl radical precursors, respectively. LFP of peroxyl radical precursors with 7,7'-dihydro-β-carotene (77DH) in different microemulsions gives rise to the formation of three distinct transients namely addition radical (λmax=460 nm), near infrared transient1 (NIR, λmax=700 nm) and 7,7'-dihydro-β-carotene radical cation (77DH(•+), λmax=770 nm). In addition, for ζ-carotene (ZETA) two transients (near infrared transient1 (NIR1, λmax=660 nm) and ζ-carotene radical cation (ZETA(•+), λmax=730-740 nm)) are generated following LFP of peroxyl radical precursors in the presence of ζ-carotene (ZETA) in different microemulsions. The results show that the composition of the microemulsion strongly influences the observed yield and kinetics of the transients formed from the reactions of peroxyl radicals (acetylperoxyl radicals) with carotenoids (77DH and ZETA). Also, the type of surfactant used in the microemulsions influences the yield of the transients formed. The dependence of the transient yields and kinetics on microemulsion composition (or the type of surfactant used in the microemulsion) can be attributed to the change of the polarity of the microenvironment of the carotenoid. Furthermore, the nature of

  19. Primary structures of three highly acidic ribosomal proteins S6, S12 and S15 from the archaebacterium Halobacterium marismortui.

    PubMed

    Kimura, J; Arndt, E; Kimura, M

    1987-11-16

    The amino acid sequences of three extremely acidic ribosomal proteins, S6, S12, and S15, from Halobacterium marismortui have been determined. The sequences were obtained by the sequence analysis of peptides derived by enzymatic digestion with trypsin. Stapylococcus aureus protease and chymotrypsin, as well as by cleavage with dilute HCl. The proteins, S6, S12 and S15, consist of 116, 147 and 102 amino acid residues, and have molecular masses of 12,251, 16,440 and 11,747 Da, respectively. Comparison of the amino acid sequences of these proteins with ribosomal protein sequences of other organisms revealed that halobacterial protein S12 has homology with the eukaryotic protein S16A from Saccharomyces cerevisiae, while S15 is significantly related to the Xenopus laevis S19 protein. No homology was found between these halobacterial proteins and any eubacterial ribosomal proteins.

  20. Functional Proteomic Analysis of Advanced Serous Ovarian Cancer using Reverse Phase Protein Array: TGFβ Pathway Signaling Indicates Response to Primary Chemotherapy

    PubMed Central

    Carey, Mark S.; Agarwal, Roshan; Gilks, Blake; Swenerton, Kenneth; Kalloger, Steve; Santos, Jennifer; Ju, Zhenlin; Lu, Yiling; Zhang, Fan; Coombes, Kevin; Miller, Dianne; Huntsman, David; Mills, Gordon B.; Hennessy, Bryan T

    2010-01-01

    Purpose: Using Reverse Phase Protein Array (RPPA) we measured protein expression associated with response to primary chemotherapy in patients with advanced-stage high-grade serous ovarian cancer. Experimental Design: Tumor samples were obtained from forty-five patients with advanced high-grade serous cancers from the Gynecology Tumor Bank at the British Columbia Cancer Agency. Treatment consisted of platinum-based chemotherapy following debulking surgery. Protein lysates were prepared from fresh frozen tumor samples and 80 validated proteins from signaling pathways implicated in ovarian carcinogenesis were measured by RPPA. Normalization of Ca-125 by the 3rd cycle of chemotherapy was chosen as the primary outcome measure of chemotherapy response. Logistic regression was used for multivariate analysis to identify protein predictors of Ca-125 normalization, and Cox regression to test for the association between protein expression and PFS. A significance level of p ≤ 0.05 was used. Results: The mean age at diagnosis was 56.8 years. EGFR, YKL-40 and several TGFβ pathway proteins (c-Jun N-terminal kinase JNK, JNK phosphorylated at residues 183 and 185, PAI-1, Smad3, TAZ) showed significant associations with Ca-125 normalization on univariate testing. On multivariate analysis, EGFR (p < 0.02), JNK (p < 0.01), and Smad3 (p < 0.04) were significantly associated with normalization of Ca-125. Contingency table analysis of pathway-classified proteins revealed that the selection of TGFβ pathway proteins was unlikely due to false discovery (p < 0.007, Bonferroni-adjusted). Conclusion: TGFβ pathway signaling likely plays an important role as a marker or mediator of chemoresistance in advanced serous ovarian cancer. On this basis, future studies to develop and validate a useful predictor of treatment failure are warranted. PMID:20460476

  1. Radical School Reform.

    ERIC Educational Resources Information Center

    Gross, Beatrice, Ed.; Gross, Ronald, Ed.

    This book provides a comprehensive examination of the nature of the school crisis and the ways in which radical thinkers and educators are dealing with it. Excerpts from the writings of Jonathan Kozol, John Holt, Kenneth Clark, and others are concerned with the realities of education in ghettos and suburbs. Paul Goodman, Marshall McLuhan, Sylvia…

  2. Influence of pH, metal chelator, free radical scavenger and interfacial characteristics on the oxidative stability of β-carotene in conjugated whey protein-pectin stabilised emulsion.

    PubMed

    Xu, Duoxia; Yuan, Fang; Gao, Yanxiang; McClements, D Julian; Decker, Eric A

    2013-08-15

    The influence of whey protein isolate (WPI)-beet pectin conjugates formed by dry-heating on the oxidative stability of β-carotene in O/W emulsions was studied. It was mainly focused on the influence of pH, metal chelator, free radical scavenger and interfacial characteristics on the degradation of β-carotene in the emulsion stabilised by conjugate. The conjugate increased the oxidative stability of β-carotene in the emulsion as compared to their unconjugated mixture at pH 7.0. The desferoxamine retarded β-carotene degradation at pH 4.0 more effectively than pH 7.0 and more effectively in the emulsion with the conjugate than the unconjugated mixture (p<0.05). The addition of 200 mg/kg α-tocopherol significantly improved the stability of β-carotene in the conjugate stabilised emulsion. The emulsions were washed to remove conjugate not absorbed to the emulsion droplet interface, indicating that unabsorbed emulsifiers could protect β-carotene. It suggested that WPI-pectin conjugate could be used to protect bioactive lipids in emulsions.

  3. Tyrosyl Radicals in Dehaloperoxidase

    PubMed Central

    Dumarieh, Rania; D'Antonio, Jennifer; Deliz-Liang, Alexandria; Smirnova, Tatyana; Svistunenko, Dimitri A.; Ghiladi, Reza A.

    2013-01-01

    Dehaloperoxidase (DHP) from Amphitrite ornata, having been shown to catalyze the hydrogen peroxide-dependent oxidation of trihalophenols to dihaloquinones, is the first oxygen binding globin that possesses a biologically relevant peroxidase activity. The catalytically competent species in DHP appears to be Compound ES, a reactive intermediate that contains both a ferryl heme and a tyrosyl radical. By simulating the EPR spectra of DHP activated by H2O2, Thompson et al. (Thompson, M. K., Franzen, S., Ghiladi, R. A., Reeder, B. J., and Svistunenko, D. A. (2010) J. Am. Chem. Soc. 132, 17501–17510) proposed that two different radicals, depending on the pH, are formed, one located on either Tyr-34 or Tyr-28 and the other on Tyr-38. To provide additional support for these simulation-based assignments and to deduce the role(s) that tyrosyl radicals play in DHP, stopped-flow UV-visible and rapid-freeze-quench EPR spectroscopic methods were employed to study radical formation in DHP when three tyrosine residues, Tyr-28, Tyr-34, and Tyr-38, were replaced either individually or in combination with phenylalanines. The results indicate that radicals form on all three tyrosines in DHP. Evidence for the formation of DHP Compound I in several tyrosine mutants was obtained. Variants that formed Compound I showed an increase in the catalytic rate for substrate oxidation but also an increase in heme bleaching, suggesting that the tyrosines are necessary for protecting the enzyme from oxidizing itself. This protective role of tyrosines is likely an evolutionary adaptation allowing DHP to avoid self-inflicted damage in the oxidative environment. PMID:24100039

  4. Haptoglobin Binding Stabilizes Hemoglobin Ferryl Iron and the Globin Radical on Tyrosine β145

    PubMed Central

    Schaer, Dominik J.; Buehler, Paul W.; Wilson, Michael T.; Reeder, Brandon J.; Silkstone, Gary; Svistunenko, Dimitri A.; Bulow, Leif; Alayash, Abdu I.

    2013-01-01

    Abstract Aim: Hemoglobin (Hb) becomes toxic when released from the erythrocyte. The acute phase protein haptoglobin (Hp) binds avidly to Hb and decreases oxidative damage to Hb itself and to the surrounding proteins and lipids. However, the molecular mechanism underpinning Hp protection is to date unclear. The aim of this study was to use electron paramagnetic resonance (EPR) spectroscopy, stopped flow optical spectrophotometry, and site-directed mutagenesis to explore the mechanism and specifically the role of specific tyrosine residues in this protection. Results: Following peroxide challenge Hb produces reactive oxidative intermediates in the form of ferryl heme and globin free radicals. Hp binding increases the steady state level of ferryl formation during Hb-catalyzed lipid peroxidation, while at the same time dramatically inhibiting the overall reaction rate. This enhanced ferryl stability is also seen in the absence of lipids and in the presence of external reductants. Hp binding is not accompanied by a decrease in the pK of ferryl protonation; the protonated ferryl species still forms, but is intrinsically less reactive. Ferryl stabilization is accompanied by a significant increase in the concentration of the peroxide-induced tyrosine free radical. EPR spectral parameters and mutagenesis studies suggest that this radical is located on tyrosine 145, the penultimate C-terminal amino acid on the beta Hb subunit. Innovation: Hp binding decreases both the ferryl iron and free radical reactivity of Hb. Conclusion: Hp protects against Hb-induced damage in the vasculature, not by preventing the primary reactivity of heme oxidants, but by rendering the resultant protein products less damaging. Antioxid. Redox Signal. 18, 2264–2273. PMID:22702311

  5. Forty percent methionine restriction decreases mitochondrial oxygen radical production and leak at complex I during forward electron flow and lowers oxidative damage to proteins and mitochondrial DNA in rat kidney and brain mitochondria.

    PubMed

    Caro, Pilar; Gomez, Jose; Sanchez, Ines; Naudi, Alba; Ayala, Victoria; López-Torres, Monica; Pamplona, Reinald; Barja, Gustavo

    2009-12-01

    Eighty percent dietary methionine restriction (MetR) in rodents (without calorie restriction), like dietary restriction (DR), increases maximum longevity and strongly decreases mitochondrial reactive oxygen species (ROS) production and oxidative stress. Eighty percent MetR also lowers the degree of membrane fatty acid unsaturation in rat liver. Mitochondrial ROS generation and the degree of fatty acid unsaturation are the only two known factors linking oxidative stress with longevity in vertebrates. However, it is unknown whether 40% MetR, the relevant methionine restriction degree to clarify the mechanisms of action of standard (40%) DR can reproduce these effects in mitochondria from vital tissues of strong relevance for aging. Here we study the effect of 40% MetR on ROS production and oxidative stress in rat brain and kidney mitochondria. Male Wistar rats were fed during 7 weeks semipurified diets differing only in their methionine content: control or 40% MetR diets. It was found that 40% MetR decreases mitochondrial ROS production and percent free radical leak (by 62-71%) at complex I during forward (but not during reverse) electron flow in both brain and kidney mitochondria, increases the oxidative phosphorylation capacity of brain mitochondria, lowers oxidative damage to kidney mitochondrial DNA, and decreases specific markers of mitochondrial protein oxidation, lipoxidation, and glycoxidation in both tissues. Forty percent MetR also decreased the amount of respiratory complexes I, III, and IV and apoptosis-inducing factor (AIF) in brain mitochondria and complex IV in kidney mitochondria, without changing the degree of mitochondrial membrane fatty acid unsaturation. Forty percent MetR, differing from 80% MetR, did not inhibit the increase in rat body weight. These changes are very similar to the ones previously found during dietary and protein restriction in rats. We conclude that methionine is the only dietary factor responsible for the decrease in

  6. Designed metalloprotein stabilizes a semiquinone radical.

    PubMed

    Ulas, Gözde; Lemmin, Thomas; Wu, Yibing; Gassner, George T; DeGrado, William F

    2016-04-01

    Enzymes use binding energy to stabilize their substrates in high-energy states that are otherwise inaccessible at ambient temperature. Here we show that a de novo designed Zn(II) metalloprotein stabilizes a chemically reactive organic radical that is otherwise unstable in aqueous media. The protein binds tightly to and stabilizes the radical semiquinone form of 3,5-di-tert-butylcatechol. Solution NMR spectroscopy in conjunction with molecular dynamics simulations show that the substrate binds in the active site pocket where it is stabilized by metal-ligand interactions as well as by burial of its hydrophobic groups. Spectrochemical redox titrations show that the protein stabilized the semiquinone by reducing the electrochemical midpoint potential for its formation via the one-electron oxidation of the catechol by approximately 400 mV (9 kcal mol(-1)). Therefore, the inherent chemical properties of the radical were changed drastically by harnessing its binding energy to the metalloprotein. This model sets the basis for designed enzymes with radical cofactors to tackle challenging chemistry.

  7. Designed metalloprotein stabilizes a semiquinone radical

    PubMed Central

    Ulas, Gözde; Lemmin, Thomas; Wu, Yibing; Gassner, George T.; DeGrado, William F.

    2016-01-01

    Enzymes use binding energy to stabilize their substrates in high-energy states that are otherwise inaccessible at ambient temperature. Here we show that a de novo designed Zn(ii) metalloprotein stabilizes a chemically reactive organic radical that is otherwise unstable in aqueous media. The protein binds tightly to and stabilizes the radical semiquinone form of 3,5-di-tert-butylcatechol. Solution NMR spectroscopy in conjunction with molecular dynamics simulations show that the substrate binds in the active site pocket where it is stabilized by metal–ligand interactions as well as by burial of its hydrophobic groups. Spectrochemical redox titrations show that the protein stabilized the semiquinone by reducing the electrochemical midpoint potential for its formation via the one-electron oxidation of the catechol by approximately 400 mV (9 kcal mol−1). Therefore, the inherent chemical properties of the radical were changed drastically by harnessing its binding energy to the metalloprotein. This model sets the basis for designed enzymes with radical cofactors to tackle challenging chemistry. PMID:27001731

  8. Designed metalloprotein stabilizes a semiquinone radical

    NASA Astrophysics Data System (ADS)

    Ulas, Gözde; Lemmin, Thomas; Wu, Yibing; Gassner, George T.; Degrado, William F.

    2016-04-01

    Enzymes use binding energy to stabilize their substrates in high-energy states that are otherwise inaccessible at ambient temperature. Here we show that a de novo designed Zn(II) metalloprotein stabilizes a chemically reactive organic radical that is otherwise unstable in aqueous media. The protein binds tightly to and stabilizes the radical semiquinone form of 3,5-di-tert-butylcatechol. Solution NMR spectroscopy in conjunction with molecular dynamics simulations show that the substrate binds in the active site pocket where it is stabilized by metal-ligand interactions as well as by burial of its hydrophobic groups. Spectrochemical redox titrations show that the protein stabilized the semiquinone by reducing the electrochemical midpoint potential for its formation via the one-electron oxidation of the catechol by approximately 400 mV (9 kcal mol-1). Therefore, the inherent chemical properties of the radical were changed drastically by harnessing its binding energy to the metalloprotein. This model sets the basis for designed enzymes with radical cofactors to tackle challenging chemistry.

  9. Designed metalloprotein stabilizes a semiquinone radical.

    PubMed

    Ulas, Gözde; Lemmin, Thomas; Wu, Yibing; Gassner, George T; DeGrado, William F

    2016-04-01

    Enzymes use binding energy to stabilize their substrates in high-energy states that are otherwise inaccessible at ambient temperature. Here we show that a de novo designed Zn(II) metalloprotein stabilizes a chemically reactive organic radical that is otherwise unstable in aqueous media. The protein binds tightly to and stabilizes the radical semiquinone form of 3,5-di-tert-butylcatechol. Solution NMR spectroscopy in conjunction with molecular dynamics simulations show that the substrate binds in the active site pocket where it is stabilized by metal-ligand interactions as well as by burial of its hydrophobic groups. Spectrochemical redox titrations show that the protein stabilized the semiquinone by reducing the electrochemical midpoint potential for its formation via the one-electron oxidation of the catechol by approximately 400 mV (9 kcal mol(-1)). Therefore, the inherent chemical properties of the radical were changed drastically by harnessing its binding energy to the metalloprotein. This model sets the basis for designed enzymes with radical cofactors to tackle challenging chemistry. PMID:27001731

  10. Alternative radical pairs for cryptochrome-based magnetoreception.

    PubMed

    Lee, Alpha A; Lau, Jason C S; Hogben, Hannah J; Biskup, Till; Kattnig, Daniel R; Hore, P J

    2014-06-01

    There is growing evidence that the remarkable ability of animals, in particular birds, to sense the direction of the Earth's magnetic field relies on magnetically sensitive photochemical reactions of the protein cryptochrome. It is generally assumed that the magnetic field acts on the radical pair [FAD•- TrpH•+] formed by the transfer of an electron from a group of three tryptophan residues to the photo-excited flavin adenine dinucleotide cofactor within the protein. Here, we examine the suitability of an [FAD•- Z•] radical pair as a compass magnetoreceptor, where Z• is a radical in which the electron spin has no hyperfine interactions with magnetic nuclei, such as hydrogen and nitrogen. Quantum spin dynamics simulations of the reactivity of [FAD•- Z•] show that it is two orders of magnitude more sensitive to the direction of the geomagnetic field than is [FAD•- TrpH•+] under the same conditions (50 µT magnetic field, 1 µs radical lifetime). The favourable magnetic properties of [FAD•- Z•] arise from the asymmetric distribution of hyperfine interactions among the two radicals and the near-optimal magnetic properties of the flavin radical. We close by discussing the identity of Z• and possible routes for its formation as part of a spin-correlated radical pair with an FAD radical in cryptochrome. PMID:24671932

  11. Expression of S-100 protein, epithelial membrane antigen, carcinoembryonic antigen and alpha fetoprotein in normal salivary glands and primary salivary gland tumors.

    PubMed

    Günhan, O; Evren, G; Demiriz, M; Can, C; Celasun, B; Finci, R

    1992-12-01

    The distribution of S-100 protein, epithelial membrane antigen, carcinoembryonic antigen and alpha fetoprotein was studied in 38 primary salivary gland tumors. S-100 protein, a useful marker of myoepithelial cells, was demonstrated in some benign tumors. Carcinoembryonic antigen expression was consistently positive in adenoid cystic carcinoma. Demonstration of epithelial membrane antigen helped to confirm the epithelial nature of some neoplastic cells. Alpha fetoprotein was not expressed in any of the cases examined. No correlation was found between immunopositivity and tumor behavior in the present series.

  12. Salamander-Derived, Human-Optimized nAG Protein Suppresses Collagen Synthesis and Increases Collagen Degradation in Primary Human Fibroblasts

    PubMed Central

    Al-Qattan, Mohammad M.; Shier, Medhat K.; Abd-AlWahed, Mervat M.; Mawlana, Ola H.; El-Wetidy, Mohammed S.; Bagayawa, Reginald S.; Ali, Hebatallah H.; Al-Nbaheen, May S.; Aldahmash, Abdullah M.

    2013-01-01

    Unlike humans, salamanders regrow their amputated limbs. Regeneration depends on the presence of regenerating axons which upregulate the expression of newt anterior gradient (nAG) protein. We had the hypothesis that nAG might have an inhibitory effect on collagen production since excessive collagen production results in scarring, which is a major enemy to regeneration. nAG gene was designed, synthesized, and cloned. The cloned vector was then transfected into primary human fibroblasts. The results showed that the expression of nAG protein in primary human fibroblast cells suppresses the expression of collagen I and III, with or without TGF-β1 stimulation. This suppression is due to a dual effect of nAG both by decreasing collagen synthesis and by increasing collagen degradation. Furthermore, nAG had an inhibitory effect on proliferation of transfected fibroblasts. It was concluded that nAG suppresses collagen through multiple effects. PMID:24288677

  13. Data on gene and protein expression changes induced by apabetalone (RVX-208) in ex vivo treated human whole blood and primary hepatocytes.

    PubMed

    Wasiak, Sylwia; Gilham, Dean; Tsujikawa, Laura M; Halliday, Christopher; Norek, Karen; Patel, Reena G; McLure, Kevin G; Young, Peter R; Gordon, Allan; Kulikowski, Ewelina; Johansson, Jan; Sweeney, Michael; Wong, Norman C

    2016-09-01

    Apabetalone (RVX-208) inhibits the interaction between epigenetic regulators known as bromodomain and extraterminal (BET) proteins and acetyl-lysine marks on histone tails. Data presented here supports the manuscript published in Atherosclerosis "RVX-208, a BET-inhibitor for Treating Atherosclerotic Cardiovascular Disease, Raises ApoA-I/HDL and Represses Pathways that Contribute to Cardiovascular Disease" (Gilham et al., 2016) [1]. It shows that RVX-208 and a comparator BET inhibitor (BETi) JQ1 increase mRNA expression and production of apolipoprotein A-I (ApoA-I), the main protein component of high density lipoproteins, in primary human and African green monkey hepatocytes. In addition, reported here are gene expression changes from a microarray-based analysis of human whole blood and of primary human hepatocytes treated with RVX-208. PMID:27570805

  14. The cytoplasmic membrane is a primary target for the staphylocidal action of thrombin-induced platelet microbicidal protein.

    PubMed Central

    Koo, S P; Yeaman, M R; Nast, C C; Bayer, A S

    1997-01-01

    Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, cationic peptide released from rabbit platelets exposed to thrombin in vitro. tPMP-1 is microbicidal against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. Preliminary evidence suggests that tPMP-1 targets and disrupts the staphylococcal cytoplasmic membrane. However, it is not clear if the cytoplasmic membrane is a direct or indirect target of tPMP-1. Therefore, we assessed the in vitro activity of tPMP-1 versus protoplasts prepared from logarithmic-phase (LOG) or stationary-phase (STAT) cells of the genetically related S. aureus strains 19S and 19R (tPMP-1 susceptible and resistant, respectively). Protoplasts exposed to tPMP-1 (2 microg/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and ultrastructural alterations (by transmission electron microscopy [TEM]). Exposure to tPMP-1 resulted in substantial lysis of LOG but not STAT protoplasts of 19S, coinciding with protoplast membrane disruption observed by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the staphylococcal cell wall. In contrast to 19S, neither LOG nor STAT protoplasts of 19R were lysed by tPMP-1. tPMP-1-induced membrane damage was further characterized with anionic planar lipid bilayers subjected to various trans-negative voltages. tPMP-1 increased conductance across bilayers at -90 mV but not at -30 mV. Once initiated, a reduction in voltage from -90 to -30 mV diminished conductance magnitude but did not eliminate tPMP-1-mediated membrane permeabilization. Therefore, tPMP-1 appears to directly target the staphylococcal cytoplasmic membrane as a primary event in its mechanism of action. Specifically, tPMP-1 likely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner. PMID:9353067

  15. Strand specificity in the interactions of Escherichia coli primary replicative helicase DnaB protein with a replication fork.

    PubMed

    Jezewska, M J; Rajendran, S; Bujalowski, W

    1997-08-19

    The interactions of the Escherichia coli primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having either a single arm or both arms, have been studied using the thermodynamically rigorous fluorescence titration techniques. This approach allows us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal (fluorescence) and the degree of binding. Subsequently, the formation of the complexes, with different replication fork substrates, has also been characterized using the sedimentation velocity technique. To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replication fork substrates. In the presence of the ATP nonhydrolyzable analog, AMP-PNP, the E. coli DnaB helicase preferentially binds to the 5' arm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm. ATP hydrolysis is not necessary for formation of the helicase-fork complex. The asymmetric interactions are consistent with the 5' --> 3' directionality of the helicase activity of the DnaB protein and most probably reflects a preferential 5' --> 3' polarity in the helicase binding to ssDNA, with respect to the ssDNA backbone. The double-stranded part of the fork contributes little to the free energy of binding. The data indicate a rather passive role of the duplex part of the fork in the binding of the helicase. This role seems to be limited to impose steric hindrance in the formation of nonproductive complexes of the enzyme with the fork. Quantitative analysis of binding of the helicase to the two-arm fork substrate shows that two DnaB hexamers can bind to the fork, with each single hexamer associated with a single arm of the fork. In this complex, the intrinsic affinity of the DnaB hexamer for the 5' arm in a two-arm fork is not affected by the presence of the

  16. Cell Wall Proteome in the Maize Primary Root Elongation Zone. I. Extraction and Identification of Water-Soluble and Lightly Ionically Bound Proteins1

    PubMed Central

    Zhu, Jinming; Chen, Sixue; Alvarez, Sophie; Asirvatham, Victor S.; Schachtman, Daniel P.; Wu, Yajun; Sharp, Robert E.

    2006-01-01

    Cell wall proteins (CWPs) play important roles in various processes, including cell elongation. However, relatively little is known about the composition of CWPs in growing regions. We are using a proteomics approach to gain a comprehensive understanding of the identity of CWPs in the maize (Zea mays) primary root elongation zone. As the first step, we examined the effectiveness of a vacuum infiltration-centrifugation technique for extracting water-soluble and loosely ionically bound (fraction 1) CWPs from the root elongation zone. The purity of the CWP extract was evaluated by comparing with total soluble proteins extracted from homogenized tissue. Several lines of evidence indicated that the vacuum infiltration-centrifugation technique effectively enriched for CWPs. Protein identification revealed that 84% of the CWPs were different from the total soluble proteins. About 40% of the fraction 1 CWPs had traditional signal peptides and 33% were predicted to be nonclassical secretory proteins, whereas only 3% and 11%, respectively, of the total soluble proteins were in these categories. Many of the CWPs have previously been shown to be involved in cell wall metabolism and cell elongation. In addition, maize has type II cell walls, and several of the CWPs identified in this study have not been identified in previous cell wall proteomics studies that have focused only on type I walls. These proteins include endo-1,3;1,4-β-d-glucanase and α-l-arabinofuranosidase, which act on the major polysaccharides only or mainly present in type II cell walls. PMID:16377746

  17. Development of the radical-stable Coprinus cinereus peroxidase (CiP) by blocking the radical attack.

    PubMed

    Kim, Su Jin; Joo, Jeong Chan; Kim, Han Sang; Kwon, Inchan; Song, Bong Keun; Yoo, Young Je; Kim, Yong Hwan

    2014-11-10

    Despite the potential use of peroxidases as industrial biocatalysts, their practical application is often impeded due to suicide inactivation by radicals generated in oxidative reactions. Using a peroxidase from Coprinus cinereus (CiP) as a model enzyme, we revealed a dominant factor for peroxidase inactivation during phenol oxidation, and we engineered radical-stable mutants by site-directed mutagenesis of an amino acid residue susceptible to modification by phenoxyl radical. Mass spectrometry analysis of inactivated CiP identified an adduct between F230 and a phenoxyl radical, and subsequently, the F230 residue was mutated to amino acids that resisted radical coupling. Of the F230 mutants, the F230A mutant showed the highest stability against radical inactivation, retaining 80% of its initial activity, while the wild-type protein was almost completely inactivated. The F230A mutant also exhibited a 16-fold higher turnover of the phenol substrate compared with the wild-type enzyme. Furthermore, the F230A mutant was stable during the oxidation of other phenolic compounds, including m-cresol and 3-methoxyphenol. No structural changes were observed by UV-vis and CD spectra of CiP after radical coupling, implying that the F230-phenol radical adduct inactivated CiP by blocking substrate access to the active site. Our novel strategy can be used to improve the stability of other peroxidases inactivated by radicals.

  18. Trapping of neutral radicals by aromatic and heterocyclic cation radicals

    SciTech Connect

    Shine, H.J.; Soroka, M.

    1986-09-01

    A considerable amount of knowledge has been accrued during the last 15-20 years on the chemistry of polynuclear aromatic and heterocyclic cation radicals. For the most part, the reactions that have been studied have been of cation radicals with neutral and anionic nucleophiles which lead to addition or substitution products. Classic examples among these reactions are, for example, the reaction of water with the cation radical of 9,10-diphenylanthracene and perylene, and that of water with the thianthrene cation radical. Reactions such as these have been among foundation-establishing studies in the scope and mechanism of cation-radical reactions. Cation radicals can also undergo another type reaction with nucleophies, that is, electron transfer. This possibility leads to two far-reaching and connected questions: can addition and substitution reactions of aromatic cation radicals with nucleophiles be preceded by single electron transfer (SET).; and, can such cation radicals trap neutral radicals. These questions are also then related to another question having even greater impact on organic chemistry: can electrophilic aromatic substitution (ArH + E/sup +/ ..-->.. ArE + H/sup +/) be preceded by SET. The authors have been trying to answer the first two questions about SET and trapping of radicals by combining the two possibilities. That is, they have chosen the reaction of the thianthrene cation radical (represented by the symbol S/sup +./) with grignard reagents, for which two possible routes could be anticipated: either direct addition at sulfur or addition preceded by SET.

  19. Photo-induced free radicals on a simulated Martian surface

    NASA Technical Reports Server (NTRS)

    Tseng, S.-S.; Chang, S.

    1974-01-01

    Results of an electron spin resonance study of free radicals in the ultraviolet irradiation of a simulated Martian surface suggest that the ultraviolet photolysis of CO or CO2, or a mixture of both, adsorbed on silica gel at minus 170 C involves the formation of OH radicals and possibly of H atoms as the primary process, followed by the formation of CO2H radicals. It is concluded that the photochemical synthesis of organic compounds could occur on Mars if the siliceous surface dust contains enough silanol groups and/or adsorbed H2O in the form of bound water.

  20. Primary style protein expression in the self-incompatible/compatible apricot by the 2D-DIGE technique.

    PubMed

    Cao, Xiaoyan; Feng, Jianrong; Wang, Dajiang; Sun, Junli; Lu, Xiaoyan; Liu, Huaifeng

    2012-07-15

    In order to explore the molecular mechanism underlying self-incompatibility (SI) in the apricot (Prunus armeniaca L.) at the proteome level, we examined the style proteomes at different stages of flower development: small bud, big bud, 24h after self-pollination and 24h after cross-pollination with cultivar Badanshui in the SI apricot cultivar Xinshiji and the self-compatible (SC) apricot cultivar Katy by 2D fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). About 1500 style protein spots were detected; 66 were expressed differently in the four stages in Xinshiji. About 1600 style protein spots were detected; 143 were expressed differently in the four stages of flower development in Katy. In Xinshiji, one protein was expressed specifically, four proteins showed up-regulated expression and twenty-nine proteins showed down-regulated expression in the cross-pollinated style compared to the self-pollinated style. Thirteen proteins were identified unambiguously. In Katy, three proteins were expressed specifically, five proteins showed up-regulated expression and thirteen proteins showed down-regulated expression in the cross-pollinated style compared to self-pollinated style. Seven proteins were identified unambiguously. The different reactions of the style at the proteomic level were triggered in Xinshiji and Katy by self pollen and non-self pollen.

  1. Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box.

    PubMed Central

    Kiledjian, M; Dreyfuss, G

    1992-01-01

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity. Images PMID:1628625

  2. Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins: requirement for T7 RNA polymerase.

    PubMed Central

    Romano, L J; Tamanoi, F; Richardson, C C

    1981-01-01

    The primary origin of bacteriophage T7 DNA replication is located 15% of the distance from the left end of the T7 DNA molecule. This intergenic segment is A + T-rich, contains a single gene 4 protein recognition site, and is preceded by two tandem promoters for T7 RNA polymerase [RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6]. Analysis by electron microscopy shows that T7 DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] and gene 4 protein initiate DNA synthesis at randomly located nicks on duplex DNA to produce branched molecules. However, upon the addition of T7 RNA polymerase and ribonucleoside triphosphates 14% of the product molecules have replication bubbles, all of which are located near the primary origin observed in vivo; no such initiation occurs on T7 deletion mutant LG37 DNA, which lacks the primary origin. We have also studied initiation by using plasmids into which fragments of T7 DNA have been inserted. DNA synthesis on these templates is also dependent on the presence of T7 RNA polymerase and ribonucleoside triphosphates. DNA synthesis is specific for plasmids containing the primary origin, provided they are first converted to linear forms. PMID:6945573

  3. Spectroscopic characterization of radicals and radical pairs in fruit fly cryptochrome - protonated and nonprotonated flavin radical-states.

    PubMed

    Paulus, Bernd; Bajzath, Csaba; Melin, Frédéric; Heidinger, Lorenz; Kromm, Viktoria; Herkersdorf, Christoph; Benz, Ulrike; Mann, Lisa; Stehle, Patricia; Hellwig, Petra; Weber, Stefan; Schleicher, Erik

    2015-08-01

    Drosophila melanogaster cryptochrome is one of the model proteins for animal blue-light photoreceptors. Using time-resolved and steady-state optical spectroscopy, we studied the mechanism of light-induced radical-pair formation and decay, and the photoreduction of the FAD cofactor. Exact kinetics on a microsecond to minutes timescale could be extracted for the wild-type protein using global analysis. The wild-type exhibits a fast photoreduction reaction from the oxidized FAD to the FAD(•-) state with a very positive midpoint potential of ~ +125 mV, although no further reduction could be observed. We could also demonstrate that the terminal tryptophan of the conserved triad, W342, is directly involved in electron transfer; however, photoreduction could not be completely inhibited in a W342F mutant. The investigation of another mutation close to the FAD cofactor, C416N, rather unexpectedly reveals accumulation of a protonated flavin radical on a timescale of several seconds. The obtained data are critically discussed with the ones obtained from another protein, Escherichia coli photolyase, and we conclude that the amino acid opposite N(5) of the isoalloxazine moiety of FAD is able to (de)stabilize the protonated FAD radical but not to significantly modulate the kinetics of any light-inducted reactions.

  4. Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes.

    PubMed

    Xie, Yuchao; McGill, Mitchell R; Du, Kuo; Dorko, Kenneth; Kumer, Sean C; Schmitt, Timothy M; Ding, Wen-Xing; Jaeschke, Hartmut

    2015-12-01

    3'-Hydroxyacetanilide orN-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this,we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction. PMID:26431796

  5. Free radical explosive composition

    DOEpatents

    Walker, Franklin E.; Wasley, Richard J.

    1979-01-01

    An improved explosive composition is disclosed and comprises a major portion of an explosive having a detonation velocity between about 1500 and 10,000 meters per second and a minor amount of a getter additive comprising a compound or mixture of compounds capable of capturing or deactivating free radicals or ions under mechanical or electrical shock conditions and which is not an explosive. Exemplary getter additives are isocyanates, olefins and iodine.

  6. Radicalism, Marxism, and medicine.

    PubMed

    Navarro, V

    1983-01-01

    This article presents a critique of recent radical interpretations of medicine and provides an alternative explanation of such interpretations. It analyzes 1) the articulation of medical practices, knowledge, and institutions within specific modes of production and social formations; 2) the dual functions of medicine within capitalist relations of production; 3) the reproduction of power within medicine; and 4) the meaning of capitalist, socialist, and communist medicine. The political practice derived from these analyses is also elaborated.

  7. THERMOCHEMISTRY OF HYDROCARBON RADICALS

    SciTech Connect

    Kent M. Ervin, Principal Investigator

    2004-08-17

    Gas phase negative ion chemistry methods are employed to determine enthalpies of formation of hydrocarbon radicals that are important in combustion processes and to investigate the dynamics of ion-molecule reactions. Using guided ion beam tandem mass spectrometry, we measure collisional threshold energies of endoergic proton transfer and hydrogen atom transfer reactions of hydrocarbon molecules with negative reagent ions. The measured reaction threshold energies for proton transfer yield the relative gas phase acidities. In an alternative methodology, competitive collision-induced dissociation of proton-bound ion-molecule complexes provides accurate gas phase acidities relative to a reference acid. Combined with the electron affinity of the R {center_dot} radical, the gas phase acidity yields the RH bond dissociation energy of the corresponding neutral molecule, or equivalently the enthalpy of formation of the R{center_dot} organic radical, using equation: D(R-H) = {Delta}{sub acid}H(RH) + EA(R) - IE(H). The threshold energy for hydrogen abstraction from a hydrocarbon molecule yields its hydrogen atom affinity relative to the reagent anion, providing the RH bond dissociation energy directly. Electronic structure calculations are used to evaluate the possibility of potential energy barriers or dynamical constrictions along the reaction path, and as input for RRKM and phase space theory calculations. In newer experiments, we have measured the product velocity distributions to obtain additional information on the energetics and dynamics of the reactions.

  8. Free radical propulsion concept

    NASA Technical Reports Server (NTRS)

    Hawkins, C. E.; Nakanishi, S.

    1981-01-01

    The concept of a free radical propulsion system, utilizing the recombination energy of dissociated low molecular weight gases to produce thrust, is analyzed. The system, operating at a theoretical impulse with hydrogen, as high as 2200 seconds at high thrust to power ratio, is hypothesized to bridge the gap between chemical and electrostatic propulsion capabilities. A comparative methodology is outlined by which characteristics of chemical and electric propulsion for orbit raising mission can be investigated. It is noted that free radicals proposed in rockets previously met with difficulty and complexity in terms of storage requirements; the present study proposes to eliminate the storage requirements by using electric energy to achieve a continuous-flow product of free radicals which are recombined to produce a high velocity propellant. Microwave energy used to dissociate a continuously flowing gas is transferred to the propellant via three-body-recombination for conversion to propellant kinetic energy. Microwave plasma discharge was found in excess of 90 percent over a broad range of pressure in preliminary experiments, and microwave heating compared to electrothermal heating showed much higher temperatures in gasdynamic equations.

  9. [Radical prostatectomy - pro robotic].

    PubMed

    Gillitzer, R

    2012-05-01

    Anatomical radical prostatectomy was introduced in the early 1980s by Walsh and Donker. Elucidation of key anatomical structures led to a significant reduction in the morbidity of this procedure. The strive to achieve similar oncological and functional results to this gold standard open procedure but with further reduction of morbidity through a minimally invasive access led to the establishment of laparoscopic prostatectomy. However, this procedure is complex and difficult and is associated with a long learning curve. The technical advantages of robotically assisted surgery coupled with the intuitive handling of the device led to increased precision and shortening of the learning curve. These main advantages, together with a massive internet presence and aggressive marketing, have resulted in a rapid dissemination of robotic radical prostatectomy and an increasing patient demand. However, superiority of robotic radical prostatectomy in comparison to the other surgical therapeutic options has not yet been proven on a scientific basis. Currently robotic-assisted surgery is an established technique and future technical improvements will certainly further define its role in urological surgery. In the end this technical innovation will have to be balanced against the very high purchase and running costs, which remain the main limitation of this technology.

  10. Silicon based radicals, radical ions, diradicals and diradicaloids.

    PubMed

    Chandra Mondal, Kartik; Roy, Sudipta; Roesky, Herbert W

    2016-02-21

    Radicals are an important class of species which act as intermediates in numerous chemical and biological processes. Most of the radicals have short lifetimes. However, radicals with longer lifetimes can be isolated and stored in a pure form. They are called stable radicals. Over the last five decades, the syntheses of several stable radicals have been reported. Recently, highly unstable radicals have been successfully stabilized via strong σ-donation of singlet carbenes. Cyclic aklyl(amino) carbene (cAAC) is regarded as a stronger σ-donor and a better π-acceptor when compared with that of an N-heterocyclic carbene (NHC). In this article we review preferentially the results of our group to generate stable radical centers on the carbene carbon atoms by employing the so far hidden and unique ability of the cAACs. We focus on the development of new synthetic routes to stable and isolable radicals containing silicon atoms. All the compounds have been well characterized by single crystal X-ray analysis; the mono-radicals have been distinguished by EPR spectroscpy and the ground state of the diradicals has been studied by magnetic susceptibility measurements and theoretical calculations. Many of these compounds are studied by cyclic voltammetry and are often converted to their corresponding radical cations or radical anions via electron abstraction or addition processes. Some of them are stable, having long lifetimes and hence are isolated and characterized thoroughly. Not much information has been obtained on the short lived persistent radical species. Herein, we discuss some of the examples of such a type of species and focus on what kind of chemical reactions are initiated by these short-lived radical species in solution. We also briefly mention the syntheses and charaterization of the so far reported stable silicon centered radicals. PMID:26585359

  11. Primary structure of a cerulenin-binding. beta. -ketoacyl-(acyl carrier protein) synthase from barley chloroplasts

    SciTech Connect

    Siggaard-Andersen, M.; Kauppinen, S. ); von Wettstein-Knowles, P. Univ. of Copenhagen )

    1991-05-15

    The radioactively labeled {beta}-ketoacyl thioester synthase inhibitor ({sup 3}H)cerulenin was used to tag three dimeric barley chloroplast proteins ({alpha}{alpha}, {alpha}{beta}, and {beta}{beta}) from the stromal fraction. Oligonucleotides corresponding to amino acid sequences obtained from the purified proteins were used to generate with the polymerase chain reaction a probe for cDNAs encoding the {beta} subunit. cDNA sequencing revealed an open reading frame for 462 residues comprising the mature protein and a 35-amino acid transit peptide. The deduced amino acid sequence of the mature protein is homologous to the {beta}-ketoacyl-(acyl carrier protein) (ACP) synthase I (3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3.1.41) of Escherichia coli. Under analogous experimental conditions ({sup 3}H)cerulenin tagged a single dimeric protein from spinach chloroplasts.

  12. Resonant cavity spectroscopy of radical species

    NASA Astrophysics Data System (ADS)

    Ritchie, Grant

    2015-04-01

    Photo-oxidation in the troposphere is highly complex, being initiated by short lived radical species, in the daytime dominated by the hydroxyl radical, OH, with contributions from Cl atoms, and at night by either NO3 radicals or ozone. Chemical oxidation cycles, which couple OH, HO2 and peroxy (RO2) radical species, remove primary emitted trace species which are harmful to humans or to the wider environment. However, many of the secondary products produced by atmospheric photo-oxidation are also directly harmful, for example O3, NO2, acidic and multifunctional species, many of which are of low volatility and are able to partition effectively to the condensed phase, creating secondary organic aerosol (SOA), which contributes a significant fraction of tropospheric aerosol, with associated impacts on climate and human health. The accuracy of atmospheric models to predict these impacts necessarily requires accurate knowledge of the chemical oxidative cycling. Two of the simplest intermediates are the hydroperoxy radical, HO2, and the smallest and dominant organic peroxy radical, CH3O2, formed directly by the reactions of OH with CO/O2 and CH4/O2, respectively, and indirectly following the oxidation of larger VOCs. OH, HO2 and RO2 (collectively known as ROx) are rapidly cycled, being at the centre of tropospheric oxidation, and hence are some of the best targets for models to compare with field data. The reaction of HO2 and RO2 with NO constitutes the only tropospheric in-situ source of O3. Despite their importance, neither HO2 nor CH3O2 is measured directly in the atmosphere. HO2 is only measured indirectly following its conversion to OH and CH3O2 is not measured at all. Typically only the sum of RO2 radicals is measured, making no distinction between different organic peroxy radicals. This contribution will detail recent studies using (i) optical feedback cavity enhanced absorption spectroscopy with both quantum and inter-band cascade lasers in the mid-IR, and (ii

  13. Free radical tissue damage: Protective role of antioxidant nutrients

    SciTech Connect

    Machling, L.J.; Bendich, A. )

    1987-12-01

    Highly reactive molecules called free radicals can cause tissue damage by reacting with polyunsaturated fatty acids in cellular membranes, nucleotides in DNA, and critical sulfhydryl bonds in proteins. Free radicals can originate endogenously from normal metabolic reactions or exogenously as components of tobacco smoke and air pollutants and indirectly through the metabolism of certain solvents, drugs, and pesticides as well as through exposure to radiation. There is some evidence that free radical damage contributes to the etiology of many chronic health problems such as emphysema, cardiovascular and inflammatory diseases, cataracts, and cancer. The extent of tissue damage is the result of the balance between the free radicals generated and the antioxidant protective defense system. Several dietary micronutrients contribute greatly to the protective system. Based on the growing interest in free radical biology and the lack of effective therapies for many of the chronic diseases, the usefulness of essential, safe nutrients in protecting against the adverse effects of oxidative injury warrants further study.

  14. SAS1B Protein [Ovastacin] Shows Temporal and Spatial Restriction to Oocytes in Several Eutherian Orders and Initiates Translation at the Primary to Secondary Follicle Transition

    PubMed Central

    Pires, Eusebio S; Hlavin, Callie; Macnamara, Ellen; Ishola-Gbenla, Khadijat; Doerwaldt, Christa; Chamberlain, Catherine; Klotz, Kenneth; Herr, Austin K.; Khole, Aalok; Chertihin, Olga; Curnow, Eliza; Feldman, Sandford H; Mandal, Arabinda; Shetty, Jagathpala; Flickinger, Charles; Herr, John C

    2014-01-01

    Background Sperm Acrosomal SLLP1 Binding (SAS1B) protein (ovastacin) is an oolemmal binding partner for the intra-acrosomal sperm protein SLLP1. Results Immunohistochemical localization revealed that SAS1B translation is restricted among adult tissues to the ovary and oocytes, SAS1B appearing first in follicles at the primary-secondary transition. Quiescent oocytes within primordial follicles and primary follicles did not stain for SAS1B. Examination of neonatal rat ovaries revealed SAS1B expression first as faint signals in postnatal day 3 oocytes, with SAS1B protein staining intensifying with oocyte growth. Irrespective of animal age or estrus stage, SAS1B was seen only in oocytes of follicles that initiated a second granulosa cell layer. The precise temporal and spatial onset of SAS1B expression was conserved in adult ovaries in 7 eutherian species, including non-human primates. Immunoelectron micrographs localized SAS1B within cortical granules in MII oocytes. A population of SAS1B localized on the oolemma predominantly in the microvillar region anti-podal to the nucleus in ovulated MII rat oocytes and on the oolemma in macaque GV oocytes. Conclusions The restricted expression of SAS1B protein in growing oocytes, absence in the ovarian reserve, and localization on the oolemma suggest this zinc metalloprotease deserves consideration as a candidate target for reversible female contraceptive strategies. PMID:24038607

  15. Cold-Inducible RNA-Binding Protein Bypasses Replicative Senescence in Primary Cells through Extracellular Signal-Regulated Kinase 1 and 2 Activation▿ †

    PubMed Central

    Artero-Castro, Ana; Callejas, Francisco B.; Castellvi, Josep; Kondoh, Hiroshi; Carnero, Amancio; Fernández-Marcos, Pablo J.; Serrano, Manuel; Ramón y Cajal, Santiago; Lleonart, Matilde E.

    2009-01-01

    Embryonic stem cells are immortalized cells whose proliferation rate is comparable to that of carcinogenic cells. To study the expression of embryonic stem cell genes in primary cells, genetic screening was performed by infecting mouse embryonic fibroblasts (MEFs) with a cDNA library from embryonic stem cells. Cold-inducible RNA-binding protein (CIRP) was identified due to its ability to bypass replicative senescence in primary cells. CIRP enhanced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and treatment with an MEK inhibitor decreased the proliferation caused by CIRP. In contrast to CIRP upregulation, CIRP downregulation decreased cell proliferation and resulted in inhibition of phosphorylated ERK1/2 inhibition. This is the first evidence that ERK1/2 activation, through the same mechanism as that described for a Val12 mutant K-ras to induce premature senescence, is able to bypass senescence in the absence of p16INK4a, p21WAF1, and p19ARF upregulation. Moreover, these results show that CIRP functions by stimulating general protein synthesis with the involvement of the S6 and 4E-BP1 proteins. The overall effect is an increase in kinase activity of the cyclin D1-CDK4 complex, which is in accordance with the proliferative capacity of CIRP MEFs. Interestingly, CIRP mRNA and protein were upregulated in a subgroup of cancer patients, a finding that may be of relevance for cancer research. PMID:19158277

  16. Radical Cation/Radical Reactions: A Fourier Transform Ion Cyclotron Resonance Study of Allyl Radical Reacting with Aromatic Radical Cations

    PubMed Central

    Russell, Amber L.; Rohrs, Henry W.; Read, David; Giblin, Daryl E.; Gaspar, Peter P.; Gross, Michael L.

    2010-01-01

    A method for the study of reactions of open-shell neutrals (radicals) and radical cations is described. Pyrolysis (25–1500 °C) of thermally labile compounds, such as, 1,5-hexadiene via a Chen nozzle yields a seeded beam of reactive species in helium. The pyrolysis products are then analyzed by electron ionization (EI) or reacted with stored ions. Electron ionization of the pyrolysis products of 1,5-hexadiene shows that both the allyl radical and allene are generated. Reactions of benzene radical cations and the pyrolysis products of 1,5-hexadiene result in carbon-carbon bond formation. Those reactions of allyl radical with the benzene radical cation yield the C7H7+ ion of m/z 91, permitting an unusual entry into arenium ions. The reaction of allene with benzene radical cation in contrast yields C9H10+. and C9H9+. PMID:20401179

  17. UV and X-ray structural studies of a 101-residue long Tat protein from a HIV-1 primary isolate and of its mutated, detoxified, vaccine candidate.

    PubMed

    Foucault, Marine; Mayol, Katia; Receveur-Bréchot, Véronique; Bussat, Marie-Claire; Klinguer-Hamour, Christine; Verrier, Bernard; Beck, Alain; Haser, Richard; Gouet, Patrice; Guillon, Christophe

    2010-05-01

    The 101-residue long Tat protein of primary isolate 133 of the human immunodeficiency virus type 1 (HIV-1), wt-Tat(133) displays a high transactivation activity in vitro, whereas the mutant thereof, STLA-Tat(133), a vaccine candidate for HIV-1, has none. These two proteins were chemically synthesized and their biological activity was validated. Their structural properties were characterized using circular dichroism (CD), fluorescence emission, gel filtration, dynamic light scattering, and small angle X-ray scattering (SAXS) techniques. SAXS studies revealed that both proteins were extended and belong to the family of intrinsically unstructured proteins. CD measurements showed that wt-Tat(133) or STLA-Tat(133) underwent limited structural rearrangements when complexed with specific fragments of antibodies. Crystallization trials have been performed on the two forms, assuming that the Tat(133) proteins might have a better propensity to fold in supersaturated conditions, and small crystals have been obtained. These results suggest that biologically active Tat protein is natively unfolded and requires only a limited gain of structure for its function.

  18. IL12, IL10, IFNγ and TNFα Expression in Human Primary Monocytes Stimulated with Bacterial Heat Shock GroEL (Hsp64) Protein

    PubMed Central

    Nalbant, Ayten; Saygılı, Tahsin

    2016-01-01

    Actinobacillus (Aggregatibacter) actinomycetemicomitans (Aa) is a bacterium that lives in the oral cavity and plays an important role in periodontal diseases. The effect of A.actinomycetemcomitans’s heat shock family protein GroEL on host or immune cells including monocytes is quite limited. In this study, the recombinant A.actinomycetemcomitans’s GroEL protein (rAaGroEL) was used as an antigen and its effects on monocytes of peripheral blood mononuclear cells (PBMCs) was investigated. To do that, PBMCs were stimulated with rAaGroEL protein at different time points (16h to 96h) and the cytokines of CD14+ monocytes were detected with intracellular cytokine staining by Flow cytometry. Data showed that AaGroEL protein has an antigenic effect on human primary monocytes. AaGroEL protein responsive CD14 monocytes stimulates the expression of IL12, IL10, IFNγ and TNFα cytokines with different kinetics and expression profile. Therefore, A. actinomycetemcomitans’s heat shock GroEL protein can modulate innate and adaptive immune responses and contribute to an inflammatory diseases pathology. PMID:27119521

  19. The primary structures of six human salivary acidic proline-rich proteins (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f).

    PubMed Central

    Hay, D I; Bennick, A; Schlesinger, D H; Minaguchi, K; Madapallimattam, G; Schluckebier, S K

    1988-01-01

    Human glandular salivary secretions contain several acidic proline-rich phosphoproteins (PRPs). These proteins have important biological functions related to providing a protective environment for the teeth, and appear to possess other activities associated with modulation of adhesion of bacteria to oral surfaces. These functions and activities depend on the primary structures of the PRPs. Previously determined amino acid sequences of two 150-residue molecules, PRP-1 and PRP-2, and two related 106-residue proteins, PRP-3 and PRP-4, indicated that residue 4 was Asn in PRP-1 and PRP-3, and Asp in PRP-2 and PRP-4, and position 50 was Asn in all four proteins. Recent data from cDNA sequence studies and further structural studies, however, showed that the previously proposed sequences cannot be completely correct. The present work has shown that the protein previously designated as PRP-1 actually consisted of two positional isomers, PIF-s, which has Asn and Asp at positions 4 and 50 respectively, and authentic PRP-1, which has the reverse arrangement. The same isomerism is present in the smaller proteins, PIF-f and PRP-3. Since the isomeric pairs have identical compositions and charges, their presence was not previously detected. Also, by using a more highly purified preparation, it has been found that position 50 in PRP-2 and PRP-4 is Asp, rather than Asn previously reported. These new findings for the six PRPs define their complete primary structures, which are now consistent with those proposed for PRP-1 and PIF-s from cDNA data, and are also consistent with the chromatographic and electrophoretic behaviours of the six PRPs and their derived peptides. These corrected structures are important for understanding the biological functions and activities of these unusual proteins. Images Fig. 1. Fig. 4. Fig. 5. PMID:3196309

  20. Port-site metastasis as a primary complication following retroperitoneal laparoscopic radical resection of renal pelvis carcinoma or nephron-sparing surgery: A report of three cases and review of the literature

    PubMed Central

    WANG, NING; WANG, KAI; ZHONG, DACHUAN; LIU, XIA; SUN, JI; LIN, LIANXIANG; GE, LINNA; YANG, BO

    2016-01-01

    The present study reports the clinical data of two patients with renal pelvis carcinoma and one patient with renal carcinoma who developed port-site metastasis following retroperitoneal laparoscopic surgery. The current study aimed to identify the cause and prognosis of the occurrence of port-site metastasis subsequent to laparoscopic radical resection of renal pelvis carcinoma and nephron-sparing surgery. Post-operative pathology confirmed the presence of high-grade urothelial cell carcinoma in two patients and Fuhrman grade 3 renal clear cell carcinoma in one patient. Port-site metastasis was initially detected 1–7 months post-surgery. The two patients with renal pelvis carcinoma succumbed to the disease 2 and 4 months following the identification of the port-site metastasis, respectively, whereas the patient with renal carcinoma survived with no disease progression during the targeted therapy period. The occurrence of port-site metastasis may be attributed to systemic and local factors. Measures to reduce the development of this complication include strict compliance with the operating guidelines for tumor surgery, avoidance of air leakage at the port-site, complete removal of the specimen with an impermeable bag, irrigation of the laparoscopic instruments and incisional wound with povidone-iodine when necessary, and enhancement of the body's immunity. Close post-operative follow-up observation for signs of recurrence or metastasis is essential, and systemic chemotherapy may be required in patients with high-grade renal pelvis carcinoma and renal carcinoma in order to prolong life expectancy. PMID:27313720

  1. Emerging themes in radical SAM chemistry

    PubMed Central

    Shisler, Krista A; Broderick, Joan B

    2014-01-01

    Enzymes in the radical SAM (RS) superfamily catalyze a wide variety of reactions through unique radical chemistry. The characteristic markers of the superfamily include a [4Fe–4S] cluster coordinated to the protein via a cysteine triad motif, typically CX3CX2C, with the fourth iron coordinated by S-adenosylmethionine (SAM). The SAM serves as a precursor for a 5′-deoxyadenosyl radical, the central intermediate in nearly all RS enzymes studied to date. The SAM-bound [4Fe–4S] cluster is located within a partial or full triosephosphate isomerase (TIM) barrel where the radical chemistry occurs protected from the surroundings. In addition to the TIM barrel and a RS [4Fe–4S] cluster, many members of the superfamily contain additional domains and/or additional Fe–S clusters. Recently characterized superfamily members are providing new examples of the remarkable range of reactions that can be catalyzed, as well as new structural and mechanistic insights into these fascinating reactions. PMID:23141873

  2. Density functional calculations on model tyrosyl radicals.

    PubMed Central

    Himo, F; Gräslund, A; Eriksson, L A

    1997-01-01

    A gradient-corrected density functional theory approach (PWP86) has been applied, together with large basis sets (IGLO-III), to investigate the structure and hyperfine properties of model tyrosyl free radicals. In nature, these radicals are observed in, e.g., the charge transfer pathways in photosystem II (PSII) and in ribonucleotide reductases (RNRs). By comparing spin density distributions and proton hyperfine couplings with experimental data, it is confirmed that the tyrosyl radicals present in the proteins are neutral. It is shown that hydrogen bonding to the phenoxyl oxygen atom, when present, causes a reduction in spin density on O and a corresponding increase on C4. Calculated proton hyperfine coupling constants for the beta-protons show that the alpha-carbon is rotated 75-80 degrees out of the plane of the ring in PSII and Salmonella typhimurium RNR, but only 20-30 degrees in, e.g., Escherichia coli, mouse, herpes simplex, and bacteriophage T4-induced RNRs. Furthermore, based on the present calculations, we have revised the empirical parameters used in the experimental determination of the oxygen spin density in the tyrosyl radical in E. coli RNR and of the ring carbon spin densities, from measured hyperfine coupling constants. Images FIGURE 1 FIGURE 5 PMID:9083661

  3. Are free radicals involved in thiol-based redox signaling?

    PubMed

    Winterbourn, Christine C

    2015-03-01

    Cells respond to many stimuli by transmitting signals through redox-regulated pathways. It is generally accepted that in many instances signal transduction is via reversible oxidation of thiol proteins, although there is uncertainty about the specific redox transformations involved. The prevailing view is that thiol oxidation occurs by a two electron mechanism, most commonly involving hydrogen peroxide. Free radicals, on the other hand, are considered as damaging species and not generally regarded as important in cell signaling. This paper examines whether it is justified to dismiss radicals or whether they could have a signaling role. Although there is no direct evidence that radicals are involved in transmitting thiol-based redox signals, evidence is presented that they are generated in cells when these signaling pathways are activated. Radicals produce the same thiol oxidation products as two electron oxidants, although by a different mechanism, and at this point radical-mediated pathways should not be dismissed. There are unresolved issues about how radical mechanisms could achieve sufficient selectivity, but this could be possible through colocalization of radical-generating and signal-transducing proteins. Colocalization is also likely to be important for nonradical signaling mechanisms and identification of such associations should be a priority for advancing the field.

  4. Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

    PubMed Central

    Corry, Jacqueline; Johnson, Sara M.; Cornwell, Jessica

    2015-01-01

    ABSTRACT All live attenuated respiratory syncytial virus (RSV) vaccines that have advanced to clinical trials have been produced in Vero cells. The attachment (G) glycoprotein in virions produced in these cells is smaller than that produced in other immortalized cells due to cleavage. These virions are 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cell cultures. Because HAE cells are isolated directly from human airways, Vero cell-grown vaccine virus would very likely be similarly inefficient at initiating infection of the nasal epithelium following vaccination, and therefore, a larger inoculum would be required for effective vaccination. We hypothesized that Vero cell-derived virus containing an intact G protein would be more infectious for HAE cell cultures. Using protease inhibitors with increasing specificity, we identified cathepsin L to be the protease responsible for cleavage. Our evidence suggests that cleavage occurs in the late endosome or lysosome during endocytic recycling. Cathepsin L activity was 100-fold greater in Vero cells than in HeLa cells. In addition, cathepsin L was able to cleave the G protein in Vero cell-grown virions but not in HeLa cell-grown virions, suggesting a difference in G-protein posttranslational modification in the two cell lines. We identified by mutagenesis amino acids important for cleavage, and these amino acids included a likely cathepsin L cleavage site. Virus containing a modified, noncleavable G protein produced in Vero cells was 5-fold more infectious for HAE cells in culture, confirming our hypothesis and indicating the value of including such a mutation in future live attenuated RSV vaccines. IMPORTANCE Worldwide, RSV is the second leading infectious cause of infant death, but no vaccine is available. Experimental live attenuated RSV vaccines are grown in Vero cells, but during production the virion attachment (G) glycoprotein is cleaved. Virions containing a cleaved G protein

  5. a Free Radical

    NASA Astrophysics Data System (ADS)

    Carrington, Alan

    2001-10-01

    This chapter describes my research career, spanning the period from 1955 to 2000. My initial PhD work at the University of Southampton was concerned with the electronic structure and spectra of transition metal complexes and included studies of the electronic spin resonance (ESR) spectra of magnetically dilute single crystals. After a year at the University of Minnesota, I went to Cambridge University and for the next six years studied the ESR spectra of liquid phase organic free radicals. I commenced work on the microwave magnetic resonance (MMR) spectra of gaseous free radicals in 1965, and this work continued until 1975. I moved from Cambridge to Southampton in 1967. In 1975 I turned to the study of gas phase molecular ions, using ion beam methods. In the earlier years of this period I concentrated on simple fundamental species like H+2, HD+, and H+3. In the later years until my retirement in 1999, I concentrated on the observation and analysis of microwave spectra involving energy levels lying very close to a dissociation asymptote. DEDICATION This chapter is dedicated to the memory of Harry E. Radford, who died while it was being written. Harry was a quiet and shy man, who often worked alone and never indulged in self-promotion. So far as I know, he was never awarded any medals or prizes, nor elected to any academies or learned societies. Nevertheless he was an experimentalist of the highest originality and quality, a theorist of true intellectual depth, and a remarkable pioneer in many of the techniques of studying free radicals that are now commonplace.

  6. Catalysis of Radical Reactions: A Radical Chemistry Perspective.

    PubMed

    Studer, Armido; Curran, Dennis P

    2016-01-01

    The area of catalysis of radical reactions has recently flourished. Various reaction conditions have been discovered and explained in terms of catalytic cycles. These cycles rarely stand alone as unique paths from substrates to products. Instead, most radical reactions have innate chains which form products without any catalyst. How do we know if a species added in "catalytic amounts" is a catalyst, an initiator, or something else? Herein we critically address both catalyst-free and catalytic radical reactions through the lens of radical chemistry. Basic principles of kinetics and thermodynamics are used to address problems of initiation, propagation, and inhibition of radical chains. The catalysis of radical reactions differs from other areas of catalysis. Whereas efficient innate chain reactions are difficult to catalyze because individual steps are fast, both inefficient chain processes and non-chain processes afford diverse opportunities for catalysis, as illustrated with selected examples.

  7. Complications Following Radical Nephroureterectomy.

    PubMed

    Raman, Jay D; Jafri, Syed M

    2016-05-01

    Radical nephroureterectomy (RNU) is the gold standard treatment strategy for bulky, high-grade, or muscle-invasive upper tract urothelial carcinoma (UTUC). Many patients with UTUC who require RNU are elderly, comorbid, and at risk for perioperative complications. Recognition of likelihood and extent of such complications guides preoperative counseling, decision-making process for major surgery, and perioperative care. A critical review of such data is essential, given the inevitable impact of complications on hospital duration, need for readmission, resource utilization, and costs associated with management. PMID:26968416

  8. The primary structure of component 8c-1, a subunit protein of intermediate filaments in wool keratin. Relationships with proteins from other intermediate filaments.

    PubMed Central

    Dowling, L M; Crewther, W G; Inglis, A S

    1986-01-01

    Component 8c-1, one of four highly homologous component-8 subunit proteins present in the microfibrils of wool, was isolated as its S-carboxymethyl derivative and its amino acid sequence was determined. Large peptides were isolated after cleaving the protein chemically or enzymically and the sequence of each was determined with an automatic Sequenator. The peptides were ordered by sequence overlaps and, in some instances, by homology with known sequences from other component-8 subunits. The C-terminal residues were identified by three procedures. Full details of the various procedures used have been deposited as Supplementary Publication SUP 50133 (4 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5. The result showed that the protein comprises 412 residues and has an Mr, including the N-terminal acetyl group, of 48,300. The sequence of residues 98-200 of component 8c-1 was found to correspond to the partial or complete sequences of four homologous type I helical segments previously isolated from helical fragments recovered from chymotryptic digests of microfibrillar proteins of wool [Crewther & Dowling (1971) Appl. Polym. Symp. 18, 1-20; Crewther, Gough, Inglis & McKern (1978) Text. Res. J. 48, 160-162; Gough, Inglis & Crewther (1978) Biochem. J. 173, 385]. Considered in relation to amino acid sequences of other intermediate-filament proteins, the sequence is in accord with the view that keratin filament proteins are of two types [Hanukoglu & Fuchs (1983) Cell (Cambridge, Mass.) 33, 915-924]. Filament proteins from non-keratinous tissues, such as desmin, vimentin, neurofilament proteins and the glial fibrillary acidic protein, which form monocomponent filaments, constitute a third type. It is suggested that as a whole the proteins from intermediate filaments be classed as filamentins, the three types at present identified forming

  9. Discovery of hypoiodite-mediated aminyl radical cyclization lacking a nitrogen radical-stabilizing group: application to synthesis of an oxazaspiroketal-containing cephalostatin analog.

    PubMed

    Koag, Myong; Lee, Seongmin

    2011-09-16

    Synthesis of an oxazaspiroketal-containing bissteroidal pyrazine is described. The key transformation of this synthesis involves stereoselective formation of oxazaspiroketal via aminyl-radical cyclization of primary amine lacking a radical-stabilizing group by Suárez hypoiodite oxidation.

  10. The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90)

    PubMed Central

    Zhang, Hongbo

    2014-01-01

    Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes. PMID:24604735

  11. The primary structure of the ribosomal A-protein (L12) from the moderate halophile NRCC 41227.

    PubMed

    Falkenberg, P; Yaguchi, M; Roy, C; Zuker, M; Matheson, A T

    1986-07-01

    The complete amino acid sequence of the ribosomal A-protein (equivalent to L7/L12 in Escherichia coli) from a moderate halophile, NRCC 41227, has been determined using an automatic Beckman sequencer and by the manual Edman cleavage of peptides obtained from selective proteolytic cleavage of the ribosomal A-protein. The protein contains 122 amino acids and has a composition of Asp5, Asn2, Thr6, Ser6, Glu21, Gln2, Pro2, Gly12, Ala21, Val14, Met4, Ile4, Leu9, Phe2, Lys11, and Arg1, and a molecular weight of 12 537. It has a net negative charge of -14 and is, therefore, slightly more acidic than other eubacterial ribosomal A-proteins. The phylogenetic tree, obtained by computer analysis of the amino acid sequence of this and other eubacterial A-proteins, indicate these proteins form five subgroups within the eubacterial kingdom. The moderate halophile NRCC 41227 is part of a group of Gram-negative bacteria that include E. coli and another moderate halophile Vibrio costicola. The sequence data provides further evidence that the moderate and extreme halophiles have evolved by separate pathways.

  12. Augmentation of virus secretion by the human immunodeficiency virus type 1 Vpu protein is cell type independent and occurs in cultured human primary macrophages and lymphocytes.

    PubMed Central

    Schubert, U; Clouse, K A; Strebel, K

    1995-01-01

    The human immunodeficiency virus type 1-specific Vpu protein is a small integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and, independently, increases the release of progeny virions from infected cells. To address the importance of Vpu for virus replication in primary human cells such as peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM), we used three different sets of monocyte-tropic molecular clones of human immunodeficiency virus type 1: a primary isolate, AD8+, and two chimeric variants of the T-cell-tropic isolate NL4-3 carrying the env determinants of either AD8+ or SF162 monocyte-tropic primary isolates. Isogenic variants of these chimeric viruses were constructed to express either wild-type Vpu or various mutants of Vpu. The effects of these mutations in the vpu gene on virus particle secretion from infected MDM or PBMC were assessed by determination of the release of virion-associated reverse transcriptase into culture supernatants, Western blot (immunoblot) analysis of pelleted virions, and steady-state or pulse-chase metabolic labeling. Wild-type Vpu increased virus release four- to sixfold in MDM and two- to threefold in PBMC, while nonphosphorylated Vpu and a C-terminal truncation mutant of Vpu were partially active on virus release in primary cells. These results demonstrate that Vpu regulates virus release in primary lymphocyte and macrophage cultures in a similar manner and to a similar extent to those previously observed in HeLa cells or CD4+ T-cell lines. Thus, our findings provide evidence that Vpu functions in a variety of human cells, both primary cells and continuous cell lines, and mutations in Vpu affect its biological activity independent of the cell type and virus isolate used. PMID:7494279

  13. Crystal Structure of the Zorbamycin-Binding Protein ZbmA, the Primary Self-Resistance Element in Streptomyces flavoviridis ATCC21892

    SciTech Connect

    Rudolf, Jeffrey D.; Bigelow, Lance; Chang, Changsoo; Cuff, Marianne E.; Lohman, Jeremy R.; Chang, Chin-Yuan; Ma, Ming; Yang, Dong; Clancy, Shonda; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

    2015-11-17

    The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity, while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at high resolutions of 1.90 and 1.65 angstrom, respectively. A comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM family of antitumor antibiotics.

  14. Investigation of drying stresses on proteins during lyophilization: differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer.

    PubMed

    Luthra, Sumit; Obert, Jean-Philippe; Kalonia, Devendra S; Pikal, Michael J

    2007-01-01

    This article describes the design, performance testing, and application of a controlled humidity mini-freeze-dryer in studying the physical stability of lactate dehydrogenase during lyophilization. Performance evaluation of the mini-freeze-dryer was conducted with tests, namely water sublimation, radiation heat exchange, lowest achievable temperature, and leak testing. Protein stability studies were conducted by comparing protein activity at various stages of lyophilization with the initial activity. The shelf and condenser temperature were stable at <-40 degrees C, wall temperature was within 2 degrees C of the shelf temperature, and the leak rate was small. The chamber pressure was controlled by the ice on the condenser and the product temperature during sublimation was equal to the shelf temperature. Addition of Tween 80 prevented activity loss in solution and after freeze-thaw. No activity loss was observed after primary-drying even in absence of lyoprotectants and with collapse of cake structure. Five percent (w/w) sucrose concentration was required to achieve full stabilization. In conclusion, performance testing established that the mini-freeze-dryer was suitable for mechanistic freeze-drying studies. Secondary-drying was the critical step for protein stability. The concentration of sucrose required to stabilize the protein completely was several orders of magnitude higher than that required to satisfy the direct interaction requirement of the protein. PMID:17031859

  15. LIVER TYPE FATTY ACID BINDING PROTEIN (L-FABP) GENE ABLATION REDUCES NUCLEAR LIGAND DISTRIBUTION AND PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR-α ACTIVITY IN CULTURED PRIMARY HEPATOCYTES1

    PubMed Central

    McIntosh, Avery L.; Atshaves, Barbara P.; Hostetler, Heather A.; Huang, Huan; Davis, Jason; Lyuksyutova, Olga I.; Landrock, Danilo; Kier, Ann B.; Schroeder, Friedhelm

    2009-01-01

    The effect of liver type fatty acid binding protein (L-FABP) gene ablation on the uptake and distribution of long chain fatty acids (LCFA) to the nucleus by real-time laser scanning confocal imaging and peroxisome proliferator activated receptor-α (PPARα) activity was examined in cultured primary hepatocytes from livers wild-type L-FABP+/+ and gene ablated L-FABP−/− mice. Cultured primary hepatocytes from livers of L-FABP−/− mice exhibited: (i) reduced oxidation of palmitic acid, a common dietary long chain fatty acid (LCFA); (ii) reduced expression of fatty acid oxidative enzymes—proteins transcriptionally regulated by PPARα; (iii) reduced palmitic acid-induced PPARα coimmunoprecipitation with coactivator SRC1 concomitant with increased PPARα coimmunoprecipitation with coinhibitor N-CoR; (iv) reduced palmitic acid-induced PPARα. Diminished PPARα activation in L-FABP null hepatocytes was associated with lower uptake of common dietary LCFA (palmitic acid as well as its fluorescent derivative BODIPY FL C16), reduced level of total unesterified LCFA, and real-time redistribution of BODIPY FL C16 from the central nucleoplasm to the nuclear envelope. Taken together, these studies support the hypothesis that L-FABP may facilitate ligand (LCFA)-activated PPARα transcriptional activity at least in part by increasing total LCFA ligand available to PPARα for inducing PPARα-mediated transcription of proteins involved in LCFA metabolism. PMID:19285478

  16. Bursectomy at radical gastrectomy

    PubMed Central

    Kayaalp, Cuneyt

    2015-01-01

    Radical gastrectomy with extended lymph node dissection and prophylactic resection of the omentum, peritoneum over the posterior lesser sac, pancreas and/or spleen was advocated at the beginning of the 1960s in Japan. In time, prophylactic routine resections of the pancreas and/or spleen were abandoned because of the high incidence of postoperative complications. However, omentectomy and bursectomy continued to be standard parts of traditional radical gastrectomy. The bursa omentalis was thought to be a natural barrier against invasion of cancer cells into the posterior part of the stomach. The theoretical rationale for bursectomy was to reduce the risk of peritoneal recurrences by eliminating the peritoneum over the lesser sac, which might include free cancer cells or micrometastases. Over time, the indication for bursectomy was gradually reduced to only patients with posterior gastric wall tumors penetrating the serosa. Despite its theoretical advantages, its benefit for recurrence or survival has not been proven yet. The possible reasons for this inconsistency are discussed in this review. In conclusion, the value of bursectomy in the treatment of gastric cancer is still under debate and large-scale randomized studies are necessary. Until clear evidence of patient benefit is obtained, its routine use cannot be recommended. PMID:26523213

  17. Bursectomy at radical gastrectomy.

    PubMed

    Kayaalp, Cuneyt

    2015-10-27

    Radical gastrectomy with extended lymph node dissection and prophylactic resection of the omentum, peritoneum over the posterior lesser sac, pancreas and/or spleen was advocated at the beginning of the 1960s in Japan. In time, prophylactic routine resections of the pancreas and/or spleen were abandoned because of the high incidence of postoperative complications. However, omentectomy and bursectomy continued to be standard parts of traditional radical gastrectomy. The bursa omentalis was thought to be a natural barrier against invasion of cancer cells into the posterior part of the stomach. The theoretical rationale for bursectomy was to reduce the risk of peritoneal recurrences by eliminating the peritoneum over the lesser sac, which might include free cancer cells or micrometastases. Over time, the indication for bursectomy was gradually reduced to only patients with posterior gastric wall tumors penetrating the serosa. Despite its theoretical advantages, its benefit for recurrence or survival has not been proven yet. The possible reasons for this inconsistency are discussed in this review. In conclusion, the value of bursectomy in the treatment of gastric cancer is still under debate and large-scale randomized studies are necessary. Until clear evidence of patient benefit is obtained, its routine use cannot be recommended. PMID:26523213

  18. Radically innovative steelmaking technologies

    NASA Astrophysics Data System (ADS)

    Szekely, Julian

    1980-09-01

    The steel industry is faced with serious problems caused by the increasing cost of energy, labor and capital and by tough overseas competition, employing new highly efficient process plants. The very high cost of capital and of capital equipment renders the construction of new green field site plants, exemplifying the best available technology economically unattractive. For this reason, over the long term the development radically innovative steelmaking technologies appears to be the only satisfactory resolution of this dilemma. The purpose of this article is to present a critical review of some of the radically innovative steelmaking technologies that have been proposed during the past few years and to develop the argument that these indeed do deserve serious consideration at the present time. It should be stressed, however, that these innovative technologies can be implemented only as part of a carefully conceived long range plan, which contains as a subset short term solutions, such as trigger prices improved investment credits, and so forth and intermediate term solutions, such as more extensive use of continuous casting, external desulfurization and selective modernization in general.

  19. Oligorotaxane Radicals under Orders

    PubMed Central

    2016-01-01

    A strategy for creating foldameric oligorotaxanes composed of only positively charged components is reported. Threadlike components—namely oligoviologens—in which different numbers of 4,4′-bipyridinium (BIPY2+) subunits are linked by p-xylylene bridges, are shown to be capable of being threaded by cyclobis(paraquat-p-phenylene) (CBPQT4+) rings following the introduction of radical-pairing interactions under reducing conditions. UV/vis/NIR spectroscopic and electrochemical investigations suggest that the reduced oligopseudorotaxanes fold into highly ordered secondary structures as a result of the formation of BIPY•+ radical cation pairs. Furthermore, by installing bulky stoppers at each end of the oligopseudorotaxanes by means of Cu-free alkyne–azide cycloadditions, their analogous oligorotaxanes, which retain the same stoichiometries as their progenitors, can be prepared. Solution-state studies of the oligorotaxanes indicate that their mechanically interlocked structures lead to the enforced interactions between the dumbbell and ring components, allowing them to fold (contract) in their reduced states and unfold (expand) in their fully oxidized states as a result of Coulombic repulsions. This electrochemically controlled reversible folding and unfolding process, during which the oligorotaxanes experience length contractions and expansions, is reminiscent of the mechanisms of actuation associated with muscle fibers. PMID:27163033

  20. Protective effect of Pterostilbene against free radical mediated oxidative damage

    PubMed Central

    2013-01-01

    Background Pterostilbene, a methoxylated analog of Resveratrol, is gradually gaining more importance as a therapeutic drug owing to its higher lipophilicity, bioavailability and biological activity than Resveratrol. This study was undertaken to characterize its ability to scavenge free radicals such as superoxide, hydroxyl and hydrogen peroxide and to protect bio-molecules within a cell against oxidative insult. Methods Anti-oxidant activity of Pterostilbene was evaluated extensively by employing several in vitro radical scavenging/inhibiting assays and pulse radiolysis study. In addition, its ability to protect rat liver mitochondria against tertiary-butyl hydroperoxide (TBHP) and hydroxyl radical generated oxidative damage was determined by measuring the damage markers such as protein carbonyls, protein sulphydryls, lipid hydroperoxides, lipid peroxides and 8-hydroxy-2'-deoxyguanosine. Pterostilbene was also evaluated for its ability to inhibit •OH radical induced single strand breaks in pBR322 DNA. Result Pterostilbene exhibited strong anti-oxidant activity against various free radicals such as DPPH, ABTS, hydroxyl, superoxide and hydrogen peroxide in a concentration dependent manner. Pterostilbene conferred protection to proteins, lipids and DNA in isolated mitochondrial fractions against TBHP and hydroxyl radical induced oxidative damage. It also protected pBR322 DNA against oxidative assault. Conclusions Thus, present study provides an evidence for the strong anti-oxidant property of Pterostilbene, methoxylated analog of Resveratrol, thereby potentiating its role as an anti-oxidant. PMID:24070177

  1. Radical-radical interactions among oxidized guanine bases including guanine radical cation and dehydrogenated guanine radicals.

    PubMed

    Zhao, Jing; Wang, Mei; Yang, Hongfang; Zhang, Meng; Liu, Ping; Bu, Yuxiang

    2013-09-19

    We present here a theoretical investigation of the structural and electronic properties of di-ionized GG base pairs (G(•+)G(•+),G(-H1)(•)G(•+), and G(-H1)(•)G(-H1)(•)) consisting of the guanine cation radical (G(•+)) and/or dehydrogenated guanine radical (G(-H1)(•)) using density functional theory calculations. Different coupling modes (Watson-Crick/WC, Hoogsteen/Hoog, and minor groove/min hydrogen bonding, and π-π stacking modes) are considered. We infer that a series of G(•+)G(•+) complexes can be formed by the high-energy radiation. On the basis of density functional theory and complete active space self-consistent (CASSCF) calculations, we reveal that in the H-bonded and N-N cross-linked modes, (G(•+)G(•+))WC, (G(-H1)(•)G(-H1)(•))WC, (G(-H1)(•)G(-H1)(•))minI, and (G(-H1)(•)G(-H1)(•))minIII have the triplet ground states; (G(•+)G(•+))HoogI, (G(-H1)(•)G(•+))WC, (G(-H1)(•)G(•+))HoogI, (G(-H1)(•)G(•+))minI, (G(-H1)(•)G(•+))minII, and (G(-H1)(•)G(-H1)(•))minII possess open-shell broken-symmetry diradical-characterized singlet ground states; and (G(•+)G(•+))HoogII, (G(•+)G(•+))minI, (G(•+)G(•+))minII, (G(•+)G(•+))minIII, (G(•+)G(•+))HoHo, (G(-H1)(•)G(•+))minIII, (G(-H1)(•)G(•+))HoHo, and (G(-H1)(•)G(-H1)(•))HoHo are the closed-shell systems. For these H-bonded diradical complexes, the magnetic interactions are weak, especially in the diradical G(•+)G(•+) series and G(-H1)(•)G(-H1)(•) series. The magnetic coupling interactions of the diradical systems are controlled by intermolecular interactions (H-bond, electrostatic repulsion, and radical coupling). The radical-radical interaction in the π-π stacked di-ionized GG base pairs ((G(•+)G(•+))ππ, (G(-H1)(•)G(•+))ππ, and (G(-H1)(•)G(-H1)(•))ππ) are also considered, and the magnetic coupling interactions in these π-π stacked base pairs are large. This is the first theoretical prediction that some di

  2. Kinetics of Propargyl Radical Dissociation.

    PubMed

    Klippenstein, Stephen J; Miller, James A; Jasper, Ahren W

    2015-07-16

    Due to the prominent role of the propargyl radical for hydrocarbon growth within combustion environments, it is important to understand the kinetics of its formation and loss. The ab initio transition state theory-based master equation method is used to obtain theoretical kinetic predictions for the temperature and pressure dependence of the thermal decomposition of propargyl, which may be its primary loss channel under some conditions. The potential energy surface for the decomposition of propargyl is first mapped at a high level of theory with a combination of coupled cluster and multireference perturbation calculations. Variational transition state theory is then used to predict the microcanonical rate coefficients, which are subsequently implemented within the multiple-well multiple-channel master equation. A variety of energy transfer parameters are considered, and the sensitivity of the thermal rate predictions to these parameters is explored. The predictions for the thermal decomposition rate coefficient are found to be in good agreement with the limited experimental data. Modified Arrhenius representations of the rate constants are reported for utility in combustion modeling. PMID:25871530

  3. Kinetics of Propargyl Radical Dissociation.

    PubMed

    Klippenstein, Stephen J; Miller, James A; Jasper, Ahren W

    2015-07-16

    Due to the prominent role of the propargyl radical for hydrocarbon growth within combustion environments, it is important to understand the kinetics of its formation and loss. The ab initio transition state theory-based master equation method is used to obtain theoretical kinetic predictions for the temperature and pressure dependence of the thermal decomposition of propargyl, which may be its primary loss channel under some conditions. The potential energy surface for the decomposition of propargyl is first mapped at a high level of theory with a combination of coupled cluster and multireference perturbation calculations. Variational transition state theory is then used to predict the microcanonical rate coefficients, which are subsequently implemented within the multiple-well multiple-channel master equation. A variety of energy transfer parameters are considered, and the sensitivity of the thermal rate predictions to these parameters is explored. The predictions for the thermal decomposition rate coefficient are found to be in good agreement with the limited experimental data. Modified Arrhenius representations of the rate constants are reported for utility in combustion modeling.

  4. Application of 125I-labelled soluble proteins in the histoautoradiographic detection of antigen and antibodies in the spleen of rabbits during primary immune response.

    PubMed

    Rodák, L

    1975-10-01

    An autoradiographic method for detecting soluble antigen (chicken serum albumin, CSA) and specific antibodies in the spleen of rabbits during a primary immune response is described. The method consists of incubating sections from the spleen with 125I-labelled IgG2 anti CSA (for demonstration of antigen) or with 125I-labelled antigen (for demonstration of specific antibodies). This treatment of histological sections combines the advantages and principles of the immunofluorescence technique with the possibility of evaluating the exact localization of the proteins by light microscopy in preparations stained with haematoxylin or methyl green-pyronin. The sensitivity of detection is very high: both antigen and antibodies could be demonstrated in the spleen follicles for as long as 42 days after the primary intravenous injection.

  5. Molecular cloning, expression, and primary sequence of outer membrane protein P2 of Haemophilus influenzae type b.

    PubMed Central

    Munson, R; Tolan, R W

    1989-01-01

    The structural gene for the porin of Haemophilus influenzae type b, designated outer membrane protein P2, was cloned, and the DNA sequence was determined. An oligonucleotide probe generated by reverse translation of N-terminal amino acid sequence data from the purified protein was used to screen genomic DNA. The probe detected a single EcoRI fragment of approximately 1,700 base pairs which was cloned to lambda gt11 and then into M13 and partially sequenced. The derived amino acid sequence indicated that we had cloned the N-terminal portion of the P2 gene. An overlapping approximately 1,600-base-pair PvuII genomic fragment was cloned into M13, and the sequence of the remainder of the P2 gene was determined. The gene for P2 was then reconstructed under the control of the T7 promoter and expressed in Escherichia coli. The N-terminal sequence of the purified protein corresponds to residues 21 through 34 of the derived amino acid sequence. Thus, the protein is synthesized with a 20-amino-acid leader peptide. The Mr of the processed protein is 37,782, in good agreement with the estimate of 37,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:2535836

  6. Primary blast-induced traumatic brain injury in rats leads to increased prion protein in plasma: a potential biomarker for blast-induced traumatic brain injury.

    PubMed

    Pham, Nam; Sawyer, Thomas W; Wang, Yushan; Jazii, Ferdous Rastgar; Vair, Cory; Taghibiglou, Changiz

    2015-01-01

    Traumatic brain injury (TBI) is deemed the "signature injury" of recent military conflicts in Afghanistan and Iraq, largely because of increased blast exposure. Injuries to the brain can often be misdiagnosed, leading to further complications in the future. Therefore, the use of protein biomarkers for the screening and diagnosis of TBI is urgently needed. In the present study, we have investigated the plasma levels of soluble cellular prion protein (PrPC) as a novel biomarker for the diagnosis of primary blast-induced TBI (bTBI). We hypothesize that the primary blast wave can disrupt the brain and dislodge extracellular localized PrPC, leading to a rise in concentration within the systemic circulation. Adult male Sprague-Dawley rats were exposed to single pulse shockwave overpressures of varying intensities (15-30 psi or 103.4-206.8 kPa] using an advanced blast simulator. Blood plasma was collected 24 h after insult, and PrPC concentration was determined with a modified commercial enzyme-linked immunosorbent assay (ELISA) specific for PrPC. We provide the first report that mean PrPC concentration in primary blast exposed rats (3.97 ng/mL ± 0.13 SE) is significantly increased compared with controls (2.46 ng/mL ± 0.14 SE; two tailed test p < 0.0001). Furthermore, we report a mild positive rank correlation between PrPC concentration and increasing blast intensity (psi) reflecting a plateaued response at higher pressure magnitudes, which may have implications for all military service members exposed to blast events. In conclusion, it appears that plasma levels of PrPC may be a novel biomarker for the detection of primary bTBI.

  7. Primary Blast-Induced Traumatic Brain Injury in Rats Leads to Increased Prion Protein in Plasma: A Potential Biomarker for Blast-Induced Traumatic Brain Injury

    PubMed Central

    Pham, Nam; Sawyer, Thomas W.; Wang, Yushan; Jazii, Ferdous Rastgar; Vair, Cory

    2015-01-01

    Abstract Traumatic brain injury (TBI) is deemed the “signature injury” of recent military conflicts in Afghanistan and Iraq, largely because of increased blast exposure. Injuries to the brain can often be misdiagnosed, leading to further complications in the future. Therefore, the use of protein biomarkers for the screening and diagnosis of TBI is urgently needed. In the present study, we have investigated the plasma levels of soluble cellular prion protein (PrPC) as a novel biomarker for the diagnosis of primary blast-induced TBI (bTBI). We hypothesize that the primary blast wave can disrupt the brain and dislodge extracellular localized PrPC, leading to a rise in concentration within the systemic circulation. Adult male Sprague–Dawley rats were exposed to single pulse shockwave overpressures of varying intensities (15-30 psi or 103.4–206.8 kPa] using an advanced blast simulator. Blood plasma was collected 24 h after insult, and PrPC concentration was determined with a modified commercial enzyme-linked immunosorbent assay (ELISA) specific for PrPC. We provide the first report that mean PrPC concentration in primary blast exposed rats (3.97 ng/mL±0.13 SE) is significantly increased compared with controls (2.46 ng/mL±0.14 SE; two tailed test p<0.0001). Furthermore, we report a mild positive rank correlation between PrPC concentration and increasing blast intensity (psi) reflecting a plateaued response at higher pressure magnitudes, which may have implications for all military service members exposed to blast events. In conclusion, it appears that plasma levels of PrPC may be a novel biomarker for the detection of primary bTBI. PMID:25058115

  8. Common Prairie feeds with different soluble and insoluble fractions used for CPM diet formulation in dairy cattle: impact of carbohydrate-protein matrix structure on protein and other primary nutrient digestion.

    PubMed

    Peng, Quanhui; Wang, Zhisheng; Zhang, Xuewei; Yu, Peiqiang

    2014-01-01

    An experiment was conducted to investigate the relationship of carbohydrates molecular spectral characteristics to rumen degradability of primary nutrients in Prairie feeds in dairy cattle. In total, 12 different types of feeds were selected, each type of feed was from three different source with total 37 samples. Six types of them were energy-sourced feeds and the others were protein-sourced feeds. The carbohydrates molecular spectral intensity of various functional groups were collected using Fourier transform infrared attenuated total reflectance (ATR-FT/IR) spectroscopy. In the in situ study, the results showed that the rumen digestibility and digestible fractions of primary nutrients (DM, OM, NCP, and CP) were significantly different (P<0.05) among the feeds. The spectral bands features were significantly different (P<0.05) among the feeds. Spectral intensities of A_Cell, H_1415 and H_1370 were weakly positively correlated with in situ rumen digestibility and digestible fractions of DM, OM and NCP. Spectral intensities of H_1150, H_1015, A_1, and A_3 were weakly negatively associated with in situ rumen degradation of CP. Spectral intensities of A_1240 and H_1240, mainly associated with cellulosic compounds, were correlated with rumen CP degradation. The multiple regression analysis demonstrated that the spectral intensities of A_3 and H_1415 played the most important role and could be used as a potential tool to predict rumen protein degradation of feeds in dairy cattle. In conclusion, this study showed that the carbohydrates as a whole have an effect on protein rumen degradation, rather than cellulose alone, indicating carbohydrate-protein matrix structure impact protein utilization in dairy cattle. The non-invasive molecular spectral technique (ATR-FT/IR) could be used as a rapid potential tool to predict rumen protein degradation of feedstuffs by using molecular spectral bands intensities in carbohydrate fingerprint region.

  9. A new fluorescence reaction in protein cytochemistry: formation of naphthalimide fluorophores from primary amino groups and 1,8-naphthalic anhydride derivatives.

    PubMed

    Stockert, J C; Trigoso, C I; Braña, M F

    1994-01-01

    In this work we describe the formation of fluorescent naphthalimide derivatives as a new cytochemical method for revealing protein amino groups. The reaction is based on the condensation of 1,8-naphthalic anhydrides in organic solvents with primary aliphatic amines. Under optimal violet-blue (436 nm) excitation, a strong yellow-green emission is observed in specific cell components from blood smears treated with 3-amino-1,8-naphthalic anhydride in N,N-dimethylformamide, which were the most suitable reagent and solvent for microscopic studies. Cytoplasmic granules of mammalian eosinophils and avian heterophils showed the highest fluorescence reaction, which was abolished by blocking procedures for amino groups. Spectrofluorometric analysis confirmed the emission characteristics of the naphthalimides produced from n-butylamine and gelatin. Taking into account the chemical reactivity of 1,8-naphthalic anhydrides and present results, the reaction can be considered selective for lysine and arginine residues of proteins.

  10. Free radical propulsion concept

    NASA Technical Reports Server (NTRS)

    Hawkins, C. E.; Nakanishi, S.

    1981-01-01

    A free radical propulsion concept utilizing the recombination energy of dissociated low molecular weight gases to produce thrust was examined. The concept offered promise of a propulsion system operating at a theoretical impulse, with hydrogen, as high as 2200 seconds at high thrust to power ratio, thus filling the gas existing between chemical and electrostatic propulsion capabilities. Microwave energy used to dissociate a continuously flowing gas was transferred to the propellant via three body recombination for conversion to propellant kinetic energy. Power absorption by the microwave plasma discharge was in excess of 90 percent over a broad range of pressures. Gas temperatures inferred from gas dynamic equations showed much higher temperatures from microwave heating than from electrothermal heating. Spectroscopic analysis appeared to corroborate the inferred temperatures of one of the gases tested.

  11. Radicals in Berkeley?

    PubMed Central

    Linn, Stuart

    2015-01-01

    In a previous autobiographical sketch for DNA Repair (Linn, S. (2012) Life in the serendipitous lane: excitement and gratification in studying DNA repair. DNA Repair 11, 595–605), I wrote about my involvement in research on mechanisms of DNA repair. In this Reflections, I look back at how I became interested in free radical chemistry and biology and outline some of our bizarre (at the time) observations. Of course, these studies could never have succeeded without the exceptional aid of my mentors: my teachers; the undergraduate and graduate students, postdoctoral fellows, and senior lab visitors in my laboratory; and my faculty and staff colleagues here at Berkeley. I am so indebted to each and every one of these individuals for their efforts to overcome my ignorance and set me on the straight and narrow path to success in research. I regret that I cannot mention and thank each of these mentors individually. PMID:25713083

  12. Cleavable ester linked magnetic nanoparticles for labeling of solvent exposed primary amine groups of peptides/proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to study the solvent exposed lysine residues of peptides/proteins, we previously reported disulfide linked N-hydrosuccinimide ester modified silica coated iron oxide magnetic nanoparticles (NHS-SS-SiO2@Fe3O4 MNPs). The presence of a disulfide bond in the linker limits the use of disulfide r...

  13. Secretion of bioactive interleukin-6 and tumor necrosis factor-alpha proteins from primary cultured human fetal membrane chorion cells infected with influenza virus.

    PubMed

    Uchide, N; Suzuki, A; Ohyama, K; Bessho, T; Toyoda, H

    2006-01-01

    Influenza virus infection during pregnancy is implicated in one of the causes of premature delivery, abortion and stillbirth. Pro-inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha produced by fetal membranes, are postulated to facilitate premature delivery. We investigated the secretion of IL-6 and TNF-alpha from primary cultured human fetal membrane chorion and amnion cells infected with influenza virus at protein and bioactivity levels in order to understand the pathology of premature delivery during influenza virus infection. Concentrations of IL-6 and TNF-alpha proteins were significantly increased in culture supernatants of chorion cells by influenza virus infection. Culture supernatants of the virus-infected chorion cells stimulated the proliferation of IL-6-sensitive 7-TD-1 cells and induced the cytolysis of TNF-alpha-sensitive L929 cells, both activities of which were inhibited by the addition of respective antibody, whereas no such phenomena were observed in amnion cells. The results demonstrated that only chorion cells secreted significant amounts of bioactive IL-6 and TNF-alpha proteins responding to influenza virus infection. The present study suggests a possibility that the secretion of bioactive IL-6 and TNF-alpha proteins from fetal membrane chorion cells is implicated in the pathogenesis of premature delivery during influenza virus infection. PMID:16122792

  14. Familial amyloid precursor protein mutants cause caspase-6-dependent but amyloid β-peptide-independent neuronal degeneration in primary human neuron cultures.

    PubMed Central

    Sivananthan, S N; Lee, A W; Goodyer, C G; LeBlanc, A C

    2010-01-01

    Although familial Alzheimer disease (AD)-associated autosomal dominant mutants have been extensively studied, little is known about the underlying molecular mechanisms of neurodegeneration induced by these mutants in AD. Wild-type, Swedish or London amyloid precursor protein (APP) transfection in primary human neurons induced neuritic beading, in which several co-expressed proteins, such as enhanced green fluorescent protein, red fluorescent protein (RFP)-tau and RFP-ubiquitin, accumulated. APP-induced neuritic beading was dependent on caspase-6 (Casp6), because it was inhibited with 5 μM z-VEID-fmk or with dominant-negative Casp6. Neuritic beading was independent from APP-mediated amyloid β-peptide (Aβ) production, because the APPM596V (APPMV) mutant, which cannot generate Aβ, still induced Casp6-dependent neuritic beading. However, the beaded neurons underwent Casp6- and Aβ-dependent cell death. These results indicate that overexpression of wild-type or mutant APP causes Casp6-dependent but Aβ-independent neuritic degeneration in human neurons. Because Casp6 is activated early in AD and is involved in axonal degeneration, these results suggest that the inhibition of Casp6 may represent an efficient early intervention against familial forms of AD. Furthermore, these results indicate that removing Aβ without inhibiting Casp6 may have little effect in preventing the progressive dementia associated with sporadic or familial AD. PMID:21368865

  15. 3,3',5-Triiodothyronine-induced differences in water-insoluble protein synthesis in primary epidermal cell cultures from the hind limb of premetamorphic Rana catesbeiana tadpoles.

    PubMed

    Ketola-Pirie, C A; Atkinson, B G

    1988-02-01

    Epidermal cells from the hind limb of premetamorphic tadpoles of the American bullfrog Rana catesbeiana (stages IX-XI) were maintained in primary culture for 120 hr. Cultures, maintained for 36 hr in medium supplemented with L-triiodothyronine (T3; 3 x 10(-10) mol T3/ml medium), synthesize water-insoluble proteins with Mrs of 59, 50, 48, and 43. The 59 and 48 kDa proteins synthesized by T3-supplemented cultures are not detectably synthesized by 36-hr-old cell cultures without added T3, but they are synthesized by both hormone-supplemented and unsupplemented cultures after 120 hr. These two proteins have Mrs and pIs which correspond with keratins detected in stratifying mammalian epidermis and are immunoprecipitated by antibodies prepared against human keratins. These observations indicate that T3 induces epidermal cell cultures from the hind limb of premetamorphic tadpoles to precociously synthesize water-insoluble proteins which (1) have Mrs and pIs similar to keratins from differentiating amphibian epidermal tissue and (2) are biochemically and immunologically similar to keratins associated with the differentiation of mammalian epidermal tissue.

  16. RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii.

    PubMed

    Yamasaki, Tomohito; Onishi, Masayuki; Kim, Eun-Jeong; Cerutti, Heriberto; Ohama, Takeshi

    2016-09-20

    Canonical microRNAs (miRNAs) are embedded in duplexed stem-loops in long precursor transcripts and are excised by sequential cleavage by DICER nuclease(s). In this miRNA biogenesis pathway, dsRNA-binding proteins play important roles in animals and plants by assisting DICER. However, these RNA-binding proteins are poorly characterized in unicellular organisms. Here we report that a unique RNA-binding protein, Dull slicer-16 (DUS16), plays an essential role in processing of primary-miRNA (pri-miRNA) transcripts in the unicellular green alga Chlamydomonas reinhardtii In animals and plants, dsRNA-binding proteins involved in miRNA biogenesis harbor two or three dsRNA-binding domains (dsRBDs), whereas DUS16 contains one dsRBD and also an ssRNA-binding domain (RRM). The null mutant of DUS16 showed a drastic reduction in most miRNA species. Production of these miRNAs was complemented by expression of full-length DUS16, but the expression of RRM- or dsRBD-truncated DUS16 did not restore miRNA production. Furthermore, DUS16 is predominantly localized to the nucleus and associated with nascent (unspliced form) pri-miRNAs and the DICER-LIKE 3 protein. These results suggest that DUS16 recognizes pri-miRNA transcripts cotranscriptionally and promotes their processing into mature miRNAs as a component of a microprocessor complex. We propose that DUS16 is an essential factor for miRNA production in Chlamydomonas and, because DUS16 is functionally similar to the dsRNA-binding proteins involved in miRNA biogenesis in animals and land plants, our report provides insight into this mechanism in unicellular eukaryotes. PMID:27582463

  17. [Research progress on free radicals in human body].

    PubMed

    Wang, Q B; Xu, F P; Wei, C X; Peng, J; Dong, X D

    2016-08-10

    Free radicals are the intermediates of metabolism, widely exist in the human bodies. Under normal circumstances, the free radicals play an important role in the metabolic process on human body, cell signal pathway, gene regulation, induction of cell proliferation and apoptosis, so as to maintain the normal growth and development of human body and to inhibit the growth of bacteria, virus and cancer. However, when organic lesion occurs affected by external factors or when equilibrium of the free radicals is tipped in the human body, the free radicals will respond integratedly with lipids, protein or nucleic acid which may jeopardize the health of human bodies. This paper summarizes the research progress of the free radicals conducted in recent years, in relations to the perspective of the types, origins, test methods of the free radicals and their relationship with human's health. In addition, the possible mechanisms of environmental pollutants (such as polycyclic aromatic hydrocarbons) mediating oxidative stress and free radicals scavenging in the body were also summarized.

  18. Mineral dust exposure and free radical-mediated lung damage

    SciTech Connect

    Doelman, C.J.; Leurs, R.; Oosterom, W.C.; Bast, A. )

    1990-01-01

    Chronic exposure to several types of mineral dust particles induces an inflammatory reaction in the lung. Dust particles activate alveolar macrophages and prime leukocytes (neutrophils, eosinophils, and basophils), leading to an enhanced release of reactive oxygen species. Sometimes mineral dust particles also contain radicals. Reactive oxygen species (superoxide anion radical, hydrogen peroxide, hydroxyl radical, and singlet oxygen) may lead to tissue damage. These are able to break DNA strands, to destroy proteins, and to induce the process of lipid peroxidation. The effects of oxygen radicals on the beta-adrenergic and muscarinic receptor response of the guinea pig and rat tracheal strip are described. The beta-adrenergic receptor response appeared to be more susceptible to oxidative stress than the muscarinic receptor response. This may lead to an autonomic imbalance on exposure to oxygen radicals. The lipid peroxidation product 4-hydroxy-2,3-trans-nonenal diminished the beta-adrenergic responsiveness in guinea pig tracheal preparations. Histologic examinations indicated that at low concentrations of cumene hydroperoxide (10(-4) M) the epithelial layer of rat trachea was already destroyed, whereas no effect on the muscarinic response was found. Oxygen radical-mediated damage in lung tissue may lead to lung emphysema, hyperresponsiveness, and hypersensitivity. Pharmacotherapeutic interventions that prevent initiation or propagation of these free radical reactions may have a beneficial effect in mineral dust-associated lung disease. 70 references.

  19. [Research progress on free radicals in human body].

    PubMed

    Wang, Q B; Xu, F P; Wei, C X; Peng, J; Dong, X D

    2016-08-10

    Free radicals are the intermediates of metabolism, widely exist in the human bodies. Under normal circumstances, the free radicals play an important role in the metabolic process on human body, cell signal pathway, gene regulation, induction of cell proliferation and apoptosis, so as to maintain the normal growth and development of human body and to inhibit the growth of bacteria, virus and cancer. However, when organic lesion occurs affected by external factors or when equilibrium of the free radicals is tipped in the human body, the free radicals will respond integratedly with lipids, protein or nucleic acid which may jeopardize the health of human bodies. This paper summarizes the research progress of the free radicals conducted in recent years, in relations to the perspective of the types, origins, test methods of the free radicals and their relationship with human's health. In addition, the possible mechanisms of environmental pollutants (such as polycyclic aromatic hydrocarbons) mediating oxidative stress and free radicals scavenging in the body were also summarized. PMID:27539355

  20. Obesity-associated proinflammatory cytokines increase calcium sensing receptor (CaSR) protein expression in primary human adipocytes and LS14 human adipose cell line.

    PubMed

    Cifuentes, Mariana; Fuentes, Cecilia; Mattar, Pamela; Tobar, Nicolas; Hugo, Eric; Ben-Jonathan, Nira; Rojas, Cecilia; Martínez, Jorge

    2010-08-15

    Obesity-associated health complications are thought to be in part due to the low-grade proinflammatory state that characterizes this disease. The calcium sensing receptor (CaSR), which is expressed in human adipose cells, plays an important role in diseases involving inflammation. To assess the relevance of this protein in adipose pathophysiology, we evaluated its expression in adipocytes under obesity-related proinflammatory conditions. As in primary adipose cells, we established that LS14, a recently described human adipose cell line, expresses the CaSR. Differentiated LS14 and primary adipose cells were exposed overnight to cytokines typically involved in obesity-related inflammation (interleukin (IL)1beta, IL6 and tumor necrosis factor (TNF)alpha). The cytokines increased CaSR abundance in differentiated adipocytes. We incubated LS14 cells with medium previously conditioned (CM) by adipose tissue from subjects with a wide range of body mass index (BMI). Cells exposed to CM from subjects of higher BMI underwent a greater increase in CaSR protein, likely resulting from the greater proinflammatory cytokines secreted from obese tissue. Our observations that proinflammatory factors increase CaSR levels in adipocytes, and the reported ability of CaSR to elevate cytokine levels, open new aspects in the study of obesity inflammatory state pathophysiology, providing a potential novel therapeutic prevention and treatment target.

  1. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

    PubMed Central

    Eidet, J. R.; Reppe, S.; Pasovic, L.; Olstad, O. K.; Lyberg, T.; Khan, A. Z.; Fostad, I. G.; Chen, D. F.; Utheim, T. P.

    2016-01-01

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

  2. Primary structure of bovine pituitary secretory protein I (chromogranin A) deduced from the cDNA sequence

    SciTech Connect

    Ahn, T.G.; Cohn, D.V.; Gorr, S.U.; Ornstein, D.L.; Kashdan, M.A.; Levine, M.A.

    1987-07-01

    Secretory protein I (SP-I), also referred to as chromogranin A, is an acidic glycoprotein that has been found in every tissue of endocrine and neuroendocrine origin examined but never in exocrine or epithelial cells. Its co-storage and co-secretion with peptide hormones and neurotransmitters suggest that it has an important endocrine or secretory function. The authors have isolated cDNA clones from a bovine pituitary lambdagt11 expression library using an antiserum to parathyroid SP-I. The largest clone (SP4B) hybridized to a transcript of 2.1 kilobases in RNA from parathyroid, pituitary, and adrenal medulla. Immunoblots of bacterial lysates derived from SP4B lysognes demonstrated specific antibody binding to an SP4B/..beta..-galactosidase fusion protein (160 kDa) with a cDNA-derived component of 46 kDa. Radioimmunoassay of the bacterial lystates with SP-I antiserum yielded parallel displacement curves of /sup 125/I-labeled SP-I by the SP4B lysate and authentic SP-I. SP4B contains a cDNA of 1614 nucleotides that encodes a 449-amino acid protein (calculated mass, 50 kDa). The nucleotide sequences of the pituitary SP-I cDNA and adrenal medullary SP-I cDNAs are nearly identical. Analysis of genomic DNA suggests that pituitary, adrenal, and parathyroid SP-I are products of the same gene.

  3. Apolipoprotein A-I, an antimicrobial protein in Oncorhynchus mykiss: evaluation of its expression in primary defence barriers and plasma levels in sick and healthy fish.

    PubMed

    Villarroel, Franz; Bastías, Adriana; Casado, Alin; Amthauer, Rodolfo; Concha, Margarita I

    2007-07-01

    Antimicrobial proteins and peptides play an important role in the primary defence barriers in vertebrates and invertebrates. In a previous study it was shown that high-density lipoprotein (HDL) and its major apolipoproteins, ApoA-I and ApoA-II display antimicrobial activity in the carp (Cyprinus carpio L.). The aim of this study was to evaluate if ApoA-I conserves this defensive function in a salmonid fish like the rainbow trout, in spite of the low level of primary sequence conservation between fish ApoA-I. Here it is shown that trout ApoA-I displays an antimicrobial activity in the micromolar range against Gram positive and Gram negative bacteria, including some fish pathogens. In addition, its expression was also demonstrated by immunohistochemistry and RT-PCR in epidermis, gills and intestinal mucosa, which constitute the main primary defence barriers in fish. Finally, no significant difference in the hepatic expression and plasma levels of this abundant apolipoprotein was found in groups of healthy and diseased fish, in clear contrast with mammals where ApoA-I have been considered a negative acute phase reactant. These findings suggest that ApoA-I could constitute an important innate immunity effector in trout and perhaps other teleost fish. PMID:17391986

  4. Dexamethasone potentiates in vitro blood-brain barrier recovery after primary blast injury by glucocorticoid receptor-mediated upregulation of ZO-1 tight junction protein.

    PubMed

    Hue, Christopher D; Cho, Frances S; Cao, Siqi; Dale Bass, Cameron R; Meaney, David F; Morrison, Barclay

    2015-07-01

    Owing to the frequent incidence of blast-induced traumatic brain injury (bTBI) in recent military conflicts, there is an urgent need to develop effective therapies for bTBI-related pathologies. Blood-brain barrier (BBB) breakdown has been reported to occur after primary blast exposure, making restoration of BBB function and integrity a promising therapeutic target. We tested the hypothesis that treatment with dexamethasone (DEX) after primary blast injury potentiates recovery of an in vitro BBB model consisting of mouse brain endothelial cells (bEnd.3). DEX treatment resulted in complete recovery of transendothelial electrical resistance and hydraulic conductivity 1 day after injury, compared with 3 days for vehicle-treated injured cultures. Administration of RU486 (mifepristone) inhibited effects of DEX, confirming that barrier restoration was mediated by glucocorticoid receptor signaling. Potentiated recovery with DEX treatment was accompanied by stronger zonula occludens (ZO)-1 tight junction immunostaining and expression, suggesting that increased ZO-1 expression was a structural correlate to BBB recovery after blast. Interestingly, augmented ZO-1 protein expression was associated with specific upregulation of the α(+) isoform but not the α(-) isoform. This is the first study to provide a mechanistic basis for potentiated functional recovery of an in vitro BBB model because of glucocorticoid treatment after primary blast injury.

  5. Studies of radiation-produced radicals and radical ions

    SciTech Connect

    Williams, T.F.

    1991-01-01

    The radiolytic oxidation of anti-5-methylbicyclo(2.1.0)pentane gives the 1-methylcyclopentene radical cation as the sole rearrangement product H migration whereas oxidation of its syn isomer results in the highly selective formation of the 3-methylcyclopentene radical cation by methyl group migration. Since exactly the same stereoselectivity of olefin formation was observed in corresponding PET (photosensitized electron transfer) studies in the liquid phase, it is concluded that the rearrangement in this case also occurs through the intermediacy of radical cations. Clearly, the radical cation rearrangement must occur very rapidly (10{sup {minus}8}--10{sup {minus}9}s) under liquid-phase conditions at room temperature to compete with back electron transfer, and therefore the hydrogen (or methyl) migration is a fast process under these conditions. An intramolecular cycloaddition reaction was demonstrated in the radical cation rearrangement of 4-vinylcyclohexene to bicyclo(3.2.1)oct-2-ene. ESR studies show that the radiolytic oxidation of quadricyclane in Freon matrices under conditions of high substrate dilution leads to the bicyclo(3.2.0)hepta-2,6-diene radical cation as well as the previously reported norbornadiene radical cation, the former species predominating at sufficiently low concentrations.

  6. The Nectin-4/Afadin Protein Complex and Intercellular Membrane Pores Contribute to Rapid Spread of Measles Virus in Primary Human Airway Epithelia

    PubMed Central

    Singh, Brajesh K.; Hornick, Andrew L.; Krishnamurthy, Sateesh; Locke, Anna C.; Mendoza, Crystal A.; Mateo, Mathieu; Miller-Hunt, Catherine L.; Cattaneo, Roberto

    2015-01-01

    ABSTRACT The discovery that measles virus (MV) uses the adherens junction protein nectin-4 as its epithelial receptor provides a new vantage point from which to characterize its rapid spread in the airway epithelium. We show here that in well-differentiated primary cultures of airway epithelial cells from human donors (HAE), MV infectious centers form rapidly and become larger than those of other respiratory pathogens: human respiratory syncytial virus, parainfluenza virus 5, and Sendai virus. While visible syncytia do not form after MV infection of HAE, the cytoplasm of an infected cell suddenly flows into an adjacent cell, as visualized through wild-type MV-expressed cytoplasmic green fluorescent protein (GFP). High-resolution video microscopy documents that GFP flows through openings that form on the lateral surfaces between columnar epithelial cells. To assess the relevance of the protein afadin, which connects nectin-4 to the actin cytoskeleton, we knocked down its mRNA. This resulted in more-limited infectious-center formation. We also generated a nectin-4 mutant without the afadin-binding site in its cytoplasmic tail. This mutant was less effective than wild-type human nectin-4 at promoting MV infection in primary cultures of porcine airway epithelia. Thus, in airway epithelial cells, MV spread requires the nectin-4/afadin complex and is based on cytoplasm transfer between columnar cells. Since the viral membrane fusion apparatus may open the passages that allow cytoplasm transfer, we refer to them as intercellular membrane pores. Virus-induced intercellular pores may contribute to extremely efficient measles contagion by promoting the rapid spread of the virus through the upper respiratory epithelium. IMPORTANCE Measles virus (MV), while targeted for eradication, still causes about 120,000 deaths per year worldwide. The recent reemergence of measles in insufficiently vaccinated populations in Europe and North America reminds us that measles is extremely

  7. Bureaucratic Activism and Radical School Change in Tamil Nadu, India

    ERIC Educational Resources Information Center

    Niesz, Tricia; Krishnamurthy, Ramchandar

    2013-01-01

    In 2007, Activity Based Learning (ABL), a child-centered, activity-based method of pedagogical practice, transformed classrooms in all of the over 37,000 primary-level government schools in Tamil Nadu, India. The large scale, rapid pace, and radical nature of educational change sets the ABL initiative apart from most school reform efforts.…

  8. The NS1 protein of a human influenza virus inhibits type I interferon production and the induction of antiviral responses in primary human dendritic and respiratory epithelial cells.

    PubMed

    Haye, Kester; Burmakina, Svetlana; Moran, Thomas; García-Sastre, Adolfo; Fernandez-Sesma, Ana

    2009-07-01

    The NS1 protein of the influenza A virus is a potent virulence factor that inhibits type I interferon (IFN) synthesis, allowing the virus to overcome host defenses and replicate efficiently. However, limited studies have been conducted on NS1 function using human virus strains and primary human cells. We used NS1 truncated mutant influenza viruses derived from the human isolate influenza A/TX/91 (TX WT, where WT is wild type) to study the functions of NS1 in infected primary cells. Infection of primary differentiated human tracheo-bronchial epithelial cells with an NS1 truncated mutant demonstrated limited viral replication and enhanced type I IFN induction. Additionally, human dendritic cells (DCs) infected with human NS1 mutant viruses showed higher levels of activation and stimulated naïve T-cells better than TX WT virus-infected DCs. We also compared infections of DCs with TX WT and our previously characterized laboratory strain A/PR/8/34 (PR8) and its NS1 knockout strain, deltaNS1. TX WT-infected DCs displayed higher viral replication than PR8 but had decreased antiviral gene expression at late time points and reduced naïve T-cell stimulation compared to PR8 infections, suggesting an augmented inhibition of IFN production and human DC activation. Our findings show that human-derived influenza A viruses have a high capacity to inhibit the antiviral state in a human system, and here we have evaluated the possible mechanism of this inhibition. Lastly, C-terminal truncations in the NS1 protein of human influenza virus are sufficient to make the virus attenuated and more immunogenic, supporting its use as a live attenuated influenza vaccine in humans.

  9. Discovery, Primary, and Crystal Structures and Capacitation-related Properties of a Prostate-derived Heparin-binding Protein WGA16 from Boar Sperm*

    PubMed Central

    Garénaux, Estelle; Kanagawa, Mayumi; Tsuchiyama, Tomoyuki; Hori, Kazuki; Kanazawa, Takeru; Goshima, Ami; Chiba, Mitsuru; Yasue, Hiroshi; Ikeda, Akemi; Yamaguchi, Yoshiki; Sato, Chihiro; Kitajima, Ken

    2015-01-01

    Mammalian sperm acquire fertility through a functional maturation process called capacitation, where sperm membrane molecules are drastically remodeled. In this study, we found that a wheat germ agglutinin (WGA)-reactive protein on lipid rafts, named WGA16, is removed from the sperm surface on capacitation. WGA16 is a prostate-derived seminal plasma protein that has never been reported and is deposited on the sperm surface in the male reproductive tract. Based on protein and cDNA sequences for purified WGA16, it is a homologue of human zymogen granule protein 16 (ZG16) belonging to the Jacalin-related lectin (JRL) family in crystal and primary structures. A glycan array shows that WGA16 binds heparin through a basic patch containing Lys-53/Lys-73 residues but not the conventional lectin domain of the JRL family. WGA16 is glycosylated, contrary to other ZG16 members, and comparative mass spectrometry clearly shows its unique N-glycosylation profile among seminal plasma proteins. It has exposed GlcNAc and GalNAc residues without additional Gal residues. The GlcNAc/GalNAc residues can work as binding ligands for a sperm surface galactosyltransferase, which actually galactosylates WGA16 in situ in the presence of UDP-Gal. Interestingly, surface removal of WGA16 is experimentally induced by either UDP-Gal or heparin. In the crystal structure, N-glycosylated sites and a potential heparin-binding site face opposite sides. This geography of two functional sites suggest that WGA16 is deposited on the sperm surface through interaction between its N-glycans and the surface galactosyltransferase, whereas its heparin-binding domain may be involved in binding to sulfated glycosaminoglycans in the female tract, enabling removal of WGA16 from the sperm surface. PMID:25568322

  10. Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

    PubMed Central

    Maj, Magdalena; Milenkovic, Ivan; Bauer, Jan; Berggård, Tord; Veit, Martina; Ilhan-Mutlu, Aysegül; Wagner, Ludwig; Tretter, Verena

    2012-01-01

    Secretagogin is a calcium binding protein (CBP) highly expressed in neuroendocrine cells. It has been shown to be involved in insulin secretion from pancreatic beta cells and is a strong candidate as a biomarker for endocrine tumors, stroke, and eventually psychiatric conditions. Secretagogin has been hypothesized to exert a neuroprotective role in neurodegenerative diseases like Alzheimer’s disease. The expression pattern of Secretagogin is not conserved from rodents to humans. We used brain tissue and primary neuronal cell cultures from rat to further characterize this CBP in rodents and to perform a few functional assays in vitro. Immunohistochemistry on rat brain slices revealed a high density of Secretagogin-positive cells in distinct brain regions. Secretagogin was found in the cytosol or associated with subcellular compartments. We tested primary neuronal cultures for their suitability as model systems to further investigate functional properties of Secretagogin. These cultures can easily be manipulated by treatment with drugs or by transfection with test constructs interfering with signaling cascades that might be linked to the cellular function of Secretagogin. We show that, like in pancreatic beta cells and insulinoma cell lines, also in neurons the expression level of Secretagogin is dependent on extracellular insulin and glucose. Further, we show also for rat brain neuronal tissue that Secretagogin interacts with the microtubule-associated protein Tau and that this interaction is dependent on Ca2+. Future studies should aim to study in further detail the molecular properties and function of Secretagogin in individual neuronal cell types, in particular the subcellular localization and trafficking of this protein and a possible active secretion by neurons. PMID:22888312

  11. Aquaporin 4-dependent expression of glial fibrillary acidic protein and tenascin-C in activated astrocytes in stab wound mouse brain and in primary culture.

    PubMed

    Ikeshima-Kataoka, Hiroko; Abe, Yoichiro; Yasui, Masato

    2015-01-01

    We previously reported that aquaporin 4 (AQP4) has a neuroimmunological function via astrocytes and microglial cells involving osteopontin. AQP4 is a water channel localized in the endofoot of astrocytes in the brain, and its expression is upregulated after a stab wound to the mouse brain or the injection of methylmercury in common marmosets. In this study, the correlation between the expression of AQP4 and the expression of glial fibrillary acidic protein (GFAP) or tenascin-C (TN-C) in reactive astrocytes was examined in primary cultures and brain tissues of AQP4-deficient mice (AQP4/KO). In the absence of a stab wound to the brain or of any stimulation of the cells, the expressions of both GFAP and TN-C were lower in astrocytes from AQP4/KO mice than in those from wild-type (WT) mice. High levels of GFAP and TN-C expression were observed in activated astrocytes after a stab wound to the brain in WT mice; however, the expressions of GFAP and TN-C were insignificant in AQP4/KO mice. Furthermore, lipopolysaccharide (LPS) stimulation activated primary culture of astrocytes and upregulated GFAP and TN-C expression in cells from WT mice, whereas the expressions of GFAP and TN-C were slightly upregulated in cells from AQP4/KO mice. Moreover, the stimulation of primary culture of astrocytes with LPS also upregulated inflammatory cytokines in cells from WT mice, whereas modest increases were observed in cells from AQP4/KO mice. These results suggest that AQP4 expression accelerates GFAP and TN-C expression in activated astrocytes induced by a stab wound in the mouse brain and LPS-stimulated primary culture of astrocytes.

  12. Ion implanted, radical-rich surfaces for the rapid covalent immobilization of active biomolecules

    NASA Astrophysics Data System (ADS)

    Hirsh, Stacey L.; Bilek, Marcela M. M.; Bax, Daniel V.; Kondyurin, Alexey; Kosobrodova, Elena; Tsoutas, Kostadinos; Tran, Clara T. H.; Waterhouse, Anna; Yin, Yongbai; Nosworthy, Neil J.; McKenzie, David R.; dos Remedios, Christobal G.; Ng, Martin K. C.; Weiss, Anthony S.

    2013-04-01

    Protein immobilization through the use of direct radical induced covalent coupling is described. Ions implanted in a polymer surface generate a highly cross-linked surface layer that is rich in radicals. These radicals can diffuse to the surface and covalently immobilize physically adsorbed proteins, as illustrated in a kinetic model for the covalent attachment process. Radical induced covalent coupling provides rapid covalent attachment, while also retaining native protein conformation and enabling control over the composition of the adsorbed protein layer when adsorbed from a protein mixture. Advantages of using this method for improving the biocompatibility of implanted biomedical devices and for immobilizing antibodies in protein microarrays for disease diagnosis and early detection are highlighted.

  13. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  14. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  15. Interferon gamma-inducible protein 16 (IFI16) and anti-IFI16 antibodies in primary Sjögren's syndrome: findings in serum and minor salivary glands.

    PubMed

    Alunno, A; Caneparo, V; Carubbi, F; Bistoni, O; Caterbi, S; Gariglio, M; Bartoloni, E; Landolfo, S; Gerli, R

    2015-01-01

    The interferon (IFN) signature, namely the overexpression of IFN-inducible genes is a crucial aspect in the pathogenesis of primary Sjögren's syndrome (pSS). The IFN-inducible IFI16 protein, normally expressed in cell nuclei, may be overexpressed, mislocalized in the cytoplasm and secreted in the extracellular milieu in several autoimmune disorders including pSS. This leads to tolerance breaking to this self-protein and development of anti-IFI16 antibodies. The aim of this study was to identify pathogenic and clinical significance of IFI16 and anti-IFI16 autoantibodies in pSS. IFI16 and anti-IFI16 were assessed in the serum of 30 pSS patients and one-hundred healthy donors (HD) by ELISA. IFI16 was also evaluated in 5 minor salivary glands (MSGs) of pSS patients and 5 MSGs of non-pSS patients with sicca symptoms by immunohistochemistry. Normal MSGs do not constitutively express IFI16. Conversely, in pSS-MSGs a marked expression and cytoplasmic mislocalization of IFI16 by epithelial cells was observed with infiltrations in lymphocytes and peri/ intra-lesional endothelium. pSS patients display higher serum levels of both IFI16 and anti-IFI16 autoantibodies compared to HD. Our data suggest that IFI16 protein may be involved in the initiation and perpetuation of glandular inflammation occurring in pSS. PMID:26876186

  16. Contribution of the R-Ras2 GTP-binding protein to primary breast tumorigenesis and late-stage metastatic disease

    NASA Astrophysics Data System (ADS)

    Larive, Romain M.; Moriggi, Giulia; Menacho-Márquez, Mauricio; Cañamero, Marta; Álava, Enrique De; Alarcón, Balbino; Dosil, Mercedes; Bustelo, Xosé R.

    2014-05-01

    R-Ras2 is a transforming GTPase that shares downstream effectors with Ras subfamily proteins. However, little information exists about the function of this protein in tumorigenesis and its signalling overlap with classical Ras GTPases. Here we show, by combining loss- and gain-of-function studies in breast cancer cells, mammary epithelial cells and mouse models, that endogenous R-Ras2 has a role in both primary breast tumorigenesis and the late metastatic steps of cancer cells in the lung parenchyma. R-Ras2 drives tumorigenesis in a phosphatidylinostiol-3 kinase (PI3K)-dependent and signalling autonomous manner. By contrast, its prometastatic role requires other priming oncogenic signals and the engagement of several downstream elements. R-Ras2 function is required even in cancer cells exhibiting constitutive activation of classical Ras proteins, indicating that these GTPases are not functionally redundant. Our results also suggest that application of long-term R-Ras2 therapies will result in the development of compensatory mechanisms in breast tumours.

  17. Free radicals and male reproduction.

    PubMed

    Agarwal, Ashok; Allamaneni, Shyam S R

    2011-03-01

    Male factor accounts for almost 50% cases of infertility. The exact mechanism of sperm dysfunction is not known in many cases. Extensive research in the last decade has led to the identification of free radicals (reactive oxygen species) as mediators of sperm dysfunction in both specific diagnoses and idiopathic cases of male infertility. Elevated levels of reactive oxygen species are seen in up to 30-80% of men with male infertility. The role of free radicals has been studied extensively in the process of human reproduction. We know now that a certain level of free radicals is necessary for normal sperm function, whereas an excessive level of free radicals can cause detrimental effect on sperm function and subsequent fertilisation and offspring health. Oxidative stress develops when there is an imbalance between generation of free radicals and scavenging capacity of anti-oxidants in reproductive tract. Oxidative stress has been shown to affect both standard semen parameters and fertilising capacity. In addition, high levels of free radicals have been associated with lack of or poor fertility outcome after natural conception or assisted reproduction. Diagnostic techniques to quantify free radicals in infertile patients can assist physicians treating patients with infertility to plan for proper treatment strategies. In vivo anti-oxidants can be used against oxidative stress in male reproductive tract. Supplementation of in vitro anti-oxidants can help prevent the oxidative stress during sperm preparation techniques in assisted reproduction.

  18. Radiation-induced radicals in different polymorphic modifications of D-mannitol: Structure, conformations and dosimetric implications

    NASA Astrophysics Data System (ADS)

    Sosulin, Ilya S.; Shiryaeva, Ekaterina S.; Feldman, Vladimir I.

    2015-12-01

    The structure and conformation of radicals produced by X-ray irradiation of three polymorphic forms of D-mannitol were investigated using EPR spectroscopy. In all the cases, primary species were identified as radicals resulting from hydrogen abstraction from position 3 or 4 of the mannitol molecule. It was found that molecular packing in crystals of different polymorphic modifications had noticeable effect on the conformation of radicals observed after irradiation at room temperature and the dehydration of the primary radicals occurring at 400 K. The radicals trapped in stable modifications (β- and δ-forms) were found to be very stable at room temperature. Relatively high radical yields and remarkable stability of radicals suggest that D-mannitol can be used as an EPR dosimeter or irradiation marker.

  19. Primary photochemistry of the dark- and light-adapted states of the YtvA protein from Bacillus subtilis.

    PubMed

    Song, Sang-Hun; Madsen, Dorte; van der Steen, Jeroen B; Pullman, Robert; Freer, Lucy H; Hellingwerf, Klaas J; Larsen, Delmar S

    2013-11-12

    The primary (100 fs to 10 ns) and secondary (10 ns to 100 μs) photodynamics in the type II light-oxygen-voltage (LOV) domain from the blue light YtvA photoreceptor extracted from Bacillus subtilis were explored with transient absorption spectroscopy. The photodynamics of full-length YtvA were characterized after femtosecond 400 nm excitation of both the dark-adapted D447 state and the light-adapted S390 state. The S390 state relaxes on a 43 min time scale at room temperature back into D447, which is weakly accelerated by the introduction of imidazole. This is ascribed to an obstructed cavity in YtvA that hinders access to the embedded FMN chromophore and is more open in type I LOV domains. The primary photochemistry of dark-adapted YtvA is qualitatively similar to that of the type I LOV domains, including AsLOV2 from Avena sativa, but exhibits an appreciably higher (60% greater) terminal triplet yield, estimated near the maximal ΦISC value of ≈78%; the other 22% decays via non-triplet-generating fluorescence. The subsequent secondary dynamics are inhomogeneous, with three triplet populations co-evolving: the faster-decaying (I)T* population (38% occupancy) with a 200 ns decay time is nonproductive in generating the S390 adduct state, a slower (II)T* population (57% occupancy) exhibits a high yield (Φadduct ≈ 100%) in generating S390 and a third (5%) (III)T*population persists (>100 μs) with unresolved photoactivity. The ultrafast photoswitching dynamics of the S390 state appreciably differ from those previously resolved for the type I AcLOV2 domain from Adiantum capillus-veneris [Kennis, J. T., et al. (2004) J. Am. Chem. Soc. 126, 4512], with a low-yield dissociation (Φdis ≈ 2.5%) reaction, which is due to an ultrafast recombination reaction, following photodissociation, and is absent in AcLOV2, which results in the increased photoswitching activity of the latter domain.

  20. Future Directions of Structural Mass Spectrometry using Hydroxyl Radical Footprinting

    SciTech Connect

    J Kiselar; M Chance

    2011-12-31

    Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein-protein and protein-DNA interactions. Using synchrotron radiolysis, exposure of proteins to a 'white' X-ray beam for milliseconds provides sufficient oxidative modification to surface amino acid side chains, which can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time-resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium-dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular subdomains have similar rates of conformational change. These findings demonstrate that time-resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review, we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that the selected reaction monitoring (SRM)-based method can be utilized for quantification of oxidized species, improving the signal-to-noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis

  1. Peroxy radical partitioning during the AMMA radical intercomparison exercise

    NASA Astrophysics Data System (ADS)

    Andrés-Hernández, M. D.; Stone, D.; Brookes, D. M.; Commane, R.; Reeves, C. E.; Huntrieser, H.; Heard, D. E.; Monks, P. S.; Burrows, J. P.; Schlager, H.; Kartal, D.; Evans, M. J.; Floquet, C. F. A.; Ingham, T.; Methven, J.; Parker, A. E.

    2010-11-01

    Peroxy radicals were measured onboard two scientific aircrafts during the AMMA (African Monsoon Multidisciplinary Analysis) campaign in summer 2006. This paper reports results from the flight on 16 August 2006 during which measurements of HO2 by laser induced fluorescence spectroscopy at low pressure (LIF-FAGE) and total peroxy radicals (RO2* = HO2+ΣRO2, R = organic chain) by two similar instruments based on the peroxy radical chemical amplification (PeRCA) technique were subject of a blind intercomparison. The German DLR-Falcon and the British FAAM-BAe-146 flew wing tip to wing tip for about 30 min making concurrent measurements on 2 horizontal level runs at 697 and 485 hPa over the same geographical area in Burkina Faso. A full set of supporting measurements comprising photolysis frequencies, and relevant trace gases like CO, NO, NO2, NOy, O3 and a wider range of VOCs were collected simultaneously. Results are discussed on the basis of the characteristics and limitations of the different instruments used. Generally, no data bias are identified and the RO2* data available agree quite reasonably within the instrumental errors. The [RO2*]/[HO2] ratios, which vary between 1:1 and 3:1, as well as the peroxy radical variability, concur with variations in photolysis rates and in other potential radical precursors. Model results provide additional information about dominant radical formation and loss processes.

  2. Peroxy radical partitioning during the AMMA radical intercomparison exercise

    NASA Astrophysics Data System (ADS)

    Andrés-Hernández, M. D.; Stone, D.; Brookes, D. M.; Commane, R.; Reeves, C. E.; Huntrieser, H.; Heard, D. E.; Monks, P. S.; Burrows, J. P.; Schlager, H.; Kartal, D.; Evans, M. J.; Floquet, C. F. A.; Ingham, T.; Methven, J.; Parker, A. E.

    2010-04-01

    Peroxy radicals were measured onboard two scientific aircrafts during the AMMA (African Monsoon Multidisciplinary Analysis) campaign in summer 2006. This paper reports results from the flight on 16 August 2006 during which measurements of HO2 by laser induced fluorescence spectroscopy at low pressure (LIF-FAGE) and total peroxy radicals (RO2*=HO2+ΣRO2, R= organic chain) by two similar instruments based on the peroxy radical chemical amplification (PerCA) technique were subject of a blind intercomparison. The German DLR-Falcon and the British FAAM-BAe-146 flew wing tip to wing tip for about 30 min making concurrent measurements on 2 horizontal level runs at 697 and 485 hPa over the same geographical area in Burkina Faso. A full set of supporting measurements comprising photolysis frequencies, and relevant trace gases like CO, NO, NO2, NOy, O3 and a wider range of VOCs were collected simultaneously. Results are discussed on the basis of the characteristics and limitations of the different instruments used. Generally, no data bias are identified and the RO2* data available agree quite reasonably within the instrumental errors. The [RO2*]/[HO2] ratios, which vary between 1:1 and 3:1, as well as the peroxy radical variability, concur with variations in photolysis rates and in other potential radical precursors. Model results provide additional information about dominant radical formation and loss processes.

  3. Spin relaxation of radicals in cryptochrome and its role in avian magnetoreception.

    PubMed

    Worster, Susannah; Kattnig, Daniel R; Hore, P J

    2016-07-21

    Long-lived spin coherence and rotationally ordered radical pairs have previously been identified as key requirements for the radical pair mechanism of the avian magnetic compass sense. Both criteria are hard to meet in a biological environment, where thermal motion of the radicals creates dynamic disorder and drives efficient spin relaxation. This has long been cited as a major stumbling block of the radical pair hypothesis. Here we combine Redfield relaxation theory with analytical solutions to a rotational diffusion equation to assess the impact of restricted rotational motion of the radicals on the operation of the compass. The effects of such motions are first investigated generally in small, model systems and are then critically examined in the magnetically sensitive flavin-tryptophan radical pair that is formed photochemically in the proposed magnetoreceptor protein, cryptochrome. We conclude that relaxation is slowest when rotational motion of the radicals within the protein is fast and highly constrained; that in a regime of slow relaxation, the motional averaging of hyperfine interactions has the potential to improve the sensitivity of the compass; and that consideration of motional effects can significantly alter the design criteria for an optimal compass. In addition, we demonstrate that motion of the flavin radical is likely to be compatible with its role as a component of a functioning radical-pair compass, whereas the motion of the tryptophan radical is less ideal, unless it is particularly fast. PMID:27448908

  4. Spin relaxation of radicals in cryptochrome and its role in avian magnetoreception

    NASA Astrophysics Data System (ADS)

    Worster, Susannah; Kattnig, Daniel R.; Hore, P. J.

    2016-07-01

    Long-lived spin coherence and rotationally ordered radical pairs have previously been identified as key requirements for the radical pair mechanism of the avian magnetic compass sense. Both criteria are hard to meet in a biological environment, where thermal motion of the radicals creates dynamic disorder and drives efficient spin relaxation. This has long been cited as a major stumbling block of the radical pair hypothesis. Here we combine Redfield relaxation theory with analytical solutions to a rotational diffusion equation to assess the impact of restricted rotational motion of the radicals on the operation of the compass. The effects of such motions are first investigated generally in small, model systems and are then critically examined in the magnetically sensitive flavin-tryptophan radical pair that is formed photochemically in the proposed magnetoreceptor protein, cryptochrome. We conclude that relaxation is slowest when rotational motion of the radicals within the protein is fast and highly constrained; that in a regime of slow relaxation, the motional averaging of hyperfine interactions has the potential to improve the sensitivity of the compass; and that consideration of motional effects can significantly alter the design criteria for an optimal compass. In addition, we demonstrate that motion of the flavin radical is likely to be compatible with its role as a component of a functioning radical-pair compass, whereas the motion of the tryptophan radical is less ideal, unless it is particularly fast.

  5. Cadmium-induced activation of stress signaling pathways, disruption of ubiquitin-dependent protein degradation and apoptosis in primary rat Sertoli cell-gonocyte cocultures.

    PubMed

    Yu, Xiaozhong; Hong, Sungwoo; Faustman, Elaine M

    2008-08-01

    Cadmium (Cd) is a ubiquitous environmental pollutant that has been associated with male reproductive toxicity in both humans and animal models. The underlying mechanism of this response, however, is still uncharacterized. To address this issue, we employed a recently developed and optimized three-dimensional primary Sertoli cell-gonocyte coculture system and examined the time- and dose-dependent effects of Cd on morphological alterations, cell viability, activation of stress signaling pathway proteins, and the disruption of the ubiquitin proteasome system (UPS). Our results demonstrated that Cd exposure lead to time- and dose-dependent morphological changes that are associated with the induction of apoptosis. In response to Cd, we also saw a disruption of the UPS as evaluated through the accumulation of high-molecular weight polyubiquitinated proteins (HMW-polyUb) as well as alterations in proteasome activity. Robust activation of cellular stress response, measured through the increased phosphorylation of stress-activated protein kinase/c-jun N-terminal kinase and p38, paralleled the accumulation of HMW-polyUb. In addition, p53, a key regulatory protein, was upregulated and underwent increased ubiquitination in response to Cd. To further characterize the role of the UPS in Cd cellular response, we compared the above changes with two classic proteasomal inhibitors, lactacystin, and MG132. The stress response and the accumulation of HWM-polyUb induced by Cd were consistent with the response seen with MG132 but not with lactacystin. In addition, Cd treatment resulted in a dose- and time-dependent effect on proteasome activity, but the overall Cd-induced proteasomal inhibition was unique as compared to MG132 and lactacystin. Taken together, our studies further characterize Cd-induced in vitro testicular toxicity and highlight the potential role of the UPS in this response. PMID:18463101

  6. Radical-driven peptide backbone dissociation tandem mass spectrometry.

    PubMed

    Oh, Han Bin; Moon, Bongjin

    2015-01-01

    In recent years, a number of novel tandem mass spectrometry approaches utilizing radical-driven peptide gas-phase fragmentation chemistry have been developed. These approaches show a peptide fragmentation pattern quite different from that of collision-induced dissociation (CID). The peptide fragmentation features of these approaches share some in common with electron capture dissociation (ECD) or electron transfer dissociation (ETD) without the use of sophisticated equipment such as a Fourier-transform mass spectrometer. For example, Siu and coworkers showed that CID of transition metal (ligand)-peptide ternary complexes led to the formation of peptide radical ions through dissociative electron transfer (Chu et al., 2000. J Phys Chem B 104:3393-3397). The subsequent collisional activation of the generated radical ions resulted in a number of characteristic product ions, including a, c, x, z-type fragments and notable side-chain losses. Another example is the free radical initiated peptide sequencing (FRIPS) approach, in which Porter et al. and Beauchamp et al. independently introduced a free radical initiator to the primary amine group of the lysine side chain or N-terminus of peptides (Masterson et al., 2004. J Am Chem Soc 126:720-721; Hodyss et al., 2005 J Am Chem Soc 127: 12436-12437). Photodetachment of gaseous multiply charged peptide anions (Joly et al., 2008. J Am Chem Soc 130:13832-13833) and UV photodissociation of photolabile radical precursors including a C-I bond (Ly & Julian, 2008. J Am Chem Soc 130:351-358; Ly & Julian, 2009. J Am Soc Mass Spectrom 20:1148-1158) also provide another route to generate radical ions. In this review, we provide a brief summary of recent results obtained through the radical-driven peptide backbone dissociation tandem mass spectrometry approach.

  7. The dopamine transporter protein gene (SLC6A3): Primary linage mapping and linkage studies in Tourette syndrome

    SciTech Connect

    Gelernter, J.; Kruger, S.D.; Pakstis, A.J. |

    1995-12-10

    The dopamine transporter, the molecule responsible for presynaptic reuptake of dopamine and a major site of action of psychostimulant drugs, including cocaine, is encoded by locus SLC6A3 (alias DAT1). The protein`s actions and DAT`s specific localization to dopaminergic neurons make it a candidate gene for several psychiatric illnesses. SLC6A3 has been mapped to distal chromosome 5p, using physical methods. Genetic linkage methods were used to place SLC6A3 in the genetic linkage map. Four extended pedigrees (one of which overlaps with CEPH) were typed. Linkage with Tourette syndrome (TS) was also examined. SLC6A3 showed close linkage with several markers previously mapped to distal chromosome 5p, including D5S11 (Z{sub max} = 16.0, {theta}{sub M} = {theta}{sub F} = 0.03, results from four families) and D5S678 (Z{sub max} = 7.84, {theta}{sub M} = {theta}{sub F} = 0, results from two families). Observed crossovers established that SLC6A3 is a distal marker close to D5S10 and D5S678, but these three distal markers could not be ordered. Linkage between TS and SLC6A3 could be excluded independently in two branches of a large kindred segregating TS; the lod score in a third family was also negative, but not significant. Cumulative results show a lod score of -6.2 at {theta} = 0 and of -3.9 at {theta} = 0.05 (dominant model, narrow disease definition). SLC6A3 thus maps to distal chromosome 5p by linkage analysis, in agreement with previous physical mapping data. A mutation at SLC6A3 is not causative for TS in the two large families that generated significant negative lod scores (if the parameters of our analyses were correct) and is unlikely to be causative in the family that generated a negative lod score that did not reach significance. These results do not exclude a role for the dopamine transporter in influencing risk for TS in combination with other loci. 23 refs., 1 fig., 2 tabs.

  8. Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity.

    PubMed

    Khasnatinov, Maxim A; Tuplin, Andrew; Gritsun, Dmitri J; Slovak, Mirko; Kazimirova, Maria; Lickova, Martina; Havlikova, Sabina; Klempa, Boris; Labuda, Milan; Gould, Ernest A; Gritsun, Tamara S

    2016-01-01

    Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras. PMID:27341437

  9. Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity

    PubMed Central

    Khasnatinov, Maxim A.; Tuplin, Andrew; Gritsun, Dmitri J.; Slovak, Mirko; Kazimirova, Maria; Lickova, Martina; Havlikova, Sabina; Klempa, Boris; Gould, Ernest A.

    2016-01-01

    Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras. PMID:27341437

  10. Redox Properties of Free Radicals.

    ERIC Educational Resources Information Center

    Neta, P.

    1981-01-01

    Describes pulse radiolysis as a useful means in studing one-electron redox potentials. This method allows the production of radicals and the determination of their concentration and rates of reaction. (CS)

  11. Ribonucleotide Reductase-- a Radical Enzyme

    NASA Astrophysics Data System (ADS)

    Reichard, Peter; Ehrenberg, Anders

    1983-08-01

    Ribonucleotide reductases catalyze the enzymatic formation of deoxyribonucleotides, an obligatory step in DNA synthesis. The native form of the enzyme from Escherichia coli or from mammalian sources contains as part of its polypeptide structure a free tyrosyl radical, stabilized by an iron center. The radical participates in all probability in the catalytic process during the substitution of the hydroxyl group at C-2 of ribose by a hydrogen atom. A second, inactive form of the E. coli reductase lacks the tyrosyl radical. Extracts from E. coli contain activities that interconvert the two forms. The tyrosyl radical is introduced in the presence of oxygen, while anaerobiosis favors its removal, suggesting a regulatory role in DNA synthesis for oxygen.

  12. Acute activation of AMP-activated protein kinase prevents H2O2-induced premature senescence in primary human keratinocytes.

    PubMed

    Ido, Yasuo; Duranton, Albert; Lan, Fan; Cacicedo, Jose M; Chen, Tai C; Breton, Lionel; Ruderman, Neil B

    2012-01-01

    We investigated the effects of AMPK on H(2)O(2)-induced premature senescence in primary human keratinocytes. Incubation with 50 µM H(2)O(2) for 2 h resulted in premature senescence with characteristic increases in senescence-associated ß-galactosidase (SA-gal) staining 3 days later and no changes in AMPK or p38 MAPK activity. The increase in SA-gal staining was preceded by increases in both p53 phosphorylation (S15) (1 h) and transactivation (6 h) and the abundance of the cyclin inhibitor p21(CIP1) (16 h). Incubation with AICAR or resveratrol, both of which activated AMPK, prevented the H(2)O(2)-induced increases in both SA-Gal staining and p21 abundance. In addition, AICAR diminished the increase in p53 transactivation. The decreases in SA-Gal expression induced by resveratrol and AICAR were prevented by the pharmacological AMPK inhibitor Compound C, expression of a DN-AMPK or AMPK knock-down with shRNA. Likewise, both knockdown of AMPK and expression of DN-AMPK were sufficient to induce senescence, even in the absence of exogenous H(2)O(2). As reported by others, we found that AMPK activation by itself increased p53 phosphorylation at S15 in embryonic fibroblasts (MEF), whereas under the same conditions it decreased p53 phosphorylation in the keratinocytes, human aortic endothelial cells, and human HT1080 fibrosarcoma cells. In conclusion, the results indicate that H(2)O(2) at low concentrations causes premature senescence in human keratinocytes by activating p53-p21(CIP1) signaling and that these effects can be prevented by acute AMPK activation and enhanced by AMPK downregulation. They also suggest that this action of AMPK may be cell or context-specific. PMID:22514710

  13. Induction of bone formation in biphasic calcium phosphate scaffolds by bone morphogenetic protein-2 and primary osteoblasts.

    PubMed

    Strobel, L A; Rath, S N; Maier, A K; Beier, J P; Arkudas, A; Greil, P; Horch, R E; Kneser, U

    2014-03-01

    Bone tissue engineering strategies mainly depend on porous scaffold materials. In this study, novel biphasic calcium phosphate (BCP) matrices were generated by 3D-printing. High porosity was achieved by starch consolidation. This study aimed to characterise the porous BCP-scaffold properties and interactions of osteogenic cells and growth factors under in vivo conditions. Five differently treated constructs were implanted subcutaneously in syngeneic rats: plain BCP constructs (group A), constructs pre-treated with BMP-2 (group B; 1.6 µg BMP-2 per scaffold), seeded with primary osteoblasts (OB) (group C), seeded with OB and BMP-2 (group D) and constructs seeded with OB and pre-cultivated in a flow bioreactor for 6 weeks (group E). After 2, 4 and 6 weeks, specimens were explanted and subjected to histological and molecular biological analyses. Explanted scaffolds were invaded by fibrovascular tissue without significant foreign body reactions. Morphometric analysis demonstrated significantly increased bone formation in samples from group D (OB + BMP-2) compared to all other groups. Samples from groups B-E displayed significant mRNA expression of bone-specific genes after 6 weeks. Pre-cultivation in the flow bioreactor (group E) induced bone formation comparable with group B. In this study, differences in bone distribution between samples with BMP-2 or osteoblasts could be observed. In conclusion, combination of osteoblasts and BMP-2 synergistically enhanced bone formation in novel ceramic scaffolds. These results provide the basis for further experiments in orthotopic defect models with a focus on future applications in orthopaedic and reconstructive surgery.

  14. A Novel Mutation in the type Iα Regulatory Subunit of Protein Kinase A (PRKAR1A) in a Cushing's Syndrome Patient with Primary Pigmented Nodular Adrenocortical Disease.

    PubMed

    Mineo, Ryohei; Tamba, Sachiko; Yamada, Yuya; Okita, Tomonori; Kawachi, Yusuke; Mori, Reiko; Kyo, Mitsuaki; Saisho, Kenji; Kuroda, Yohei; Yamamoto, Koji; Furuya, Akiko; Mukai, Tokuo; Maekawa, Takashi; Nakamura, Yasuhiro; Sasano, Hironobu; Matsuzawa, Yuji

    2016-01-01

    A 40-year-old man presented with Cushing's syndrome due to bilateral adrenal hyperplasia with multiple nodules. Computed tomography scan results were atypical demonstrating an enlargement of the bilateral adrenal glands harboring multiple small nodules, but the lesion was clinically diagnosed to be primary pigmented nodular adrenocortical disease (PPNAD) based on both endocrinological test results and his family history. We performed bilateral adrenalectomy and confirmed the diagnosis histologically. An analysis of the patient and his mother's genomic DNA identified a novel mutation in the type Iα regulatory subunit of protein kinase A (PRKAR1A) gene; p.E17X (c.49G>T). This confirmed the diagnosis of PPNAD which is associated with Carney Complex. PMID:27580546

  15. Interleukin 1β Regulation of the System xc- Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR.

    PubMed

    Shi, Jingxue; He, Yan; Hewett, Sandra J; Hewett, James A

    2016-01-22

    System xc(-) is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the CNS, where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1β, increases the activity of system xc(-) in CNS astrocytes secondary to an up-regulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3'-UTR identified numerous copies of adenine- and uridine-rich elements, raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1β increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1β induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3'-UTR of xCT in an IL-1β-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1β-mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels, as well as a functional increase in system xc(-) activity. Altogether, these data support a critical role for HuR in mediating the IL-1β-induced stabilization of astrocyte xCT mRNA.

  16. [Carriage of Streptococcus pyogenes in primary school children: M-protein types, pyrogenic toxin genes, and investigation of the clonal relationships between the isolates].

    PubMed

    Otlu, Barış; Karakurt, Cemşit; Bayındır, Yaşar; Kayabaş, Üner; Yakupoğulları, Yusuf; Gözükara Bağ, Harika

    2015-07-01

    M-protein and pyrogenic toxins are the most important virulence factors of Streptococcus pyogenes, and they play significant role in the pathophysiology of acute rheumatoid fever and scarlet fever, respectively. In this study, the pharyngeal carriage of S.pyogenes of the primary school children, clonal relationship of the strains, M-protein types, and the presence of pyrogenic toxin genes were aimed to be investigated. A total of 668 throat cultures obtained from children (age range: 6-16 years) in two primary schools in our region, were included in the study. The clonal relationships of the isolated group A streptococci (GAS) strains were investigated by DiversiLab assay (BioMérieux, France), and the clonal relatedness was confirmed by pulsed-field gel electrophoresis (PFGE) method. M-protein (emm) typing was performed by DNA sequencing as suggested by Centers for Disease Control and Prevention (CDC). The genes encoding pyrogenic toxins, speA and speC, were investigated by an in-house multiplex polymerase chain reaction (PCR) method. S.pyogenes was isolated from 134 (20.05%) of the throat samples. The GAS carriage rate of the students aged ≥10 was statistically higher than those 7-9 years age group (%22 vs %16.4, p<0.05). The M protein gene could be characterized only among 123 isolates by DNA sequencing, and 20 different emm types were detected. The most frequent emm type was emm1 (n=38, 30.9%) followed by emm12 (n=18, 14.6%), emm89 (n=10, 8.1%), emm118 (n=9, 7.3%), and emm4 (n=7, 5.7%). Pyrogenic toxin genes were found in 25 (18.6%) of the isolates, including speA in 11 isolates (8.2%) and speC in 12 isolates (8.9%) and both genes were detected in 2 isolates (1.5%). Sixty-two different Rep (Repetitive extragenic palindromic)-PCR profiles were detected in 134 S.pyogenes isolates by DiversiLab method. Thirteen different clusters were formed by a total of clonally related 36 isolates revealing a strain clustering ratio of 26.9%. Clonal relationship of all

  17. VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells.

    PubMed

    Takahashi, T; Ueno, H; Shibuya, M

    1999-04-01

    KDR/FIk-1 tyrosine kinase, one of the two VEGF receptors induces mitogenesis and differentiation of vascular endothelial cells. We have previously reported that a major target molecule of KDR/Flk-1 kinase is PLC-gamma, and that VEGF induces activation of MAP kinase, mainly mediated by protein kinase C (PKC) in the NIH3T3 cells overexpressing KDR/FIk-1 (Takahashi and Shibuya, 1997). However, the signal transduction initiated from VEGF in endothelial cells remains to be elucidated. In primary sinusoidal endothelial cells which showed strictly VEGF-dependent growth, we found that VEGF stimulated the activation of Raf-1-MEK-MAP kinase cascade. To our surprise, an important regulator, Ras was not efficiently activated to a significant level in response to VEGF. Consistent with this, dominant-negative Ras did not block the VEGF-induced phosphorylation of MAP kinase. On the other hand, PKC-specific inhibitors severely reduced VEGF-dependent phosphorylation of MEK, activation of MAP kinase and subsequent DNA synthesis. A potent PI3 kinase inhibitor, Wortmannin, could not inhibit either of them. These results suggest that in primary endothelial cells, VEGF-induced activation of Raf-MEK-MAP kinase and DNA synthesis are mainly mediated by PKC-dependent pathway, much more than by Ras-dependent or PI3 kinase-dependent pathway.

  18. Auxiliary iron-sulfur cofactors in radical SAM enzymes.

    PubMed

    Lanz, Nicholas D; Booker, Squire J

    2015-06-01

    A vast number of enzymes are now known to belong to a superfamily known as radical SAM, which all contain a [4Fe-4S] cluster ligated by three cysteine residues. The remaining, unligated, iron ion of the cluster binds in contact with the α-amino and α-carboxylate groups of S-adenosyl-l-methionine (SAM). This binding mode facilitates inner-sphere electron transfer from the reduced form of the cluster into the sulfur atom of SAM, resulting in a reductive cleavage of SAM to methionine and a 5'-deoxyadenosyl radical. The 5'-deoxyadenosyl radical then abstracts a target substrate hydrogen atom, initiating a wide variety of radical-based transformations. A subset of radical SAM enzymes contains one or more additional iron-sulfur clusters that are required for the reactions they catalyze. However, outside of a subset of sulfur insertion reactions, very little is known about the roles of these additional clusters. This review will highlight the most recent advances in the identification and characterization of radical SAM enzymes that harbor auxiliary iron-sulfur clusters. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

  19. Complex of Escherichia coli primary replicative helicase DnaB protein with a replication fork: recognition and structure.

    PubMed

    Jezewska, M J; Rajendran, S; Bujalowski, W

    1998-03-01

    Interactions of the Escherichia coli replicative helicase DnaB protein, with DNA replication fork substrates, have been studied using rigorous fluorescence titration, fluorescence energy transfer, and analytical ultracentrifugation methods. DnaB binds the 5' single-arm fork, the 3' single-arm fork, and the two-arm fork with stoichiometries of 1, 1, and 2 DnaB hexamers per fork, independent of the length of the duplex part of the fork. Within the structurally heterogeneous binding site, the helicase accesses most of the 20 nucleotide residues of an arm. The dsDNA of the fork does not contribute to the affinity; however, it affects the positioning of the enzyme on the 5' or 3' arm. Fluorescence energy transfer experiments provide direct evidence that the DnaB helicase binds the 5' arm of the fork in a single orientation, with respect to the duplex part of the fork. The 33-kDa domains of the hexamer face the dsDNA, while the small 12-kDa domains face the 5' end of the arm. In the complex with the 3' arm, the helicase is bound in an opposite orientation when compared to the 5' arm. This is the first determination of the strict, single orientation of a helicase in the complex with a replication fork. The 3' arm accommodates a DnaB hexamer, while another hexamer is associated with the 5' arm. The complex of two DnaB hexamers bound in opposite orientations with each arm of the fork may play an important role during bidirectional replication of the E. coli DNA.

  20. Proteins

    NASA Astrophysics Data System (ADS)

    Regnier, Fred E.; Gooding, Karen M.

    Because of the complexity of cellular material and body fluids, it is seldom possible to analyze a natural product directly. Qualitative and quantitative analyses must often be preceded by some purification step that separates the molecular species being examined from interfering materials. In the case of proteins, column liquid chromatography has been used extensively for these fractionations. With the advent of gel permeation, cation exchange, anion exchange, hydrophobic, and affinity chromatography, it became possible to resolve proteins through their fundamental properties of size, charge, hydrophobicity, and biological affinity. The chromatographic separations used in the early isolation and characterization of many proteins later became analytical tools in their routine analysis. Unfortunately, these inherently simple and versatile column chromatographic techniques introduced in the 50s and 60s have a severe limitation in routine analysis-separation time. It is common to encounter 1-24 h separation times with the classical gel-type supports.

  1. Selection for low or high primary dormancy in Lolium rigidum Gaud seeds results in constitutive differences in stress protein expression and peroxidase activity

    PubMed Central

    Goggin, Danica E.; Powles, Stephen B.; Steadman, Kathryn J.

    2011-01-01

    Seed dormancy in wild Lolium rigidum Gaud (annual ryegrass) populations is highly variable and not well characterized at the biochemical level. To identify some of the determinants of dormancy level in these seeds, the proteomes of subpopulations selected for low and high levels of primary dormancy were compared by two-dimensional polyacrylamide gel electrophoresis of extracts from mature, dry seeds. High-dormancy seeds showed higher expression of small heat shock proteins, enolase, and glyoxalase I than the low-dormancy seeds. The functional relevance of these differences in protein expression was confirmed by the fact that high-dormancy seeds were more tolerant to high temperatures imposed at imbibition and had consistently higher glyoxalase I activity over 0–42 d dark stratification. Higher expression of a putative glutathione peroxidase in low-dormancy seeds was not accompanied by higher activity, but these seeds had a slightly more oxidized glutathione pool and higher total peroxidase activity. Overall, these biochemical and physiological differences suggest that L. rigidum seeds selected for low dormancy are more prepared for rapid germination via peroxidase-mediated cell wall weakening, whilst seeds selected for high dormancy are constitutively prepared to survive environmental stresses, even in the absence of stress during seed development. PMID:20974739

  2. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

    SciTech Connect

    Yoshino, Yuta; Yuan, Bo; Kaise, Toshikazu; Takeichi, Makoto; Tanaka, Sachiko; Hirano, Toshihiko; Kroetz, Deanna L.; Toyoda, Hiroo

    2011-12-15

    Arsenic trioxide (arsenite, As{sup III}) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As{sup III} on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As{sup III} on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As{sup III}-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As{sup III} were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As{sup III} than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As{sup III} in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As{sup III}-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As{sup III} cytotoxicity between these cells. -- Highlights: Black-Right-Pointing-Pointer Examination of effect of As{sup III} on primary cultured chorion (C) and amnion

  3. Primary Cutaneous Follicle Center Lymphomas Expressing BCL2 Protein Frequently Harbor BCL2 Gene Break and May Present 1p36 Deletion: A Study of 20 Cases.

    PubMed

    Szablewski, Vanessa; Ingen-Housz-Oro, Saskia; Baia, Maryse; Delfau-Larue, Marie-Helene; Copie-Bergman, Christiane; Ortonne, Nicolas

    2016-01-01

    The classification of cutaneous follicular lymphoma (CFL) into primary cutaneous follicle center lymphoma (PCFCL) or secondary cutaneous follicular lymphoma (SCFL) is challenging. SCFL is suspected when tumor cells express BCL2 protein, reflecting a BCL2 translocation. However, BCL2 expression is difficult to assess in CFLs because of numerous BCL2+ reactive T cells. To investigate these issues and to further characterize PCFCL, we studied a series of 25 CFLs without any extracutaneous disease at diagnosis, selected on the basis of BCL2 protein expression using 2 BCL2 antibodies (clones 124 and E17) and BOB1/BCL2 double immunostaining. All cases were studied using interphase fluorescence in situ hybridization with BCL2, BCL6, IGH, IGK, IGL breakapart, IGH-BCL2 fusion, and 1p36/1q25 dual-color probes. Nineteen CFLs were BCL2 positive, and 6 were negative. After a medium follow-up of 24 (6 to 96) months, 5 cases were reclassified as SCFL and were excluded from a part of our analyses. Among BCL2+ PCFCLs, 60% (9/15) demonstrated a BCL2 break. BCL2-break-positive cases had a tendency to occur in the head and neck and showed the classical phenotype of nodal follicular lymphoma (CD10+, BCL6+, BCL2+, STMN+) compared with BCL2-break-negative PCFCLs. Del 1p36 was observed in 1 PCFCL. No significant clinical differences were observed between BCL2+ or BCL2- PCFCL. In conclusion, we show that a subset of PCFCLs harbor similar genetic alterations, as observed in nodal follicular lymphomas, including BCL2 breaks and 1p36 deletion. As BCL2 protein expression is usually associated with the presence of a BCL2 translocation, fluorescence in situ hybridization should be performed to confirm this hypothesis.

  4. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes.

    PubMed

    Yoshino, Yuta; Yuan, Bo; Kaise, Toshikazu; Takeichi, Makoto; Tanaka, Sachiko; Hirano, Toshihiko; Kroetz, Deanna L; Toyoda, Hiroo

    2011-12-01

    Arsenic trioxide (arsenite, As(III)) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As(III) on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As(III) on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As(III)-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As(III) were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As(III) than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As(III) in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As(III)-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As(III) cytotoxicity between these cells.

  5. Crystalline bipyridinium radical complexes and uses thereof

    SciTech Connect

    Fahrenbach, Albert C.; Barnes, Jonathan C.; Li, Hao; Stoddart, J. Fraser; Basuray, Ashish Neil; Sampath, Srinivasan

    2015-09-01

    Described herein are methods of generating 4,4'-bipyridinium radical cations (BIPY.sup..cndot.+), and methods for utilizing the radical-radical interactions between two or more BIPY.sup..cndot.+ radical cations that ensue for the creation of novel materials for applications in nanotechnology. Synthetic methodologies, crystallographic engineering techniques, methods of physical characterization, and end uses are described.

  6. Social Radicalism as a Framework for Education.

    ERIC Educational Resources Information Center

    Nelson, Jack L.

    This paper examines the nature of radicalism, proposes definitions and dimensions, and suggests the dynamics of radical thoughts and their interrelation with schools. A radical idea is one which is highly divergent from the normative values, behaviors, ideals or traditions of a culture at a point in time. This paper views radical ideas as…

  7. Sulfite-mediated oxidation of myeloperoxidase to a free radical: immuno-spin trapping detection in human neutrophils.

    PubMed

    Ranguelova, Kalina; Rice, Annette B; Lardinois, Olivier M; Triquigneaux, Mathilde; Steinckwich, Natacha; Deterding, Leesa J; Garantziotis, Stavros; Mason, Ronald P

    2013-07-01

    Previous studies focused on catalyzed oxidation of (bi)sulfite, leading to the formation of the reactive sulfur trioxide ((•)SO3(-)), peroxymonosulfate ((-)O3SOO(•)), and sulfate (SO4(•-)) anion radicals, which can damage target proteins and oxidize them to protein radicals. It is known that these very reactive sulfur- and oxygen-centered radicals can be formed by oxidation of (bi)sulfite by peroxidases. Myeloperoxidase (MPO), an abundant heme protein secreted from activated neutrophils that play a central role in host defense mechanisms, allergic reactions, and asthma, is a likely candidate for initiating the respiratory damage caused by sulfur dioxide. The objective of this study was to examine the oxidative damage caused by (bi)sulfite-derived free radicals in human neutrophils through formation of protein radicals. We used immuno-spin trapping and confocal microscopy to study the protein oxidations driven by sulfite-derived radicals. We found that the presence of sulfite can cause MPO-catalyzed oxidation of MPO to a protein radical in phorbol 12-myristate 13-acetate-activated human neutrophils. We trapped the MPO-derived radicals in situ using the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide and detected them immunologically as nitrone adducts in cells. Our present study demonstrates that myeloperoxidase initiates (bi)sulfite oxidation leading to MPO radical damage, possibly leading to (bi)sulfite-exacerbated allergic reactions.

  8. Possible mediators of the ``living'' radical polymerization

    NASA Astrophysics Data System (ADS)

    Motyakin, M. V.; Wasserman, A. M.; Stott, P. E.; Zaikov, G. E.

    2006-03-01

    The stable radicals derived from different compounds were detected in process of styrene autopolymerization. The nitroxide radicals are produced from nitrosocompound, hindered hydroxylamine, nitrophenols and nitroanisoles. The phenoxyl radicals are formed from quinine methides, and naphtoxyl radicals are generated from 2-nitro-1-naphtol. The radicals are identified, the kinetics of their formation and follow-up evolution are studied. These radicals can participate in process of living radical polymerization as the mediators and can effect significantly on kinetics of polymerization and structure of the resulting polymer.

  9. Mechanisms of radical formation in beef and chicken meat during high pressure processing evaluated by electron spin resonance detection and the addition of antioxidants.

    PubMed

    Bolumar, Tomas; Andersen, Mogens L; Orlien, Vibeke

    2014-05-01

    The generation of radicals during high pressure (HP) processing of beef loin and chicken breast was studied by spin trapping and electron spin resonance detection. The pressurization resulted in a higher level of spin adducts in the beef loin than in the chicken breast. It was shown that radicals were formed in the sarcoplasmic and myofibrillar fractions as well as in the non-soluble protein fraction due to the HP treatment, indicating that other radicals than iron-derived radicals were formed, and most likely protein-derived radicals. The addition of iron as well as the natural antioxidants caffeic acid, rosemary extract, and ascorbic acid resulted in an increased formation of radicals during the HP treatment, whereas addition of ethylendiamintetraacetic acid (EDTA) reduced the radical formation. This suggests that iron-species (protein-bound or free) catalyses the formation of radicals when meat systems are submitted to HP.

  10. Standard Electrode Potentials Involving Radicals in Aqueous Solution: Inorganic Radicals

    SciTech Connect

    Armstrong, David A.; Huie, Robert E.; Koppenol, Willem H.; Lymar, Sergei V.; Merenyi, Gabor; Neta, Pedatsur; Ruscic, Branko; Stanbury, David M.; Steenken, Steen; Wardman, Peter

    2015-12-01

    Recommendations are made for standard potentials involving select inorganic radicals in aqueous solution at 25 °C. These recommendations are based on a critical and thorough literature review and also by performing derivations from various literature reports. The recommended data are summarized in tables of standard potentials, Gibbs energies of formation, radical pKa’s, and hemicolligation equilibrium constants. In all cases, current best estimates of the uncertainties are provided. An extensive set of Data Sheets is appended that provide original literature references, summarize the experimental results, and describe the decisions and procedures leading to each of the recommendations

  11. Mechanically Stabilized Tetrathiafulvalene Radical Dimers

    SciTech Connect

    Coskun, Ali; Spruell, Jason M.; Barin, Gokhan; Fahrenbach, Albert C.; Forgan, Ross S.; Colvin, Michael T.; Carmieli, Raanan; Benitez, Diego; Tkatchouk, Ekaterina; Friedman, Douglas C.; Sarjeant, Amy A.; Wasielewski, Michael R.; Goddard, William A.; Stoddart, J. Fraser

    2011-01-01

    Two donor-acceptor [3]catenanes—composed of a tetracationic molecular square, cyclobis(paraquat-4,4'-biphenylene), as the π-electron deficient ring and either two tetrathiafulvalene (TTF) and 1,5-dioxynaphthalene (DNP) containing macrocycles or two TTF-butadiyne-containing macrocycles as the π-electron rich components—have been investigated in order to study their ability to form TTF radical dimers. It has been proven that the mechanically interlocked nature of the [3]catenanes facilitates the formation of the TTF radical dimers under redox control, allowing an investigation to be performed on these intermolecular interactions in a so-called “molecular flask” under ambient conditions in considerable detail. In addition, it has also been shown that the stability of the TTF radical-cation dimers can be tuned by varying the secondary binding motifs in the [3]catenanes. By replacing the DNP station with a butadiyne group, the distribution of the TTF radical-cation dimer can be changed from 60% to 100%. These findings have been established by several techniques including cyclic voltammetry, spectroelectrochemistry and UV-vis-NIR and EPR spectroscopies, as well as with X-ray diffraction analysis which has provided a range of solid-state crystal structures. The experimental data are also supported by high-level DFT calculations. The results contribute significantly to our fundamental understanding of the interactions within the TTF radical dimers.

  12. Missing Peroxy Radical Sources Within a Rural Forest Canopy

    NASA Technical Reports Server (NTRS)

    Wolfe, G. M.; Cantrell, C.; Kim, S.; Mauldin, R. L., III; Karl, T.; Harley, P.; Turnipseed, A.; Zheng, W.; Flocke, F.; Apel, E. C.; Hornbrook, R. S.; Hall, S. R.; Ullmann, K.; Henry, S. B.; DiGangi, J. P.; Boyle, E. S.; Kaser, L.; Schnitzhofer, R.; Hansel, A.; Graus, M.; Nakashima, Y.; Kajii, Y.; Guenther, A.; Keutsch, F. N.

    2013-01-01

    Organic peroxy (RO2) and hydroperoxy (HO2) radicals are key intermediates in the photochemical processes that generate ozone, secondary organic aerosol and reactive nitrogen reservoirs throughout the troposphere. In regions with ample biogenic hydrocarbons, the richness and complexity of peroxy radical chemistry presents a significant challenge to current-generation models, especially given the scarcity of measurements in such environments. We present peroxy radical observations acquired within a Ponderosa pine forest during the summer 2010 Bio-hydro-atmosphere interactions of Energy, Aerosols, Carbon, H2O, Organics and Nitrogen - Rocky Mountain Organic Carbon Study (BEACHON-ROCS). Total peroxy radical mixing ratios reach as high as 180 pptv and are among the highest yet recorded. Using the comprehensive measurement suite to constrain a near-explicit 0-D box model, we investigate the sources, sinks and distribution of peroxy radicals below the forest canopy. The base chemical mechanism underestimates total peroxy radicals by as much as a factor of 3. Since primary reaction partners for peroxy radicals are either measured (NO) or under-predicted (HO2 and RO2, i.e. self-reaction), missing sources are the most likely explanation for this result. A close comparison of model output with observations reveals at least two distinct source signatures. The first missing source, characterized by a sharp midday maximum and a strong dependence on solar radiation, is consistent with photolytic production of HO2. The diel profile of the second missing source peaks in the afternoon and suggests a process that generates RO2 independently of sun-driven photochemistry, such as ozonolysis of reactive hydrocarbons. The maximum magnitudes of these missing sources (approximately 120 and 50 pptv min-1, respectively) are consistent with previous observations alluding to unexpectedly intense oxidation within forests. We conclude that a similar mechanism may underlie many such observations.

  13. Development of a Functional Contrast Agent for Targeting Lipid-derived Radicals.

    PubMed

    Yamada, Ken-Ichi

    2016-01-01

    Lipid derived radicals and their metabolic products are closely involved in the pathogenesis of oxidative stress diseases, such as inflammation and angiogenesis, through the formation of a protein or DNA complex. The starting point of lipid peroxide generation is lipid-derived radicals, which increase explosively via radical chain reaction. Therefore, the trapping of lipid-derived radicals is useful in understanding the mechanism of the formation of oxidative stress diseases, and in suppressing the following chain reaction. On the other hand, nitroxides with a stable unpaired electron allow for spin trapping with carbon-centered radicals. Hence, we focused on the following points to develop lipid radical detection methods. 1) Fluorescence will be quenched through interaction with nitroxide's unpaired electron. 2) Nitroxide can react with lipid-derived radicals via radical-radical reaction. 3) Fluorescence will recover from the loss of an unpaired electron in nitroxide, after reaction with the lipid-derived radicals, by using a profluorescent nitroxide. In this paper, I will discuss the development of a lipid-derived detection method using profluorescent nitroxide switching methods, and discuss its application to cell imaging. PMID:27477723

  14. The role of free radicals in traumatic brain injury.

    PubMed

    O'Connell, Karen M; Littleton-Kearney, Marguerite T

    2013-07-01

    Traumatic brain injury (TBI) is a significant cause of death and disability in both the civilian and the military populations. The primary impact causes initial tissue damage, which initiates biochemical cascades, known as secondary injury, that expand the damage. Free radicals are implicated as major contributors to the secondary injury. Our review of recent rodent and human research reveals the prominent role of the free radicals superoxide anion, nitric oxide, and peroxynitrite in secondary brain injury. Much of our current knowledge is based on rodent studies, and the authors identified a gap in the translation of findings from rodent to human TBI. Rodent models are an effective method for elucidating specific mechanisms of free radical-induced injury at the cellular level in a well-controlled environment. However, human TBI does not occur in a vacuum, and variables controlled in the laboratory may affect the injury progression. Additionally, multiple experimental TBI models are accepted in rodent research, and no one model fully reproduces the heterogeneous injury seen in humans. Free radical levels are measured indirectly in human studies based on assumptions from the findings from rodent studies that use direct free radical measurements. Further study in humans should be directed toward large samples to validate the findings in rodent studies. Data obtained from these studies may lead to more targeted treatment to interrupt the secondary injury cascades.

  15. Radical-Mediated Enzymatic Polymerizations

    PubMed Central

    Zavada, Scott R.; Battsengel, Tsatsral; Scott, Timothy F.

    2016-01-01

    Polymerization reactions are commonly effected by exposing monomer formulations to some initiation stimulus such as elevated temperature, light, or a chemical reactant. Increasingly, these polymerization reactions are mediated by enzymes―catalytic proteins―owing to their reaction efficiency under mild conditions as well as their environmental friendliness. The utilization of enzymes, particularly oxidases and peroxidases, for generating radicals via reduction-oxidation mechanisms is especially common for initiating radical-mediated polymerization reactions, including vinyl chain-growth polymerization, atom transfer radical polymerization, thiol–ene step-growth polymerization, and polymerization via oxidative coupling. While enzyme-mediated polymerization is useful for the production of materials intended for subsequent use, it is especially well-suited for in situ polymerizations, where the polymer is formed in the place where it will be utilized. Such polymerizations are especially useful for biomedical adhesives and for sensing applications. PMID:26848652

  16. Epistemological barriers to radical behaviorism

    PubMed Central

    O'Donohue, William T.; Callaghan, Glenn M.; Ruckstuhl, L. E.

    1998-01-01

    The historian and philosopher of science Gaston Bachelard proposed the concept of epistemological barriers to describe the intellectual challenges encountered by scientists in their work. In order to embrace novel ways of approaching a problem in science, scientists must overcome barriers or obstacles posed by their prior views. For example, Einsteinian physics presents scientists with claims that space is curved and that time and space are on the same continuum. We utilize Bachelard's concept of epistemological barriers to describe the differences between the intellectual journeys students pursuing advanced studies face when attempting to accept cognitive psychology or radical behaviorism. We contend that the folk psychological beliefs that students typically hold when entering these studies pose less challenge to cognitive psychology than to radical behaviorism. We also suggest that these barriers may also partly be involved in the problematic exegesis that has plagued radical behaviorism. In close, we offer some suggestions for dealing with these epistemological barriers. PMID:22478314

  17. Radical SAM enzymes involved in the biosynthesis of purine-based natural products

    PubMed Central

    Bandarian, Vahe

    2013-01-01

    The radical S-adenosyl-L-methionine (SAM) superfamily is a widely distributed group of iron-sulfur containing proteins that exploit the reactivity of the high energy intermediate, 5’-deoxyadenosyl radical, which is produced by reductive cleavage of SAM, to carry-out complex radical-mediated transformations. The reactions catalyzed by radical SAM enzymes range from simple group migrations to complex reactions in protein and RNA modification. This review will highlight three radical SAM enzymes that catalyze reactions involving modified guanosines in the biosynthesis pathways of the hypermodified tRNA base wybutosine; secondary metabolites of 7-deazapurine structure, including the hypermodified tRNA base queuosine; and the redox cofactor F420. PMID:22902275

  18. Interleukin-1-mediated febrile responses in mice and interleukin-1 beta activation of NFkappaB in mouse primary astrocytes, involves the interleukin-1 receptor accessory protein.

    PubMed

    Zetterström, M; Lundkvist, J; Malinowsky, D; Eriksson, G; Bartfai, T

    1998-06-01

    The endogenous pyrogen interleukin-1 (IL-1) is considered as one of the key molecules in orchestrating the host response of injury and inflammation. IL-1 exerts its effects upon binding to the type I IL-1 receptor (IL-1RI). The IL-1-IL-1RI complex is further thought to associate with the IL-1 receptor accessory protein (IL-1RAcP), which is suggested to be important for most IL-1 signal transduction pathways. With the aim of investigating the importance of the IL-1RAcP in IL-1 signalling, IL-1alpha and IL-1beta induced febrile responses and IL-1beta-mediated activation of NFkappaB in primary astrocyte cultures were examined using IL-1RAcP-deficient (IL-1RAcP KO) and wild type mice, respectively. It was shown that neither recombinant rat IL-1alpha (rrIL-1alpha, 25 microg/kg), recombinant rat IL-1beta (rrIL-1beta, 40 microg/kg) nor recombinant human IL-1beta (rhIL-1beta, 50 microg/kg) injected i.p. could elicit febrile responses in the IL-1RAcP-deficient mice, while the same doses of rrIL-1alpha/beta or rhIL-1beta injected into wild type mice caused normal fever responses. A febrile response could be induced in the IL-1RAcP-deficient mice by i.p. administration of E. coli lipopolysaccharide (LPS, 50 microg/kg) and this response was similar to that obtained in wild type mice. Furthermore, it was shown that rhIL-1beta activated, in a concentration-dependent manner, nuclear translocation of the transcriptional nuclear factor kappa B (NFkappaB) in primary astrocyte cultures prepared from wild type mice, whereas no IL-1beta-induced translocation of NFkappaB could be detected in cultures prepared from IL-1RAcP-deficient mice, as revealed by electrophoretic mobility shift assay (EMSA). The rhIL-1beta-induced NFkappaB complexes were shown to contain p50 but no, or very little, p65 and cRel immunoreactive proteins.

  19. Green fluorescent protein tagging of extracellular signal-regulated kinase and p38 pathways reveals novel dynamics of pathway activation during primary and metastatic growth.

    PubMed

    Aguirre-Ghiso, Julio A; Ossowski, Liliana; Rosenbaum, Sarah K

    2004-10-15

    We describe a novel approach that allows detection of primary and metastatic cells in vivo in which either the extracellular signal-regulated kinase (ERK) or the p38 pathway is activated. Our recent findings showed that ERK and p38 kinases regulate, respectively, programs dictating cell proliferation (high ERK-to-p38 ratio) or growth arrest and dormancy (low ERK-to-p38 ratio) in vivo. Thus, we were able to use green fluorescent protein (GFP) to reflect ERK and p38 activities and, consequently, the proliferative state of cancer cells. This was accomplished by transfecting tumorigenic T-HEp3 and HT1080 cells, and dormant D-HEp3 cells, with plasmids coding for Elk-GAL4 or CHOP-GAL4 fusion proteins that, when phosphorylated by either ERK or p38, respectively, transactivated a GFP-reporter gene. The fate of these cells was examined in culture, in primary sites, and in spontaneous metastasis in chick embryos and nude mice. In culture GFP level was directly proportional to the previously established levels of ERK or p38 activation. In contrast, during the first 24 hours of in vivo inoculation, both the tumorigenic and the dormant cells strongly activated the p38 pathway. However, in the tumorigenic cells, p38 activity was rapidly silenced, correcting the ERK/p38 imbalance and contributing to high ERK activity throughout the entire period of tumor growth. In contrast, in the small nodules formed by dormant cells, the level of ERK activity was dramatically reduced, whereas p38 activity remained high. Strong activation of ERK was evident in metastatic sites, whereas p38 activation was silenced in this anatomic location as well. These results show that it is possible to directly measure cancer cell response to microenvironment with this reporter system and that only proliferation-competent cells have the ability to rapidly adapt ERK and p38 signaling for proliferative success. This approach allows isolation and further characterization of metastatic cells with specific

  20. Vaginal radical trachelectomy: an update.

    PubMed

    Plante, Marie

    2008-11-01

    The vaginal radical trachelectomy has emerged as a valuable fertility-preserving treatment option for young women with early-stage disease. Cancer-related infertility is associated with feelings of depression, grief, stress, and sexual dysfunction. Data have shown that the overall oncological outcome is safe and that the obstetrical outcome is promising. In this article, we analyze the data on the vaginal radical trachelectomy published over the last 10 years in the context of what we have learned, what issues remain unclear, and what the future holds.

  1. Donor free radical explosive composition

    DOEpatents

    Walker, Franklin E. [15 Way Points Rd., Danville, CA 94526; Wasley, Richard J. [4290 Colgate Way, Livermore, CA 94550

    1980-04-01

    An improved explosive composition is disclosed and comprises a major portion of an explosive having a detonation velocity between about 1500 and 10,000 meters per second and a minor amount of a donor additive comprising an organic compound or mixture of organic compounds capable of releasing low molecular weight free radicals or ions under mechanical or electrical shock conditions and which is not an explosive, or an inorganic compound or mixture of inorganic compounds capable of releasing low molecular weight free radicals or ions under mechanical or electrical shock conditions and selected from ammonium or alkali metal persulfates.

  2. Nitric oxide rapidly scavenges tyrosine and tryptophan radicals.

    PubMed Central

    Eiserich, J P; Butler, J; van der Vliet, A; Cross, C E; Halliwell, B

    1995-01-01

    By utilizing a pulse-radiolytic technique, we demonstrate for the first time that the rate constant for the reaction of nitric oxide (.NO) with biologically relevant tyrosine and tryptophan radicals (Tyr. and Trp. respectively) in amino acids, peptides and proteins is of the order of (1-2) x 10(9) M-1.s-1. We also show that .NO effectively interferes with electron-transfer processes between tryptophan and tyrosine residues in proteins subjected to pulse radiolysis. The near diffusion-controlled rates of these reactions, coupled with the increasingly recognized role of protein radicals in enzyme catalysis and oxidative damage, suggest that Tyr. and Trp. are likely and important targets for .NO generated in vivo. PMID:7575405

  3. Increased mRNA expression and protein secretion of interleukin-6 in primary human osteoblasts differentiated in vitro from rheumatoid and osteoarthritic bone.

    PubMed

    Chenoufi, H L; Diamant, M; Rieneck, K; Lund, B; Stein, G S; Lian, J B

    2001-01-01

    We have investigated the expression and synthesis of potential bone-resorbing cytokines, interleukin-6 (IL-6), interleukin-1 (IL-1), and tumor necrosis factor (TNF) in rheumatoid arthritic (RA) and osteoarthritic (OA) bone, two common diseases which are associated with bone loss. Primary human osteoblast (hOB) cultures were established to determine the temporal mRNA expression of IL-6, IL-1 (alpha and beta), and TNF (alpha and beta) in relation to osteoblast growth and phenotypic genes. IL-6 mRNA levels were found to be significantly higher (P < 0.04) in both OA hOB (17 patients) and RA hOB (10 patients) compared to normal (NO) hOB (9 patients) and reached five-fold increases in OA hOB and 13-fold increases in RA hOB. Maximal levels of IL-6 are expressed at Day 21 which corresponds to the mineralization stage reflected by decreasing collagen I (alpha(1)), osteopontin, bone sialoprotein, alkaline phosphatase mRNA levels, while osteocalcin (OC) mRNA levels increased. IL-6 protein levels also were significantly higher (P < 0.05) in OA hOB and RA hOB compared to NO hOB. These increases were not attributable to sex or age of the donor bone. Neither the mRNA encoding IL-1(alpha and beta) and TNF(alpha and beta) nor the related proteins were detectable. These results indicate that differentiated OA hOB and RA hOB within a bone tissue-like matrix constitutively express and secrete high levels of IL-6. This inherent property suggests that these osteoblasts, independent of local inflammatory parameters, can contribute to enhanced recruitment of osteoclast progenitors and thereby bone resorption.

  4. Mutations in Radial Spoke Head Protein Genes RSPH9 and RSPH4A Cause Primary Ciliary Dyskinesia with Central-Microtubular-Pair Abnormalities

    PubMed Central

    Castleman, Victoria H.; Romio, Leila; Chodhari, Rahul; Hirst, Robert A.; de Castro, Sandra C.P.; Parker, Keith A.; Ybot-Gonzalez, Patricia; Emes, Richard D.; Wilson, Stephen W.; Wallis, Colin; Johnson, Colin A.; Herrera, Rene J.; Rutman, Andrew; Dixon, Mellisa; Shoemark, Amelia; Bush, Andrew; Hogg, Claire; Gardiner, R. Mark; Reish, Orit; Greene, Nicholas D.E.; O'Callaghan, Christopher; Purton, Saul; Chung, Eddie M.K.; Mitchison, Hannah M.

    2009-01-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous inherited disorder arising from dysmotility of motile cilia and sperm. This is associated with a variety of ultrastructural defects of the cilia and sperm axoneme that affect movement, leading to clinical consequences on respiratory-tract mucociliary clearance and lung function, fertility, and left-right body-axis determination. We performed whole-genome SNP-based linkage analysis in seven consanguineous families with PCD and central-microtubular-pair abnormalities. This identified two loci, in two families with intermittent absence of the central-pair structure (chromosome 6p21.1, Zmax 6.7) and in five families with complete absence of the central pair (chromosome 6q22.1, Zmax 7.0). Mutations were subsequently identified in two positional candidate genes, RSPH9 on chromosome 6p21.1 and RSPH4A on chromosome 6q22.1. Haplotype analysis identified a common ancestral founder effect RSPH4A mutation present in UK-Pakistani pedigrees. Both RSPH9 and RSPH4A encode protein components of the axonemal radial spoke head. In situ hybridization of murine Rsph9 shows gene expression restricted to regions containing motile cilia. Investigation of the effect of knockdown or mutations of RSPH9 orthologs in zebrafish and Chlamydomonas indicate that radial spoke head proteins are important in maintaining normal movement in motile, “9+2”-structure cilia and flagella. This effect is rescued by reintroduction of gene expression for restoration of a normal beat pattern in zebrafish. Disturbance in function of these genes was not associated with defects in left-right axis determination in humans or zebrafish. PMID:19200523

  5. Mutations in radial spoke head protein genes RSPH9 and RSPH4A cause primary ciliary dyskinesia with central-microtubular-pair abnormalities.

    PubMed

    Castleman, Victoria H; Romio, Leila; Chodhari, Rahul; Hirst, Robert A; de Castro, Sandra C P; Parker, Keith A; Ybot-Gonzalez, Patricia; Emes, Richard D; Wilson, Stephen W; Wallis, Colin; Johnson, Colin A; Herrera, Rene J; Rutman, Andrew; Dixon, Mellisa; Shoemark, Amelia; Bush, Andrew; Hogg, Claire; Gardiner, R Mark; Reish, Orit; Greene, Nicholas D E; O'Callaghan, Christopher; Purton, Saul; Chung, Eddie M K; Mitchison, Hannah M

    2009-02-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous inherited disorder arising from dysmotility of motile cilia and sperm. This is associated with a variety of ultrastructural defects of the cilia and sperm axoneme that affect movement, leading to clinical consequences on respiratory-tract mucociliary clearance and lung function, fertility, and left-right body-axis determination. We performed whole-genome SNP-based linkage analysis in seven consanguineous families with PCD and central-microtubular-pair abnormalities. This identified two loci, in two families with intermittent absence of the central-pair structure (chromosome 6p21.1, Zmax 6.7) and in five families with complete absence of the central pair (chromosome 6q22.1, Zmax 7.0). Mutations were subsequently identified in two positional candidate genes, RSPH9 on chromosome 6p21.1 and RSPH4A on chromosome 6q22.1. Haplotype analysis identified a common ancestral founder effect RSPH4A mutation present in UK-Pakistani pedigrees. Both RSPH9 and RSPH4A encode protein components of the axonemal radial spoke head. In situ hybridization of murine Rsph9 shows gene expression restricted to regions containing motile cilia. Investigation of the effect of knockdown or mutations of RSPH9 orthologs in zebrafish and Chlamydomonas indicate that radial spoke head proteins are important in maintaining normal movement in motile, "9+2"-structure cilia and flagella. This effect is rescued by reintroduction of gene expression for restoration of a normal beat pattern in zebrafish. Disturbance in function of these genes was not associated with defects in left-right axis determination in humans or zebrafish.

  6. Effect of controlled ice nucleation on primary drying stage and protein recovery in vials cooled in a modified freeze-dryer.

    PubMed

    Passot, Stéphanie; Tréléa, Ioan Cristian; Marin, Michèle; Galan, Miquel; Morris, G John; Fonseca, Fernanda

    2009-07-01

    The freezing step influences lyophilization efficiency and protein stability. The main objective of this work was to investigate the impact on the primary drying stage of an ultrasound controlled ice nucleation technology, compared with usual freezing protocols. Lyophilization cycles involving different freezing protocols (applying a constant shelf cooling rate of 1 degrees C/min or 0.2 degrees C/min, putting vials on a precooled shelf, and controlling nucleation by ultrasounds or by addition of a nucleating agent) were performed in a prototype freeze-dryer. Three protective media including sucrose or maltodextrin and differing by their thermal properties and their ability to preserve a model protein (catalase) were used. The visual aspect of the lyophilized cake, residual water content, and enzymatic activity recovery of catalase were assessed after each lyophilization cycle and after 1 month of storage of the lyophilized product at 4 degrees C and 25 degrees C. The freezing protocols allowing increasing nucleation temperature (precooled shelf and controlled nucleation by using ultrasounds or a nucleating agent) induced a faster sublimation step and higher sublimation rate homogeneity. Whatever the composition of the protective medium, applying the ultrasound technology made it possible to decrease the sublimation time by 14%, compared with the freezing method involving a constant shelf cooling rate of 1 degrees C/min. Concerning the enzyme activity recovery, the impact of the freezing protocol was observed only for the protective medium involving maltodextrin, a less effective protective agent than sucrose. Higher activity recovery results were obtained after storage when the ultrasound technology or the precooled shelf method was applied. Controlling ice nucleation during the freezing step of the lyophilization process improved the homogeneity of the sublimation rates, which will, in turn, reduce the intervial heterogeneity. The freeze-dryer prototype including

  7. Observation of the widetilde{A} - widetilde{X} Electronic Transition of C_6-C_{10} Peroxy Radicals

    NASA Astrophysics Data System (ADS)

    Kline, Neal D.; Miller, Terry A.

    2013-06-01

    The widetilde{A} - widetilde{X} electronic transition of straight chain C_6-C_{10} peroxy radicals and of the isooctyl peroxy radical have been observed and analyzed. These larger hydrocarbons are significant constituents of gasoline with heptane (octane rating of 0) and isooctane (2,2,4 trimethylpentane; octane rating of 100) being the two standards on which the octane rating scale is based. Spectra were obtained by abstraction of hydrogen atoms from the hydrocarbons using chlorine atoms. The origin and -OO stretch regions of the straight chain peroxy radicals are easily identifiable. It is relatively easy to uniquely identify hexyl peroxy, but differentiation among the spectra of the larger straight chain peroxy radicals has proven difficult. However, isooctyl peroxy is easily distinguished and the observation of the tertiary peroxy radical along with the primary and/or secondary peroxy radical(s) is discussed.

  8. Paramagnetic Intermediates Generated by Radical S-Adenosylmethionine (SAM) Enzymes

    PubMed Central

    2015-01-01

    Conspectus A [4Fe–4S]+ cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5′-deoxyadenosyl radical (5′-dA•). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5′-dA• is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, l-tyrosine in the [FeFe] hydrogenase maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms. In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5′-dA• to the product radical l-β-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the l-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN– ligands for the [FeFe] cluster of hydrogenase. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp2-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA. These four systems provide just a brief survey of the ever-growing set

  9. The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate.

    PubMed

    Buettner, G R

    1993-02-01

    Free radicals vary widely in their thermodynamic properties, ranging from very oxidizing to very reducing. These thermodynamic properties can be used to predict a pecking order, or hierarchy, for free radical reactions. Using one-electron reduction potentials, the predicted pecking order is in agreement with experimentally observed free radical electron (hydrogen atom) transfer reactions. These potentials are also in agreement with experimental data that suggest that vitamin E, the primary lipid soluble small molecule antioxidant, and vitamin C, the terminal water soluble small molecule antioxidant, cooperate to protect lipids and lipid structures against peroxidation. Although vitamin E is located in membranes and vitamin C is located in aqueous phases, vitamin C is able to recycle vitamin E; i.e., vitamin C repairs the tocopheroxyl (chromanoxyl) radical of vitamin E, thereby permitting vitamin E to function again as a free radical chain-breaking antioxidant. This review discusses: (i) the thermodynamics of free radical reactions that are of interest to the health sciences; (ii) the fundamental thermodynamic and kinetic properties that are associated with chain-breaking antioxidants; (iii) the unique interfacial nature of the apparent reaction of the tocopherol free radical (vitamin E radical) and vitamin C; and (iv) presents a hierarchy, or pecking order, for free radical electron (hydrogen atom) transfer reactions.

  10. Characterization of Kevlar 49 fibers by electron paramagnetic resonance. Final report, 20 May 1981-20 June 1982. [Radicals induced by ultraviolet or fracture

    SciTech Connect

    Brown, I.M.; Sandreczki, T.C.

    1982-06-20

    EPR was used to investigate the free radicals created in Kevlar 49 fibers by stress-induced and photo-induced macromolecular chain scissions. Mn/sup +2/ ions were identified from the EPR spectrum of frozen solutions of concentrated sulfuric acid containing Kevlar 49. Other ions present are Cu/sup +2/, and possibly Fe/sup +3/, Cr/sup +3/, and Ti/sup +3/. EPR lineshape anisotropy indicates that some of the metal ions and first coordinate spheres are oriented. The concentration of stress-induced radicals (2 x 10/sup 10/ per filament) suggest that chain scission occurs in more weak planes than are estimated to exist in the fracture surfaces of the fiber core. These radicals are unstable in air and have some aromatic character. Several different types of radicals were obtained following uv irradiations of the Kevlar 49 fibers in vacuum (photodegradative radicals) and in air (photo-oxidative radicals). The photodegradative radicals are identified with primary radicals involved in the photo-Fries rearrangement reaction, secondary radicals formed as a result of a hydrogen atom abstraction by the primary radical, and/or ketyl radicals produced as a result of uv irradiation of the photo-Fries rearrangement product. The photo-oxidative radicals are identified with the uv irradiation products of a peroxide intermediate. Lineshape anisotropy indicates that both radical types are oriented. 31 figures.

  11. Free radicals, antioxidants and functional foods: Impact on human health

    PubMed Central

    Lobo, V.; Patil, A.; Phatak, A.; Chandra, N.

    2010-01-01

    In recent years, there has been a great deal of attention toward the field of free radical chemistry. Free radicals reactive oxygen species and reactive nitrogen species are generated by our body by various endogenous systems, exposure to different physiochemical conditions or pathological states. A balance between free radicals and antioxidants is necessary for proper physiological function. If free radicals overwhelm the body's ability to regulate them, a condition known as oxidative stress ensues. Free radicals thus adversely alter lipids, proteins, and DNA and trigger a number of human diseases. Hence application of external source of antioxidants can assist in coping this oxidative stress. Synthetic antioxidants such as butylated hydroxytoluene and butylated hydroxyanisole have recently been reported to be dangerous for human health. Thus, the search for effective, nontoxic natural compounds with antioxidative activity has been intensified in recent years. The present review provides a brief overview on oxidative stress mediated cellular damages and role of dietary antioxidants as functional foods in the management of human diseases. PMID:22228951

  12. Effect of brain-derived neurotrophic factor on activity-regulated cytoskeleton-associated protein gene expression in primary frontal cortical neurons. Comparison with NMDA and AMPA.

    PubMed

    El-Sayed, Mona; Hofman-Bang, Jacob; Mikkelsen, Jens D

    2011-06-25

    The effect of brain-derived neurotrophic factor (BDNF) on activity-regulated cytoskeleton-associated protein (Arc) mRNA levels in primary neuronal cultures of rat frontal cortex was characterized pharmacologically and compared to the effect on expression of c-fos, bdnf, neuritin, cox-2 as examples of other immediate early genes. BDNF induced a very strong increase (around 100 fold) in Arc mRNA and the maximal effect seen at 25 ng/ml. The effect was dose-dependent with EC50 around 1.6 ng/ml. The time profile revealed a significant effect after 25 min. BDNF also increased levels of c-Fos, neuritin and BDNF mRNA, but not COX-2 mRNA. The pharmacological profile of NMDA and AMPA-induced arc gene expression in frontal cortical neurons was compared to BDNF. NMDA and AMPA increased Arc mRNA but their maximal effect did not exceed 20-fold. The effect of AMPA was completely blocked by the NMDA receptor antagonist MK-801. Further, the relative amount of Arc mRNA compared to c-Fos mRNA was higher for BDNF, equal for NMDA and lower for AMPA. These results demonstrate BDNF to be a highly potent and efficient inducer of arc gene expression in vitro, emphasizing the role of this growth factor in synaptic plasticity in the frontal cortex. PMID:21515256

  13. Fine-tuning of a radical-based reaction by radical S-adenosyl-L-methionine tryptophan lyase.

    PubMed

    Sicoli, Giuseppe; Mouesca, Jean-Marie; Zeppieri, Laura; Amara, Patricia; Martin, Lydie; Barra, Anne-Laure; Fontecilla-Camps, Juan C; Gambarelli, Serge; Nicolet, Yvain

    2016-03-18

    The radical S-adenosyl-L-methionine tryptophan lyase NosL converts L-tryptophan into 3-methylindolic acid, which is a precursor in the synthesis of the thiopeptide antibiotic nosiheptide. Using electron paramagnetic resonance spectroscopy and multiple L-tryptophan isotopologues, we trapped and characterized radical intermediates that indicate a carboxyl fragment migration mechanism for NosL. This is in contrast to a proposed fragmentation-recombination mechanism that implied Cα-Cβ bond cleavage of L-tryptophan. Although NosL resembles related tyrosine lyases, subtle substrate motions in its active site are responsible for a fine-tuned radical chemistry, which selects the Cα-C bond for disruption. This mechanism highlights evolutionary adaptation to structural constraints in proteins as a route to alternative enzyme function.

  14. Students' Ideas and Radical Constructivism

    ERIC Educational Resources Information Center

    Sánchez Gómez, Pedro J.

    2016-01-01

    In this article, I study, from the point of view of the analytic philosophy of mind, the compatibility of students' ideas studies (SIS) with radical constructivism (RC). I demonstrate that RC is based on a psychology of "narrow mental states"; that is, the idea that the mental content of an individual can be fully characterised without…

  15. The Other Women: Radicalizing Feminism.

    ERIC Educational Resources Information Center

    Puigvert, Lidia; Darder, Antonia; Merrill, Barbara; de los Reyes, Eileen; Stromquist, Nelly

    A recent international symposium on radicalizing feminism explored ways of developing a dialogic feminism that emphasizes working in different settings under the common goal of including women who have been invisible in the dominant feminist literature by furthering theories and practices based on the principles of dialogic feminism. The seminar…

  16. The Kjeldahl method as a primary reference procedure for total protein in certified reference materials used in clinical chemistry. II. Selection of direct Kjeldahl analysis and its preliminary performance parameters.

    PubMed

    Vinklárková, Bára; Chromý, Vratislav; Šprongl, Luděk; Bittová, Miroslava; Rikanová, Milena; Ohnútková, Ivana; Žaludová, Lenka

    2015-01-01

    To select a Kjeldahl procedure suitable for the determination of total protein in reference materials used in laboratory medicine, we reviewed in our previous article Kjeldahl methods adopted by clinical chemistry and found an indirect two-step analysis by total Kjeldahl nitrogen corrected for its nonprotein nitrogen and a direct analysis made on isolated protein precipitates. In this article, we compare both procedures on various reference materials. An indirect Kjeldahl method gave falsely lower results than a direct analysis. Preliminary performance parameters qualify the direct Kjeldahl analysis as a suitable primary reference procedure for the certification of total protein in reference laboratories.

  17. Comparative proteomic analysis of primary schwann cells and a spontaneously immortalized schwann cell line RSC 96: a comprehensive overview with a focus on cell adhesion and migration related proteins.

    PubMed

    Ji, Yuhua; Shen, Mi; Wang, Xin; Zhang, Shuqiang; Yu, Shu; Chen, Gang; Gu, Xiaosong; Ding, Fei

    2012-06-01

    Schwann cells (SCs) are the principal glial cells of the peripheral nervous system (PNS). As a result of tissue heterogeneity and difficulties in the isolation and culture of primary SCs, a considerable understanding of SC biology is obtained from SC lines. However, the differences between the primary SCs and SC lines remain uncertain. In the present study, quantitative proteomic analysis based on isobaric tags for relative and absolute quantitation (iTRAQ) labeling was conducted to obtain an unbiased view of the proteomic profiles of primary rat SCs and RSC96, a spontaneously immortalized rat SC line. Out of 1757 identified proteins (FDR < 1%), 1702 were quantified, while 61 and 78 were found to be, respectively, up- or down-regulated (90% confidence interval) in RSC96. Bioinformatics analysis indicated the unique features of spontaneous immortalization, illustrated the dedifferentiated state of RSC96, and highlighted a panel of novel proteins associated with cell adhesion and migration including CADM4, FERMT2, and MCAM. Selected proteomic data and the requirement of these novel proteins in SC adhesion and migration were properly validated. Taken together, our data collectively revealed proteome differences between primary SCs and RSC96, validated several differentially expressed proteins with potential biological significance, and generated a database that may serve as a useful resource for studies of SC biology and pathology.

  18. Intravenous immunoglobulin treatment preserves and protects primary rat hippocampal neurons and primary human brain cultures against oxidative insults.

    PubMed

    Lahiri, Debomoy K; Ray, Balmiki

    2014-01-01

    Alzheimer's disease (AD) is characterized by deleterious accumulation of amyloid-β (Aβ) peptide into senile plaque, neurofibrillary tangles formed from hyperphosphorylated tau protein, and loss of cholinergic synapses in the cerebral cortex. The deposition of Aβ-loaded plaques results in microglial activation and subsequent production of reactive oxygen species (ROS), including free radicals. Neurons in aging and AD brains are particularly vulnerable to ROS and other toxic stimuli. Therefore, agents that decrease the vulnerability of neurons against ROS may provide therapeutic values for the treatment or prevention of AD. In the present study, our goal was to test whether intravenous immunoglobulin (IVIG) treatment could preserve as well as protect neurons from oxidative damage. We report that treatment with IVIG protects neuronal viability and synaptic proteins in primary rat hippocampal neurons. Further, we demonstrate the tolerability of IVIG treatment in the primary human fetal mixed brain cultures. Indeed, a high dose (20 mg/ml) of IVIG treatment was well-tolerated by primary human brain cultures that exhibit a normal neuronal phenotype. We also observed a potent neuropreservatory effect of IVIG against ROS-mediated oxidative insults in these human fetal brain cultures. These results indicate that IVIG treatment has great potential to preserve and protect primary human neuronal-enriched cultures and to potentially rescue dying neurons from oxidative insults. Therefore, our findings suggest that IVIG treatment may represent an important therapeutic agent for clinical trials designed to prevent and delay the onset of neurodegeneration as well as AD pathology. PMID:25115544

  19. New Evidence for Hydroxyalkyl Radicals and Light- and Thermally Induced Trapped Electron Reactions in Rhamnose.

    PubMed

    Aalbergsjø, Siv G; Sagstuen, Einar

    2015-08-01

    Radical formation and trapping of radicals in X-irradiated crystals of rhamnose at 6 K were investigated using electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and ENDOR-induced EPR (EIE) techniques, complemented with periodic density functional theory (DFT) calculations. The two major radical species at 6 K were the O4-centered alkoxy radical and the intermolecularly trapped electron (IMTE), previously also detected by other authors. The current experimental results provided hyperfine coupling constants for these two species in good agreement with the previous data, thus providing a consistency check that improves their credibility. In addition to the O4-centered alkoxy radical and the IMTE, the C3-centered and C5-centered hydroxyalkyl radicals are the most prominent primary species at 6 K. The C3-centered radical appears in two slightly different conformations at 6 K, designated C and D. The C5-centered radical exhibits a coupling to a methyl group with tunneling rotation at 6 K, and analysis of one of the rotational substates (A) of the spin system yielded an understanding of the structure of this radical. Visible light bleaching of the IMTE at 6 K led to the C3-centered radical C, and thermal annealing above 6 K resulted in a conversion of the C to the D conformation. In addition, thermal annealing releases the IMTE, apparently resulting in the formation of the C2-centered radical. It is possible that the thermal decay of the IMTE also contributes to a small part of the C3-centered radical (D) population at 85 K. There are several other products trapped in rhamnose crystals directly after irradiation at 6 K, among which are resonance lines due to the C2 H-abstraction product. However, these other products are minority species and were not fully characterized in the current work.

  20. Observation of OH radicals produced by pulsed discharges on the surface of a liquid

    NASA Astrophysics Data System (ADS)

    Kanazawa, Seiji; Kawano, Hirokazu; Watanabe, Satoshi; Furuki, Takashi; Akamine, Shuichi; Ichiki, Ryuta; Ohkubo, Toshikazu; Kocik, Marek; Mizeraczyk, Jerzy

    2011-06-01

    The hydroxyl radical (OH) plays an important role in plasma chemistry at atmospheric pressure. OH radicals have a higher oxidation potential compared with other oxidative species such as free radical O, atomic oxygen, hydroperoxyl radical (HO2), hydrogen peroxide(H2O2) and ozone. In this study, surface discharges on liquids (water and its solutions) were investigated experimentally. A pulsed streamer discharge was generated on the liquid surface using a point-to-plane electrode geometry. The primary generation process of OH radicals is closely related to the streamer propagation, and the subsequent secondary process after the discharge has an influence on the chemical reaction. Taking into account the timescale of these processes, we investigated the behavior of OH radicals using two different diagnostic methods. Time evolution of the ground-state OH radicals above the liquid surface after the discharge was observed by a laser-induced fluorescence (LIF) technique. In order to observe the ground-state OH, an OH [A 2∑+(v' = 1) <-- X 2Π(v'' = 0)] system at 282 nm was used. As the secondary process, a portion of OH radicals diffused from gas phase to the liquid surface and dissolved in the liquid. These dissolved OH radicals were measured by a chemical probe method. Terephthalic acid was used as an OH radical trap and fluorescence of the resulting 2-hydroxyterephthalic acid was measured. This paper directly presents visualization of OH radicals over the liquid surface by means of LIF, and indirectly describes OH radicals dissolved in water by means of a chemical method.

  1. Photoisomerization and photodissociation dynamics of reactive free radicals

    SciTech Connect

    Bise, Ryan T.

    2000-08-24

    The photofragmentation pathways of chemically reactive free radicals have been examined using the technique of fast beam photofragment translational spectroscopy. Measurements of the photodissociation cross-sections, product branching ratios, product state energy distributions, and angular distributions provide insight into the excited state potential energy surfaces and nonadiabatic processes involved in the dissociation mechanisms. Photodissociation spectroscopy and dynamics of the predissociative {tilde A}{sup 2}A{sub 1} and {tilde B}{sup 2}A{sub 2} states of CH{sub 3}S have been investigated. At all photon energies, CH{sub 3} + S({sup 3}P{sub j}), was the main reaction channel. The translational energy distributions reveal resolved structure corresponding to vibrational excitation of the CH{sub 3} umbrella mode and the S({sup 3}P{sub j}) fine-structure distribution from which the nature of the coupled repulsive surfaces is inferred. Dissociation rates are deduced from the photofragment angular distributions, which depend intimately on the degree of vibrational excitation in the C-S stretch. Nitrogen combustion radicals, NCN, CNN and HNCN have also been studied. For all three radicals, the elimination of molecular nitrogen is the primary reaction channel. Excitation to linear excited triplet and singlet electronic states of the NCN radical generates resolved vibrational structure of the N{sub 2} photofragment. The relatively low fragment rotational excitation suggests dissociation via a symmetric C{sub 2V} transition state. Resolved vibrational structure of the N{sub 2} photofragment is also observed in the photodissociation of the HNCN radical. The fragment vibrational and rotational distributions broaden with increased excitation energy. Simple dissociation models suggest that the HNCN radical isomerizes to a cyclic intermediate (c-HCNN) which then dissociates via a tight cyclic transition state. In contrast to the radicals mentioned above, resolved

  2. [Yield of pigment cation-radicals in the reaction of quinone photooxidation of chlorophyll].

    PubMed

    Kostikov, A P; Sadovnikova, N A; Evstigneev, V B

    1976-01-01

    Photoinduced transfer of electrons in alkohol solutions of chlorophyll and its deuterated analog, deuterochlorophyll containing the quinoses: p-benzoquinone, chloranyl, duroquinone, 1,4-naftoquinone and ubiquinone (coenzyme Q6) is studied. It is shown that pigment cation-radical and quinone anion-radical are the primary products of photoreaction. A relationship between stationary concentrations of deuterochlorophyll and p-benzoquinone radicals and quinone concentration in solution is obtained. The reaction mechanism and causes of other authors' (G. Tollin et al.) failure in finding pigment cation-radicals which are formed in the reaction of the latter with quinoses are discussed. It is shown that optimal conditions for accumulating photoinduced cation-radicals of the pigment in pigment solutions of chlorophyll with quinones are lowered temperature, high viscosity of the solvent, low pH of the solution, careful purification of the quinone from hydroquinone admixture.

  3. Platelet-derived growth factor and reactive oxygen species (ROS) regulate Ras protein levels in primary human fibroblasts via ERK1/2. Amplification of ROS and Ras in systemic sclerosis fibroblasts.

    PubMed

    Svegliati, Silvia; Cancello, Raffaella; Sambo, Paola; Luchetti, Michele; Paroncini, Paolo; Orlandini, Guido; Discepoli, Giancarlo; Paterno, Roberto; Santillo, Mariarosaria; Cuozzo, Concetta; Cassano, Silvana; Avvedimento, Enrico V; Gabrielli, Armando

    2005-10-28