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Sample records for profiler adp nwhi

  1. CRED Acoustic Doppler Profiler (ADP); NWHI, MID; Long: -177.42181, Lat: 28.21826 (WGS84); Sensor Depth: 1.83m; Data Range: 20080926-20090321.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Data from Coral Reef Ecosystem Division (CRED), NOAA Pacific Island Fisheries Science Center Acoustic Doppler Profilers (ADP) provide a time series of water current...

  2. Cost Earnings Survey of NWHI Bottomfish Fleets (2003)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — During May-August 2004, face-to-face interviews with owners and/or captains of all NWHI bottomfish vessels were conducted to collect primary information on costs of...

  3. NWHI Basking Green Turtle Data (Turtle Sightings from Seal Surveys)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set contains records of green turtle sightings in the Northwestern Hawaiian Islands (NWHI) since 1982 at Lisianski Island, and since 1983 for most other...

  4. EX1504L1 Water Column Summary Report and Profile Data Collection

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — A complete set of water column profile data and CTD Summary Report (if generated) generated by the Okeanos Explorer during EX1504L1: CAPSTONE NWHI & Johnston...

  5. Hydrofluoric Acid-Based Derivatization Strategy To Profile PARP-1 ADP-Ribosylation by LC-MS/MS.

    Science.gov (United States)

    Gagné, Jean-Philippe; Langelier, Marie-France; Pascal, John M; Poirier, Guy G

    2018-06-11

    Despite significant advances in the development of mass spectrometry-based methods for the identification of protein ADP-ribosylation, current protocols suffer from several drawbacks that preclude their widespread applicability. Given the intrinsic heterogeneous nature of poly(ADP-ribose), a number of strategies have been developed to generate simple derivatives for effective interrogation of protein databases and site-specific localization of the modified residues. Currently, the generation of spectral signatures indicative of ADP-ribosylation rely on chemical or enzymatic conversion of the modification to a single mass increment. Still, limitations arise from the lability of the poly(ADP-ribose) remnant during tandem mass spectrometry, the varying susceptibilities of different ADP-ribose-protein bonds to chemical hydrolysis, or the context dependence of enzyme-catalyzed reactions. Here, we present a chemical-based derivatization method applicable to the confident identification of site-specific ADP-ribosylation by conventional mass spectrometry on any targeted amino acid residue. Using PARP-1 as a model protein, we report that treatment of ADP-ribosylated peptides with hydrofluoric acid generates a specific +132 Da mass signature that corresponds to the decomposition of mono- and poly(ADP-ribosylated) peptides into ribose adducts as a consequence of the cleavage of the phosphorus-oxygen bonds.

  6. Deciphering of ADP-induced, phosphotyrosine-dependent signaling networks in human platelets by Src-homology 2 region (SH2)-profiling.

    Science.gov (United States)

    Schweigel, Hardy; Geiger, Jörg; Beck, Florian; Buhs, Sophia; Gerull, Helwe; Walter, Ulrich; Sickmann, Albert; Nollau, Peter

    2013-03-01

    Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Activity-based assay for human mono-ADP-ribosyltransferases ARTD7/PARP15 and ARTD10/PARP10 aimed at screening and profiling inhibitors.

    Science.gov (United States)

    Venkannagari, Harikanth; Fallarero, Adyary; Feijs, Karla L H; Lüscher, Bernhard; Lehtiö, Lari

    2013-05-13

    Poly(ADP-ribose) polymerases (PARPs) or diphtheria toxin like ADP-ribosyl transferases (ARTDs) are enzymes that catalyze the covalent modification of proteins by attachment of ADP-ribose units to the target amino acid residues or to the growing chain of ADP-ribose. A subclass of the ARTD superfamily consists of mono-ADP-ribosyl transferases that are thought to modify themselves and other substrate proteins by covalently adding only a single ADP-ribose moiety to the target. Many of the ARTD enzymes are either established or potential drug targets and a functional activity assay for them will be a valuable tool to identify selective inhibitors for each enzyme. Existing assays are not directly applicable for screening of inhibitors due to the different nature of the reaction and different target molecules. We modified and applied a fluorescence-based assay previously described for PARP1/ARTD1 and tankyrase/ARTD5 for screening of PARP10/ARTD10 and PARP15/ARTD7 inhibitors. The assay measures the amount of NAD(+) present after chemically converting it to a fluorescent analog. We demonstrate that by using an excess of a recombinant acceptor protein the performance of the activity-based assay is excellent for screening of compound libraries. The assay is homogenous and cost effective, making it possible to test relatively large compound libraries. This method can be used to screen inhibitors of mono-ARTDs and profile inhibitors of the enzyme class. The assay was optimized for ARTD10 and ARTD7, but it can be directly applied to other mono-ARTDs of the ARTD superfamily. Profiling of known ARTD inhibitors against ARTD10 and ARTD7 in a validatory screening identified the best inhibitors with submicromolar potencies. Only few of the tested ARTD inhibitors were potent, implicating that there is a need to screen new compound scaffolds. This is needed to create small molecules that could serve as biological probes and potential starting points for drug discovery projects against

  8. The Level of AdpA Directly Affects Expression of Developmental Genes in Streptomyces coelicolor ▿ †

    OpenAIRE

    Wolański, Marcin; Donczew, Rafał; Kois-Ostrowska, Agnieszka; Masiewicz, Paweł; Jakimowicz, Dagmara; Zakrzewska-Czerwińska, Jolanta

    2011-01-01

    AdpA is a key regulator of morphological differentiation in Streptomyces. In contrast to Streptomyces griseus, relatively little is known about AdpA protein functions in Streptomyces coelicolor. Here, we report for the first time the translation accumulation profile of the S. coelicolor adpA (adpASc) gene; the level of S. coelicolor AdpA (AdpASc) increased, reaching a maximum in the early stage of aerial mycelium formation (after 36 h), and remained relatively stable for the next several hour...

  9. CRED Acoustic Doppler Profiler (ADP); AMSM, ROS; Long: -168.15481, Lat: -14.53510 (WGS84); Sensor Depth: 7.01m; Data Range: 20080311-20080314.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Data from Coral Reef Ecosystem Division (CRED), NOAA Pacific Island Fisheries Science Center Acoustic Doppler Profilers (ADP) provide a time series of water current...

  10. French Frigate Shoals, NWHI, 2005 Sea Surface Temperature and Meteorological Enhanced (Iridium) Mooring - CRED CREWS Near Real Time and Historical Data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Site - French Frigate Shoals, NWHI (23.85678, -166.27183 ) ARGOS ID 261-003 Time series data from this mooring provide high resolution sea surface temperature, and...

  11. ADP-ribosyl-N₃: A Versatile Precursor for Divergent Syntheses of ADP-ribosylated Compounds.

    Science.gov (United States)

    Li, Lingjun; Li, Qianqian; Ding, Shengqiang; Xin, Pengyang; Zhang, Yuqin; Huang, Shenlong; Zhang, Guisheng

    2017-08-14

    Adenosine diphosphate-ribose (ADP-ribose) and its derivatives play important roles in a series of complex physiological procedures. The design and synthesis of artificial ADP-ribosylated compounds is an efficient way to develop valuable chemical biology tools and discover new drug candidates. However, the synthesis of ADP-ribosylated compounds is currently difficult due to structural complexity, easily broken pyrophosphate bond and high hydrophilicity. In this paper, ADP-ribosyl-N₃ was designed and synthesized for the first time. With ADP-ribosyl-N₃ as the key precursor, a divergent post-modification strategy was developed to prepare structurally diverse ADP-ribosylated compounds including novel nucleotides and peptides bearing ADP-ribosyl moieties.

  12. Studying Catabolism of Protein ADP-Ribosylation.

    Science.gov (United States)

    Palazzo, Luca; James, Dominic I; Waddell, Ian D; Ahel, Ivan

    2017-01-01

    Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the β-nicotinamide adenine dinucleotide (β-NAD + ) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.

  13. Toward a unified nomenclature for mammalian ADP-ribosyltransferases.

    Science.gov (United States)

    Hottiger, Michael O; Hassa, Paul O; Lüscher, Bernhard; Schüler, Herwig; Koch-Nolte, Friedrich

    2010-04-01

    ADP-ribosylation is a post-translational modification of proteins catalyzed by ADP-ribosyltransferases. It comprises the transfer of the ADP-ribose moiety from NAD+ to specific amino acid residues on substrate proteins or to ADP-ribose itself. Currently, 22 human genes encoding proteins that possess an ADP-ribosyltransferase catalytic domain are known. Recent structural and enzymological evidence of poly(ADP-ribose)polymerase (PARP) family members demonstrate that earlier proposed names and classifications of these proteins are no longer accurate. Here we summarize these new findings and propose a new consensus nomenclature for all ADP-ribosyltransferases (ARTs) based on the catalyzed reaction and on structural features. A unified nomenclature would facilitate communication between researchers both inside and outside the ADP-ribosylation field. 2009 Elsevier Ltd. All rights reserved.

  14. 26 CFR 1.401(k)-2 - ADP test.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 5 2010-04-01 2010-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2 Internal... TAXES Pension, Profit-Sharing, Stock Bonus Plans, Etc. § 1.401(k)-2 ADP test. (a) Actual deferral percentage (ADP) test—(1) In general—(i) ADP test formula. A cash or deferred arrangement satisfies the ADP...

  15. Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

    International Nuclear Information System (INIS)

    Okita, Naoyuki; Ashizawa, Daisuke; Ohta, Ryo; Abe, Hideaki; Tanuma, Sei-ichi

    2010-01-01

    Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V max ) and the michaelis constant (k m ) of PARG reaction were 4.46 μM and 128.33 μmol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 μM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.

  16. ADP-ribosylation of dinitrogenase reductase in Rhodobacter capsulatus

    International Nuclear Information System (INIS)

    Jouanneau, Y.; Roby, C.; Meyer, C.M.; Vignais, P.M.

    1989-01-01

    In the photosynthetic bacterium Rhodobacter capsulatus, nitrogenase is regulated by a reversible covalent modification of Fe protein or dinitrogenase reductase (Rc2). The linkage of the modifying group to inactive Rc2 was found to be sensitive to alkali and to neutral hydroxylamine. Complete release of the modifying group was achieved by incubation of inactive Rc2 in 0.4 or 1 M hydroxylamine. After hydroxylamine treatment of the Rc2 preparation, the modifying group could be isolated and purified by affinity chromatography and ion-exchange HPLC. The modifying group comigrated with ADP-ribose on both ion-exchange HPLC and thin-layer chromatography. Analyses by 31 P NMR spectroscopy and mass spectrometry provided further evidence that the modifying group was ADP-ribose. The NMR spectrum of inactive Rc2 exhibited signals characteristic of ADP-ribose; integration of these signals allowed calculation of a molar ration ADP-ribose/Rc2 of 0.63. A hexapeptide carrying the ADP-ribose moiety was purified from a subtilisin digest of inactive Rc2. The structure of this peptide, determined by amino acid analysis and sequencing, is Gly-Arg(ADP-ribose)-Gly-Val-Ile-Thr. This structure allows identification of the binding site for ADP-ribose as Arg 101 of the polypeptide chain of Rc2. It is concluded that nitrogenase activity in R. capsulatus is regulated by reversible ADP-ribosylation of a specific arginyl residue of dinitrogenase reductase

  17. Symposium cellular response to DNA damage the role of poly(ADP-ribose) poly(ADP-ribose) in the cellular response to DNA damage

    International Nuclear Information System (INIS)

    Berger, N.A.

    1985-01-01

    Poly(ADP-ribose) polymerase is a chromatin-bound enzyme which, on activation by DNA strand breaks, catalyzes the successive transfer of ADP-ribose units from NAD to nuclear proteins. Poly(ADP-ribose) synthesis is stimulated by DNA strand breaks, and the polymer may alter the structure and/or function of chromosomal proteins to facilitate the DNA repair process. Inhibitors of Poly(ADP-ribose) polymerase or deficiencies of the substrate, NAD, lead to retardation of the DNA repair process. When DNA strand breaks are extensive or when breaks fail to be repaired, the stimulus for activation of Poly(ADP-ribose) persists and the activated enzyme is capable of totaly consuming cellular pools of NAD. Depletion of NAD and consequent lowering of cellular ATP pools, due to activation of Poly(ADP-ribose) polymerase, may account for rapid cell death before DNA repair takes place and before the genetic effects of DNA damage become manifest

  18. Poly-ADP-ribosylation of proteins responds to cellular perturbations

    International Nuclear Information System (INIS)

    Schneeweiss, F.H.A.; Sharan, R.N.

    1999-01-01

    From the results presented above it is quite obvious that poly-ADP-ribosylation reaction is a sensitive parameter to monitor cellular responses to a wide variety of perturbations. Having developed a monolayer assay system using 32 P-NAD + as a marker, it has become possible to measure levels of cellular ADP-ribosylation more precisely. It has been demonstrated that the trigger of poly-ADP-ribosylation reaction may involve different cellular components for different perturbations. In this, membrane has been found to be important. The study has been particularly informative in the realm of DNA damage and repair following qualitatively different radiation assaults. As poly-ADP-ribosylation in eukaryotic cells primarily affects chromosomal proteins, notably histones, the reaction is strongly triggered in response to single and double strand breaks in DNA. Therefore, level of cellular poly-ADP-ribosylation can potentially be used as a biosensor of radiation induced strand breaks and can be specially useful in clinical monitoring of progress of radiotherapy. The assay of poly-ADP-ribosylation, however, requires use of radiolabelled tracer, e.g. 32 P-NAD + . Due to this, study of poly-ADP-ribosylation can not be extended to monitor effects of incorporated radionuclides. In order to overcome this shortcoming and to make the assay more sensitive and quick, a Western blot immunoassay has been developed. The preliminary indications are that the immunoassay of poly-ADP-ribosylation will fulfil the requirements to use poly-ADP-ribosylation as a sensitive, convenient and clinically applicable biosensor of cell response not only to radiations but also to different perturbations. (orig.)

  19. High-resolution velocimetry in energetic tidal currents using a convergent-beam acoustic Doppler profiler

    Science.gov (United States)

    Sellar, Brian; Harding, Samuel; Richmond, Marshall

    2015-08-01

    An array of single-beam acoustic Doppler profilers has been developed for the high resolution measurement of three-dimensional tidal flow velocities and subsequently tested in an energetic tidal site. This configuration has been developed to increase spatial resolution of velocity measurements in comparison to conventional acoustic Doppler profilers (ADPs) which characteristically use divergent acoustic beams emanating from a single instrument. This is achieved using geometrically convergent acoustic beams creating a sample volume at the focal point of 0.03 m3. Away from the focal point, the array is also able to simultaneously reconstruct three-dimensional velocity components in a profile throughout the water column, and is referred to herein as a convergent-beam acoustic Doppler profiler (C-ADP). Mid-depth profiling is achieved through integration of the sensor platform with the operational commercial-scale Alstom 1 MW DeepGen-IV Tidal Turbine deployed at the European Marine Energy Center, Orkney Isles, UK. This proof-of-concept paper outlines the C-ADP system configuration and comparison to measurements provided by co-installed reference instrumentation. Comparison of C-ADP to standard divergent ADP (D-ADP) velocity measurements reveals a mean difference of 8 mm s-1, standard deviation of 18 mm s-1, and an order of magnitude reduction in realisable length scale. C-ADP focal point measurements compared to a proximal single-beam reference show peak cross-correlation coefficient of 0.96 over 4.0 s averaging period and a 47% reduction in Doppler noise. The dual functionality of the C-ADP as a profiling instrument with a high resolution focal point make this configuration a unique and valuable advancement in underwater velocimetry enabling improved quantification of flow turbulence. Since waves are simultaneously measured via profiled velocities, pressure measurements and surface detection, it is expected that derivatives of this system will be a powerful tool in

  20. Poly(ADP-ribose) Glycohydrolase and Poly(ADP-ribose)-interacting Protein Hrp38 Regulate Pattern Formation during Drosophila Eye Development

    Science.gov (United States)

    Ji, Yingbiao; Jarnik, Michael; Tulin, Alexei V.

    2013-01-01

    Drosophila Hrp38, a homolog of human hnRNP A1, has been shown to regulate splicing, but its function can be modified by poly(ADP-ribosyl)ation. Notwithstanding such findings, our understanding of the roles of poly(ADP-ribosyl)ated Hrp38 on development is limited. Here, we have demonstrated that Hrp38 is essential for fly eye development based on a rough-eye phenotype with disorganized ommatidia observed in adult escapers of the hrp38 mutant. We also observed that Poly(ADP-ribose) Glycohydrolase (Parg) loss-of-function, which caused increased Hrp38 poly(ADP-ribosyl)ation, also resulted in the rough-eye phenotype with disrupted ommatidial lattice and reduced number of photoreceptor cells. In addition, ectopic expression of DE-cadherin, which is required for retinal morphogenesis, fully rescued the rough-eye phenotype of the hrp38 mutant. Similarly, Parg mutant eye clones had decreased expression level of DE-cadherin with orientation defects, which is reminiscent of DE-cadherin mutant eye phenotype. Therefore, our results suggest that Hrp38 poly(ADP-ribosyl)ation controls eye pattern formation via regulation of DE-cadherin expression, a finding which has implications for understanding the pathogenic mechanisms of Hrp38-related Fragile X syndrome and PARP1-related retinal degeneration diseases. PMID:23711619

  1. Fine-tuning of Smad protein function by poly(ADP-ribose polymerases and poly(ADP-ribose glycohydrolase during transforming growth factor β signaling.

    Directory of Open Access Journals (Sweden)

    Markus Dahl

    Full Text Available Initiation, amplitude, duration and termination of transforming growth factor β (TGFβ signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose polymerase 1 (PARP-1 negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose glycohydrolase (PARG can remove poly(ADP-ribose chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFβ signaling.A robust cell model of TGFβ signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFβ stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.TGFβ signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFβ target genes (fibronectin, Smad7 and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFβ-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.Nuclear Smad function is negatively

  2. Dielectric, thermal and mechanical properties of ADP doped PVA composites

    Science.gov (United States)

    Naik, Jagadish; Bhajantri, R. F.; Ravindrachary, V.; Rathod, Sunil G.; Sheela, T.; Naik, Ishwar

    2015-06-01

    Polymer composites of poly(vinyl alcohol) (PVA), doped with different concentrations of ammonium dihydrogen phosphate (ADP) has been prepared by solution casting. The formation of complexation between ADP and PVA was confirmed with the help of Fourier transforms infrared (FTIR) spectroscopy. Thermogravimetric analysis (TGA) shows thermal stability of the prepared composites. Impedance analyzer study revealed the increase in dielectric constant and loss with increase the ADP concentration and the strain rate of the prepared composites decreases with ADP concentration.

  3. ADP Analysis project for the Human Resources Management Division

    Science.gov (United States)

    Tureman, Robert L., Jr.

    1993-01-01

    The ADP (Automated Data Processing) Analysis Project was conducted for the Human Resources Management Division (HRMD) of NASA's Langley Research Center. The three major areas of work in the project were computer support, automated inventory analysis, and an ADP study for the Division. The goal of the computer support work was to determine automation needs of Division personnel and help them solve computing problems. The goal of automated inventory analysis was to find a way to analyze installed software and usage on a Macintosh. Finally, the ADP functional systems study for the Division was designed to assess future HRMD needs concerning ADP organization and activities.

  4. Molecular characterization of a novel intracellular ADP-ribosyl cyclase.

    Directory of Open Access Journals (Sweden)

    Dev Churamani

    2007-08-01

    Full Text Available ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates.Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1 is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained.Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized.

  5. Topographic study of the ADP/ATP transport protein. Localization of ADP and atractyloside fixation sites. Identification of the antigenic domains

    International Nuclear Information System (INIS)

    Boulay, Francois

    1983-01-01

    The objectives of this research thesis were: to determine the intramolecular localisation of binding sites of atractyloside and adenine-nucleotides; to determine whether antibodies obtained against the ADP/ATP carrier protein and isolated from beef heart mitochondria possess a reactivity specific to the organ or the species, where antigenic determinants are localized and whether there is conservation of the antigenic structure from one species to the other; to study how to follow and interpret conformational changes of the protein under the effect of ADP and inhibitors (carboxy-atractyloside or bongkrekic acid), and where the SH group unmasked by ADP and bongkrekic acid is localized [fr

  6. Regulation of pyruvate oxidation in blowfly flight muscle mitochondria: requirement for ADP.

    Science.gov (United States)

    Bulos, B A; Thomas, B J; Shukla, S P; Sacktor, B

    1984-11-01

    Blowfly (Phormia regina) flight muscle mitochondria oxidized pyruvate ( + proline) in the presence of either ADP (coupled respiration) or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP-uncoupled respiration). There was an absolute requirement for ADP (Km = 8.0 microM) when pyruvate oxidation was stimulated by FCCP in the presence of oligomycin. This requirement for ADP was limited to the oxidation of pyruvate; uncoupled alpha-glycerolphosphate oxidation proceeded maximally even in the absence of added ADP. Atractylate inhibited uncoupled pyruvate oxidation whether added before (greater than 99%) or after (95%) initiation of respiration with FCCP. In the presence of FCCP, oligomycin, and limiting concentrations of ADP (less than 110 microM), there was a shutoff in the uptake of oxygen. This inhibition of respiration was completely reversed by the addition of more ADP. Plots of net oxygen uptake as a function of the limiting ADP concentration were linear; the observed ADP/O ratio was 0.22 +/- 0.025. An ADP/O ratio of 0.2 was predicted if phosphorylation occurred only at the succinyl-CoA synthetase step of the tricarboxylate cycle. Experiments performed in the presence of limiting concentrations of ADP, and designed to monitor changes in the mitochondrial content of ADP and ATP, demonstrated that the shutoff in oxygen uptake was not due to the presence of a high intramitochondrial concentration of ATP. Indeed, ATP, added to the medium prior to the addition of FCCP, inhibited uncoupled pyruvate oxidation; the apparent KI was 0.8 mM. These results are consistent with the hypothesis that it is the intramitochondrial ATP/ADP ratio that is one of the controlling factors in determining the rate of flux through the tricarboxylate cycle. Changes in the mitochondrial content of citrate, isocitrate, alpha-ketoglutarate, and malate during uncoupled pyruvate oxidation in the presence of a limiting concentration of ADP were consistent with the hypothesis that the

  7. CRED Ocean Data Platform (ODP), Acoustic Doppler Profiler (ADP); NWHI, PHR; Long: -175.88112, Lat: 27.78204 (WGS84); Sensor Depth: 21.34m; Data Range: 20060922-20070805.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Ocean Data Platform (ODP) is placed on the sea floor to measure water current profiles, waves, temperature and conductivity. The ODP consists of an upward...

  8. CRED Ocean Data Platform (ODP), Acoustic Doppler Profiler (ADP); NWHI, PHR; Long: -175.88110, Lat: 27.78209 (WGS84); Sensor Depth: 21.34m; Data Range: 20070806-20070912.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Ocean Data Platform (ODP) is placed on the sea floor to measure water current profiles, waves, temperature and conductivity. The ODP consists of an upward...

  9. Poly(ADP-ribose) and the response of cells to ionizing radiation

    International Nuclear Information System (INIS)

    Oleinick, N.L.; Evans, H.H.

    1985-01-01

    The activity of poly(ADP-ribose) polymerase is stimulated by DNA damage resulting from treatment of cells with ionizing radiation, as well as with DNA-damaging chemicals. The elevated polymerase activity can be observed at doses lower than those necessary for measurable reduction in cellular NAD concentration. Several nuclear proteins, including the polymerase itself, are poly(ADP-ribosylated) at elevated levels in irradiated Chinese hamster cells. The addition of inhibitors of poly(ADP-ribose) polymerase to irradiated cells has been found to sensitize the cells to the lethal effects of the radiation, to inhibit the repair of potentially lethal damage, and to delay DNA strand break rejoining. Because of the nonspecificity of the inhibitors, however, it is as yet unknown whether their effects are directly related to the inhibition of poly(ADP-ribose) polymerase, to interference with the poly(ADP-ribosylation) of one or more chromosomal proteins, or to effects unrelated to the poly(ADP-ribosylation) process. The data are consistent with the involvement of poly(ADP-ribose) in the repair of radiation damage, but the nature of this involvement remains to be elucidated

  10. The Multiple Effect of Agricultural Development Programme's (ADP's ...

    African Journals Online (AJOL)

    This paper determined the multiplier effects of the use of Small Plot Adoption Technique (SPAT) by the Abia ADP on the income of Smallholder farmers in Abia State. The choice of Abia ADP for this research was purposive. A multi-stage random sampling technique was used in the selection of blocks, circles, 300 contact ...

  11. ADP-ribosylation of membrane components by pertussis and cholera toxin

    International Nuclear Information System (INIS)

    Ribeiro-Neto, F.A.P.; Mattera, F.; Hildebrandt, J.D.; Codina, J.; Field, J.B.; Birnbaumer, L.; Sekura, R.D.

    1985-01-01

    Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (N/sub s/) and inhibitory (N/sub i/) regulatory components of adenylyl cyclase. Cholera toxin acts on N/sub s/ by ADP-ribosylating its α/sub s/ subunit; pertussis toxin acts on N/sub i/ by ADP-ribosylating its α; subunit. By using [ 32 P]NAD + and determining the transfer of its [ 32 P]ADP-ribose moiety to membrane components, it is possible to obtain information on N/sub s/ and N/sub i/. A set of protocols is presented that can be used to study simultaneously and comparatively the susceptibility of N/sub s/ and N/sub i/ to be ADP-ribosylated by cholera and pertussis toxin

  12. Effects of epinephrine on ADP-induced changes in platelet inositol phosphates

    International Nuclear Information System (INIS)

    Vickers, J.D.; Keraly, C.L.; Kinlough-Rathbone, R.L.; Mustard, J.F.

    1986-01-01

    Epinephrine (EPI) does not aggregate rabbit platelets, but it does increase the labelling of inositol phosphate (IP) at 60s (21%, p + , in platelets prelabelled with [ 3 H] inositol. In contrast, 0.5 μM ADP which causes aggregation, increases the labelling of inositol bisphosphate (IP 2 ) by 30% (p 2 by 154% (p 2 stimulated by ADP + EPI was greater than the increase caused by ADP (p 2 due to 0.2 μM ADP + 0.6 μM EPI by 70% (p 2 by 108% (0 2 metabolism stimulated via the α-adrenergic receptor

  13. Crystallographic and biochemical analysis of the mouse poly(ADP-ribose glycohydrolase.

    Directory of Open Access Journals (Sweden)

    Zhizhi Wang

    Full Text Available Protein poly(ADP-ribosylation (PARylation regulates a number of important cellular processes. Poly(ADP-ribose glycohydrolase (PARG is the primary enzyme responsible for hydrolyzing the poly(ADP-ribose (PAR polymer in vivo. Here we report crystal structures of the mouse PARG (mPARG catalytic domain, its complexes with ADP-ribose (ADPr and a PARG inhibitor ADP-HPD, as well as four PARG catalytic residues mutants. With these structures and biochemical analysis of 20 mPARG mutants, we provide a structural basis for understanding how the PAR polymer is recognized and hydrolyzed by mPARG. The structures and activity complementation experiment also suggest how the N-terminal flexible peptide preceding the PARG catalytic domain may regulate the enzymatic activity of PARG. This study contributes to our understanding of PARG catalytic and regulatory mechanisms as well as the rational design of PARG inhibitors.

  14. Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

    Energy Technology Data Exchange (ETDEWEB)

    Scaife, R.M. (Fred Hutchinson Cancer Research Center, Seattle, WA (United States)); Wilson, L. (Univ. of California, Santa Barbara (United States)); Purich, D.L. (Univ. of Florida, Gainesville (United States))

    1992-01-14

    Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

  15. Spectrographic study of neodymium complexing with ATP and ADP

    International Nuclear Information System (INIS)

    Svetlova, I.E.; Dobrynina, N.A.; Martynenko, L.N.

    1989-01-01

    By spectrographic method neodymium complexing with ATP and ADP in aqueous solutions at different pH values has been studied. The composition of the complexes was determined by the method of isomolar series. On the basis of analysis of absorption spectra it has been ascertained that at equimolar ratio of Nd 3+ and ATP absorption band of L278A corresponds to monocomplex, and the band of 4290 A - to biscomplex. For the complexes with ADP the absorption band of 4288 A is referred to bicomplexes. The character of ATP and ADP coordination by Nd 3+ ion is considered. Stability constants of the complexes are calculated

  16. ADP-ribosylation of transducin by pertussis toxin

    International Nuclear Information System (INIS)

    Watkins, P.A.; Burns, D.L.; Kanaho, Y.; Liu, T.Y.; Hewlett, E.L.; Moss, J.

    1985-01-01

    Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T alpha and T beta gamma. T alpha (39 kDa), which is [ 32 P]ADP-ribosylated by pertussis toxin and [ 32 P]NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T beta gamma (36 kDa and approximately 10 kDa); neither component of T beta gamma was a pertussis toxin substrate. Labeling of T alpha was enhanced by T beta gamma and was maximal at approximately 1:1 molar ratio of T alpha : T beta gamma. Limited proteolysis by trypsin of T alpha in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and 32 kDa. The amino terminus of both 38- and 32 -kDa proteins was leucine, whereas that of T alpha could not be identified and was assumed to be blocked. The 32 -kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T beta gamma. Trypsin treatment of [ 32 P]ADP-ribosyl-T alpha produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T alpha (in the presence of T beta gamma) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTP gamma S), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDP beta S) but was increased by ATP. When photolyzed rhodopsin and T beta gamma were present, Gpp(NH)p and GTP gamma S decreased [ 32 P]ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed [ 32 P]ADP-ribosylation of T alpha was affected by nucleotides, rhodopsin and light in addition to T beta gamma

  17. Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors.

    Science.gov (United States)

    Thorsell, Ann-Gerd; Ekblad, Torun; Karlberg, Tobias; Löw, Mirjam; Pinto, Ana Filipa; Trésaugues, Lionel; Moche, Martin; Cohen, Michael S; Schüler, Herwig

    2017-02-23

    Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.

  18. ADP1 Affects Plant Architecture by Regulating Local Auxin Biosynthesis

    Science.gov (United States)

    Li, Shibai; Qin, Genji; Novák, Ondřej; Pěnčík, Aleš; Ljung, Karin; Aoyama, Takashi; Liu, Jingjing; Murphy, Angus; Gu, Hongya; Tsuge, Tomohiko; Qu, Li-Jia

    2014-01-01

    Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs. PMID:24391508

  19. Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

    Science.gov (United States)

    Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

    2008-12-26

    ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

  20. REDUCED THROMBOGENICITY OF VASCULAR PROSTHESES BY COATING WITH ADP-ASE

    NARCIS (Netherlands)

    VANDERLEI, B; ROBINSON, PH; BAKKER, WW; Bartels, H.

    1992-01-01

    In this pilot study ADP-ase coated polyurethane (PL) vascular prostheses and noncoated (control) PU vascular prostheses (all vascular prostheses: ID 1.5 mm, length 1,5 cm) were implanted into the carotid artery of the rabbit to test wheter ADP-ase might function as an adequate anti-thrombogenic

  1. The Dichotomy of the Poly(ADP-Ribose Polymerase-Like Thermozyme from Sulfolobus solfataricus

    Directory of Open Access Journals (Sweden)

    Maria Rosaria Faraone Mennella

    2018-01-01

    Full Text Available The first evidence of an ADP-ribosylating activity in Archaea was obtained in Sulfolobus solfataricus(strain MT-4 where a poly(ADP-ribose polymerase (PARP-like thermoprotein, defined with the acronymous PARPSso, was found. Similarly to the eukaryotic counterparts PARPSso cleaves beta-nicotinamide adenine dinucleotide to synthesize oligomers of ADP-ribose; cross-reacts with polyclonal anti-PARP-1 catalytic site antibodies; binds DNA. The main differences rely on the molecular mass (46.5 kDa and the thermophily of PARPSso which works at 80 °C. Despite the biochemical properties that allow correlating it to PARP enzymes, the N-terminal and partial amino acid sequences available suggest that PARPSso belongs to a different group of enzymes, the DING proteins, an item discussed in detail in this review.This finding makes PARPSso the first example of a DING protein in Archaea and extends the existence of DING proteins into all the biological kingdoms. PARPSsohas a cell peripheral localization, along with the edge of the cell membrane. The ADP-ribosylation reaction is reverted by a poly(ADP-ribose glycohydrolase-like activity, able to use the eukaryotic poly(ADP-ribose as a substrate too. Here we overview the research of (ADP-ribosylation in Sulfolobus solfataricus in the past thirty years and discuss the features of PARPSso common with the canonical poly(ADP-ribose polymerases, and the structure fitting with that of DING proteins.

  2. Poly (ADP-ribose) catabolism in mammalian cells exposed to DNA-damaging agents

    International Nuclear Information System (INIS)

    Alvarez-Gonzalez, R.; Althaus, F.R.

    1989-01-01

    DNA damage inflicted by the alkylating agens N-methyl-N-nitro-N-nitrosoquanidine, or by UV stimulated the catabolism of protein-bound poly (ADP-ribose) in the chromatin of cultured hepatocytes. The stimulation was highest at the largest doses of DNA-damaging treatment. As a consequence, the half-life of ADP-ribosyl polymers may drop to less than 41 s. This rapid turnover contrasts with the slow catabolism of a constitutive fraction of polymers exhibiting a half-life of 7.7 h. These data suggest that post-incisional stimulation of poly (ADP-ribose) biosynthesis in DNA-excision repair is coupled with an adaptation of poly (ADP-ribose) catabolism in mammalian cells. (Author). 37 refs.; 3 figs

  3. File list: Oth.Adp.10.RELA.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.10.RELA.AllCell hg19 TFs and others RELA Adipocyte SRX813772,SRX813773,SRX8...13775,SRX813774 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Adp.10.RELA.AllCell.bed ...

  4. File list: Oth.Adp.20.RELA.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.20.RELA.AllCell hg19 TFs and others RELA Adipocyte SRX813773,SRX813775,SRX8...13774,SRX813772 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Adp.20.RELA.AllCell.bed ...

  5. Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.

    Directory of Open Access Journals (Sweden)

    Gilles Gouspillou

    Full Text Available BACKGROUND: Mitochondrial dysfunctions appear strongly implicated in a wide range of pathologies. Therefore, there is a growing need in the determination of the normal and pathological integrated response of oxidative phosphorylation to cellular ATP demand. The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria. METHODOLOGY/PRINCIPAL FINDINGS: The proposed method is based on the simultaneous monitoring of substrate oxidation (determined polarographically and phosphorylation (determined using the glucose-hexokinase glucose-6-phosphate dehydrogenase-NADP(+ enzymatic system rates, coupled to the determination of actual ADP and ATP concentrations by bioluminescent assay. This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations. We demonstrate how the application of this method allows an accurate determination of mitochondrial affinity for ADP from both oxidation (K(mVox and phosphorylation (K(mVp rates. We also demonstrate that determination of K(mVox leads to an important overestimation of the mitochondrial affinity for ADP, indicating that mitochondrial affinity for ADP should be determined using phosphorylation rate. Finally, we show how this method allows the direct and precise determination of the mitochondrial coupling efficiency. Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method. CONCLUSIONS/SIGNIFICANCE: Because the proposed method allows the accurate determination of mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria, it also opens the route to a better understanding of functional consequences of mitochondrial adaptations/dysfunctions arising in various physiological/pathophysiological conditions.

  6. Poly (ADP-ribose) metabolism in alkylated mouse L5178Y cells

    International Nuclear Information System (INIS)

    Boyle, J.M.

    1985-01-01

    Poly ADP-ribosylation of two mouse lymphoma cell lines, L5178Y (LS) and the radiation and alkylating agent resistant derivative AII, was investigated by uptake of [ 3 H]NAD by permeabilised cells into acid-precipitable material that was sensitive to phosphodiesterase but insensitive to DNase and RNase. Basal activities in both lymphoma lines were 3-4-fold greater than in mouse L1210 leukaemia cells. However, total endogenous poly (ADP-R) polymerase activity in both L5178Y cell lines, stimulated by a large excess of DNase in the presence of Triton X-100, was less than half the activity in L1210 cells. Doses of N-methyl-N-nitrosourea (MNU) that produced 20-50% survival of colony-forming units increased poly (ADP-R) in both lymphoma lines by only 25% compared with 377% in L1210 cells when synthesis was measured immediately after a 30-min exposure of MNU. Concentrations of 3-aminobenzamide (3AB) above 2.5 mM inhibited colony-forming ability of lymphoma cells and equally inhibited uptake of [ 14 C]formate into protein, RNA and DNA indicating that 3AB behaves as a general metabolic poison. Non-toxic concentrations of 3AB potentiated cell killing by MNU to a similar degree in both lymphoma cell lines. In conclusion, the authors have found little evidence to support the hypothesis that the differential sensitivity of LS and AII is related to poly ADP-ribosylation. Compared with other mouse cells, L5178Y cells appear deficient in poly (ADP-R) polymerase and poly (ADP-R) glycohydrolase activities

  7. Effect of ADP on slow-twitch muscle fibres of the rat: implications for muscle fatigue.

    Science.gov (United States)

    Macdonald, W A; Stephenson, D G

    2006-05-15

    Slow-twitch mechanically skinned fibres from rat soleus muscle were bathed in solutions mimicking the myoplasmic environment but containing different [ADP] (0.1 microm to 1.0 mm). The effect of ADP on sarcoplasmic reticulum (SR) Ca2+-content was determined from the magnitude of caffeine-induced force responses, while temporal changes in SR Ca2+-content allowed determination of the effective rates of the SR Ca2+-pump and of the SR Ca2+-leak. The SR Ca2+-pump rate, estimated at pCa (-log10[Ca2+]) 7.8, was reduced by 20% as the [ADP] was increased from 0.1 to 40 microm, with no further alteration when the [ADP] was increased to 1.0 mm. The SR Ca2+-leak rate constant was not altered by increasing [ADP] from 0.1 to 40 microm, but was increased by 26% when the [ADP] was elevated to 1.0 mm. This ADP-induced SR Ca2+-leak was insensitive to ruthenium red but was abolished by 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), indicating that the leak pathway is via the SR Ca2+-pump and not the SR Ca2+-release channel. The decrease in SR Ca2+-pump rate and SR Ca2+-leak rate when [ADP] was increased led to a 40% decrease in SR Ca2+-loading capacity. Elevation of [ADP] had only minor direct effects on the contractile apparatus of slow-twitch fibres. These results suggest that ADP has only limited depressing effects on the contractility of slow-twitch muscle fibres. This is in contrast to the marked effects of ADP on force responses in fast-twitch muscle fibres and may contribute to the fatigue-resistant nature of slow-twitch muscle fibres.

  8. Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

    Science.gov (United States)

    De Maio, Anna; Natale, Emiliana; Rotondo, Sergio; Di Cosmo, Anna; Faraone-Mennella, Maria Rosaria

    2013-09-01

    Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity. © 2013 Elsevier Inc. All rights reserved.

  9. Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

    Science.gov (United States)

    Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

    2008-01-01

    ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain. PMID:18977760

  10. Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx.

    Science.gov (United States)

    Suárez, Gabriel A; Renda, Brian A; Dasgupta, Aurko; Barrick, Jeffrey E

    2017-09-01

    The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS 1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS 1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS 1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase ( dinP ) and a DNA damage response regulator ( umuD Ab [the umuD gene of A. baylyi ]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves

  11. File list: His.Adp.50.AllAg.White_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.50.AllAg.White_adipocytes mm9 Histone Adipocyte White adipocytes SRX800009 http://dbarchi...ve.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Adp.50.AllAg.White_adipocytes.bed ...

  12. File list: His.Adp.05.AllAg.White_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: His.Adp.10.AllAg.White_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.10.AllAg.White_adipocytes mm9 Histone Adipocyte White adipocytes SRX800009 http://dbarchi...ve.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Adp.10.AllAg.White_adipocytes.bed ...

  14. File list: His.Adp.20.AllAg.White_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.20.AllAg.White_adipocytes mm9 Histone Adipocyte White adipocytes SRX800009 http://dbarchi...ve.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Adp.20.AllAg.White_adipocytes.bed ...

  15. File list: ALL.Adp.50.AllAg.Adipose_Tissue [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.50.AllAg.Adipose_Tissue hg19 All antigens Adipocyte Adipose Tissue SRX13473...2 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.50.AllAg.Adipose_Tissue.bed ...

  16. File list: ALL.Adp.20.AllAg.Adipose_Tissue [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.20.AllAg.Adipose_Tissue hg19 All antigens Adipocyte Adipose Tissue SRX13473...2 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.20.AllAg.Adipose_Tissue.bed ...

  17. File list: ALL.Adp.10.AllAg.Adipose_Tissue [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.10.AllAg.Adipose_Tissue hg19 All antigens Adipocyte Adipose Tissue SRX13473...2 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.10.AllAg.Adipose_Tissue.bed ...

  18. File list: ALL.Adp.05.AllAg.Adipose_Tissue [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.05.AllAg.Adipose_Tissue hg19 All antigens Adipocyte Adipose Tissue SRX13473...2 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.05.AllAg.Adipose_Tissue.bed ...

  19. The effect of pH and ADP on ammonia affinity for human glutamate dehydrogenases

    DEFF Research Database (Denmark)

    Zaganas, Ioannis; Pajecka, Kamilla; Nielsen, Camilla Wendel

    2013-01-01

    Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two...... human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their Km for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.......0) increased the Km for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K0.5 [NH4 (+)] of hGDH2, but also introduced...

  20. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    Energy Technology Data Exchange (ETDEWEB)

    Nolan, N.L.; Kidwell, W.R.

    1982-04-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

  1. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    International Nuclear Information System (INIS)

    Nolan, N.L.; Kidwell, W.R.

    1982-01-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37 0 C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of γ-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of γ-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels

  2. The ARTT motif and a unified structural understanding of substraterecognition in ADP ribosylating bacterial toxins and eukaryotic ADPribosyltransferases

    Energy Technology Data Exchange (ETDEWEB)

    Han, S.; Tainer, J.A.

    2001-08-01

    ADP-ribosylation is a widely occurring and biologically critical covalent chemical modification process in pathogenic mechanisms, intracellular signaling systems, DNA repair, and cell division. The reaction is catalyzed by ADP-ribosyltransferases, which transfer the ADP-ribose moiety of NAD to a target protein with nicotinamide release. A family of bacterial toxins and eukaryotic enzymes has been termed the mono-ADP-ribosyltransferases, in distinction to the poly-ADP-ribosyltransferases, which catalyze the addition of multiple ADP-ribose groups to the carboxyl terminus of eukaryotic nucleoproteins. Despite the limited primary sequence homology among the different ADP-ribosyltransferases, a central cleft bearing NAD-binding pocket formed by the two perpendicular b-sheet core has been remarkably conserved between bacterial toxins and eukaryotic mono- and poly-ADP-ribosyltransferases. The majority of bacterial toxins and eukaryotic mono-ADP-ribosyltransferases are characterized by conserved His and catalytic Glu residues. In contrast, Diphtheria toxin, Pseudomonas exotoxin A, and eukaryotic poly-ADP-ribosyltransferases are characterized by conserved Arg and catalytic Glu residues. The NAD-binding core of a binary toxin and a C3-like toxin family identified an ARTT motif (ADP-ribosylating turn-turn motif) that is implicated in substrate specificity and recognition by structural and mutagenic studies. Here we apply structure-based sequence alignment and comparative structural analyses of all known structures of ADP-ribosyltransfeases to suggest that this ARTT motif is functionally important in many ADP-ribosylating enzymes that bear a NAD binding cleft as characterized by conserved Arg and catalytic Glu residues. Overall, structure-based sequence analysis reveals common core structures and conserved active sites of ADP-ribosyltransferases to support similar NAD binding mechanisms but differing mechanisms of target protein binding via sequence variations within the ARTT

  3. Interaction of ADP, atractyloside, and gummiferin on the ADP translocase of the inner mitochondrial membrane

    Energy Technology Data Exchange (ETDEWEB)

    Vignais, P V; Vignais, P M; Defaye, G; Lauquin, G; Doussiere, J; Chabert, J; Brandolin, G

    1972-05-01

    From international conference on mechanism in bioenergetica; Bari, Italy (1 May 1972). Two specific inhibitors of the adenine nucleotide translocation, gummiferin (GUM), identified to 4-carboxyatractyloside and atractyloside (ATR), were labeled with /sup 35/S and their binding properties to whole mitochondria and inner mitochondrial membrane vesicles used to monitor changes of membrane conformation induced by ADP. (auth)

  4. Molecular structure of human KATP in complex with ATP and ADP.

    Science.gov (United States)

    Lee, Kenneth Pak Kin; Chen, Jue; MacKinnon, Roderick

    2017-12-29

    In many excitable cells, KATP channels respond to intracellular adenosine nucleotides: ATP inhibits while ADP activates. We present two structures of the human pancreatic KATP channel, containing the ABC transporter SUR1 and the inward-rectifier K + channel Kir6.2, in the presence of Mg 2+ and nucleotides. These structures, referred to as quatrefoil and propeller forms, were determined by single-particle cryo-EM at 3.9 Å and 5.6 Å, respectively. In both forms, ATP occupies the inhibitory site in Kir6.2. The nucleotide-binding domains of SUR1 are dimerized with Mg 2+ -ATP in the degenerate site and Mg 2+ -ADP in the consensus site. A lasso extension forms an interface between SUR1 and Kir6.2 adjacent to the ATP site in the propeller form and is disrupted in the quatrefoil form. These structures support the role of SUR1 as an ADP sensor and highlight the lasso extension as a key regulatory element in ADP's ability to override ATP inhibition. © 2017, Lee et al.

  5. Analytical Design Package (ADP2): A computer aided engineering tool for aircraft transparency design

    Science.gov (United States)

    Wuerer, J. E.; Gran, M.; Held, T. W.

    1994-01-01

    The Analytical Design Package (ADP2) is being developed as a part of the Air Force Frameless Transparency Program (FTP). ADP2 is an integrated design tool consisting of existing analysis codes and Computer Aided Engineering (CAE) software. The objective of the ADP2 is to develop and confirm an integrated design methodology for frameless transparencies, related aircraft interfaces, and their corresponding tooling. The application of this methodology will generate high confidence for achieving a qualified part prior to mold fabrication. ADP2 is a customized integration of analysis codes, CAE software, and material databases. The primary CAE integration tool for the ADP2 is P3/PATRAN, a commercial-off-the-shelf (COTS) software tool. The open architecture of P3/PATRAN allows customized installations with different applications modules for specific site requirements. Integration of material databases allows the engineer to select a material, and those material properties are automatically called into the relevant analysis code. The ADP2 materials database will be composed of four independent schemas: CAE Design, Processing, Testing, and Logistics Support. The design of ADP2 places major emphasis on the seamless integration of CAE and analysis modules with a single intuitive graphical interface. This tool is being designed to serve and be used by an entire project team, i.e., analysts, designers, materials experts, and managers. The final version of the software will be delivered to the Air Force in Jan. 1994. The Analytical Design Package (ADP2) will then be ready for transfer to industry. The package will be capable of a wide range of design and manufacturing applications.

  6. Unscheduled synthesis of DNA and poly(ADP-ribose) in human fibroblasts following DNA damage

    International Nuclear Information System (INIS)

    McCurry, L.S.; Jacobson, M.K.

    1981-01-01

    Unscheduled DNA synthesis has been measured in human fibroblasts under conditions of reduced rates of conversion of NAD to poly)ADP-ribose). Cells heterozygous for the xeroderma pigmentosum genotype showed normal rates of uv induced unscheduled DNA synthesis under conditions in which the rate of poly(ADP-ribose) synthesis was one-half the rate of normal cells. The addition of theophylline, a potent inhibitor of poly(ADP-ribose) polymerase, to the culture medium of normal cells blocked over 90% of the conversion of NAD to poly(ADP-ribose) following treatment with uv or N-methyl-N'-nitro-N-nitro-soguanidine but did not affect the rate of unscheduled DNA synthesis

  7. The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

    Science.gov (United States)

    Suszynski, T.M.; Wildey, G.M.; Falde, E.J.; Cline, G.W.; Maynard, K. Stewart; Ko, N.; Sotiris, J.; Naji, A.; Hering, B.J.; Papas, K.K.

    2009-01-01

    Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio. PMID:18374063

  8. Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3

    International Nuclear Information System (INIS)

    Yoshida, M.; Allison, W.S.

    1986-01-01

    Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with [ 3 H]ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. [ 3 H]ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with [ 3 H]ADP in 30 min with a Kd of 30 microM. [ 3 H]ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of [ 3 H]ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. [ 3 H]ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits

  9. File list: His.Adp.05.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

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    Lifescience Database Archive (English)

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  16. File list: His.Adp.10.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. Monitoring of radon isotopes and affiliated disintegration products (ADP) in soil air and water

    International Nuclear Information System (INIS)

    Anshakov, O. M.; Bogacheva, E. S.; Bouchawach, Fauzi Hadji; Chudakov, V. A.

    2009-01-01

    The subject of research is a physic and mathematical model of the process of radon determining in soil air and water by the way of its sampling for absorbent, preparation of a sample to measurement taking, ADP radiometry: Pb- 214, Bi-214 in a sample, calculation of radon activity concentration in an initial medium. The target of research is experimental determining of assignment parameters of devices, used for radon sampling and measurement of its ADP activity in relation to the methods being developed with estimation of their expected metrological performance, analysis of radon and ADP content for ecological research in relation to objectives of radon and ADP monitoring in environmental objects. (author)

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  19. File list: DNS.Adp.20.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

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  20. Involvement of oxygen free radicals in the respiratory uncoupling induced by free calcium and ADP-magnesium in isolated cardiac mitochondria: comparing reoxygenation in cultured cardiomyocytes.

    Science.gov (United States)

    Meynier, Alexandra; Razik, Hafida; Cordelet, Catherine; Grégoire, Stéphane; Demaison, Luc

    2003-01-01

    during Reox, cultured cardiomyocytes were subjected to hypoxia and Reox. 14C-palmitate was added during Reox to determine the KCI profile. Succinate had not increased during Reox. In conclusion, calcium- and ADP-Mg-induced respiratory uncoupling is due to oxygen free radical generation through excess matrix accumulation of succinate. The phenomenon does not occur during reoxygenation because of a total restoration of mitochondrial magnesium and/or ADP concentration.

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    Lifescience Database Archive (English)

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  7. File list: NoD.Adp.05.AllAg.Adipose_Tissue [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adp.05.AllAg.Adipose_Tissue hg19 No description Adipocyte Adipose Tissue SRX134...732 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Adp.05.AllAg.Adipose_Tissue.bed ...

  8. File list: InP.Adp.05.Input_control.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.05.Input_control.AllCell mm9 Input control Input control Adipocyte SRX99775...27370 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.05.Input_control.AllCell.bed ...

  9. Plasma kallikrein enhances platelet aggregation response by subthreshold doses of ADP.

    Science.gov (United States)

    Ottaiano, Tatiana F; Andrade, Sheila S; de Oliveira, Cleide; Silva, Mariana C C; Buri, Marcus V; Juliano, Maria A; Girão, Manoel J B C; Sampaio, Misako U; Schmaier, Alvin H; Wlodawer, Alexander; Maffei, Francisco H A; Oliva, Maria Luiza V

    2017-04-01

    Human plasma kallikrein (huPK) potentiates platelet responses to subthreshold doses of ADP, although huPK itself, does not induce platelet aggregation. In the present investigation, we observe that huPK pretreatment of platelets potentiates ADP-induced platelet activation by prior proteolysis of the G-protein-coupled receptor PAR-1. The potentiation of ADP-induced platelet activation by huPK is mediated by the integrin α IIb β 3 through interactions with the KGD/KGE sequence motif in huPK. Integrin α IIb β 3 is a cofactor for huPK binding to platelets to support PAR-1 hydrolysis that contributes to activation of the ADP signaling pathway. This activation pathway leads to phosphorylation of Src, AktS 473 , ERK1/2, and p38 MAPK, and to Ca 2+ release. The effect of huPK is blocked by specific antagonists of PAR-1 (SCH 19197) and α IIb β 3 (abciximab) and by synthetic peptides comprising the KGD and KGE sequence motifs of huPK. Further, recombinant plasma kallikrein inhibitor, rBbKI, also blocks this entire mechanism. These results suggest a new function for huPK. Formation of plasma kallikrein lowers the threshold for ADP-induced platelet activation. The present observations are consistent with the notion that plasma kallikrein promotes vascular disease and thrombosis in the intravascular compartment and its inhibition may ameliorate cardiovascular disease and thrombosis. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  10. NADP+ enhances cholera and pertussis toxin-catalyzed ADP-ribosylation of membrane proteins

    International Nuclear Information System (INIS)

    Kawai, Y.; Whitsel, C.; Arinze, I.J.

    1986-01-01

    Cholera or pertussis toxin-catalyzed [ 32 P]ADP-ribosylation is frequently used to estimate the concentration of the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins which modulate the activity of adenylate cyclase. With this assay, however, the degradation of the substrate, NAD + , by endogenous enzymes such as NAD + -glycohydrolase (NADase) present in the test membranes can influence the results. In this study the authors show that both cholera and pertussis toxin-catalyzed [ 32 P]ADP-ribosylation of liver membrane proteins is markedly enhanced by NADP + . The effect is concentration dependent; with 20 μM [ 32 P]NAD + as substrate maximal enhancement is obtained at 0.5-1.0 mM NADP + . The enhancement of [ 32 P]ADP-ribosylation by NADP + was much greater than that by other known effectors such as Mg 2+ , phosphate or isoniazid. The effect of NADP + on ADP-ribosylation may occur by inhibition of the degradation of NAD + probably by acting as an alternate substrate for NADase. Among inhibitors tested (NADP + , isoniazid, imidazole, nicotinamide, L-Arg-methyl-ester and HgCl 2 ) to suppress NADase activity, NADP + was the most effective and, 10 mM, inhibited activity of the enzyme by about 90%. In membranes which contain substantial activities of NADase the inclusion of NADP + in the assay is necessary to obtain maximal ADP-ribosylation

  11. Regulation of chromatin structure by poly(ADP-ribosylation

    Directory of Open Access Journals (Sweden)

    Sascha eBeneke

    2012-09-01

    Full Text Available The interaction of DNA with proteins in the context of chromatin has to be tightly regulated to achieve so different tasks as packaging, transcription, replication and repair. The very rapid and transient post-translational modification of proteins by poly(ADP-ribose has been shown to take part in all four. Originally identified as immediate cellular answer to a variety of genotoxic stresses, already early data indicated the ability of this highly charged nucleic acid-like polymer to modulate nucleosome structure, the basic unit of chromatin. At the same time the enzyme responsible for synthesizing poly(ADP-ribose, the zinc-finger protein poly(ADP-ribose polymerase-1 (PARP1, was shown to control transcription initiation as basic factor TFIIC within the RNA-polymerase II machinery. Later research focused more on PARP-mediated regulation of DNA repair and cell death, but in the last few years, transcription as well as chromatin modulation has re-appeared on the scene. This review will discuss the impact of PARP1 on transcription and transcription factors, its implication in chromatin remodeling for DNA repair and probably also replication, and its role in controlling epigenetic events such as DNA methylation and the functionality of the insulator protein CCCTC-binding factor.

  12. Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

    Energy Technology Data Exchange (ETDEWEB)

    Jorgensen, Rene; Purdy, Alexandra E.; Fieldhouse, Robert J.; Kimber, Matthew S.; Bartlett, Douglas H.; Merrill, A. Rod (Guelph); (NIH); (UCSD)

    2008-07-15

    The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC50 = 4.6 {+-} 0.4 ng/ml) and crustaceans (Artemia nauplii LD50 = 10 {+-} 2 {mu}g/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-{angstrom} resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.

  13. Further evidence for poly-ADP-ribosylated histones as DNA suppressors

    International Nuclear Information System (INIS)

    Yu, F.L.; Geronimo, I.H.; Bender, W.; Meginniss, K.E.

    1986-01-01

    For many years histones have been considered to be the gene suppressors in eukaryotic cells. Recently, the authors have found strong evidence indicating that poly-ADP-ribosylated histones, rather than histones, are the potent inhibitors of DNA-dependent RNA synthesis. They now report additional evidence for this concept: 1) using histone inhibitor isolated directly from nuclei, the authors are able to confirm their earlier findings that the inhibitor substances are sensitive to pronase, snake venom phosphodiesterase digestion and 0.1N KOH hydrolysis, and are resistant to DNase I and RNase A digestion, 2) the O.D. 260/O.D.280 ratio of the histone inhibitor is between pure protein and nuclei acid, suggesting the inhibitor substance is a nucleoprotein hybrid. This result directly supports the fact that the isolated histone inhibitor is radioactive poly (ADP-ribose) labeled, 3) commercial histones show big differences in inhibitor activity. The authors believe this reflects the variation in poly-ADP-ribosylation among commercial histones, and 4) 0.1N KOH hydrolysis eliminates the poly (ADP-ribose) radioactivity from the acceptor proteins as well as histone inhibitor activity. Yet, on gel, the inhibitor shows identical histone bands and stain intensity before and after hydrolysis, indicating the histones per se are qualitatively and quantitatively unaffected by alkaline treatment. This result strongly suggests that histones themselves are not capable of inhibiting DNA-dependent RNA synthesis

  14. A guide for developing an ADP security plan for Navy Finance Center, Cleveland, Ohio

    OpenAIRE

    Barber, Daniel E.; Hodnett, Elwood Thomas, Jr.

    1982-01-01

    Approved for public release; distribution is unlimited This paper is intended to be used as a guide by personnel at the Navy Finance Center (NFC) Cleveland, Ohio in developing an Automatic Data Processing (ADP) Security Plan. An effort has been made to combine the requirements for an ADP security plan established by OPNAVINST5239.1A with pertinent information from other selected readings. The importance of the devotion of personnel, time and funds to ADP security planning has been emphas...

  15. Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

    International Nuclear Information System (INIS)

    Zwelling, L.A.; Kerrigan, D.; Mattern, M.R.

    1983-01-01

    The synthesis of poly(adenosine diphosphoribose [poly(ADP-R)] follows the DNA strand breakage produced by a number of physical and chemical agents, including X-radiation, and may be important for repair of several types of DNA damage. The reduction or abolition of its synthesis following X-irradiation might explain the enhanced sensitivity of ataxia-telangiectasia (A-T) cells to X-ray. We have examined 8 lines of human fibroblasts (including 4 A-T lines) for stimulation of the synthesis of poly(ADP-R) by X-irradiation. Similar amounts of X-ray-stimulated synthesis of poly(ADP-R) were detected in 4 lines of A-T fibroblasts, and in fibrolasts from a xeroderma pigmentosum (XP) patient, a Fanconi's anemia (FA) patient and 2 normal patients. 6 lines of human lymphoblastoid lines were also examined for X-ray-stimulated poly(ADP-R) synthesis. 4 A-T lines displayed an unusually high synthesis of poly(ADP-R) in unirradiated cells compared with 2 normal lines. (orig./AJ)

  16. Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Zwelling, L.A.; Kerrigan, D. (National Cancer Inst., Bethesda, MD (USA). Lab. of Molecular Pharmacology); Mattern, M.R. (National Cancer Inst., Bethesda, MD (USA). Lab. of Molecular Carcinogenesis)

    1983-04-01

    The synthesis of poly(adenosine diphosphoribose (poly(ADP-R)) follows the DNA strand breakage produced by a number of physical and chemical agents, including X-radiation, and may be important for repair of several types of DNA damage. The reduction or abolition of its synthesis following X-irradiation might explain the enhanced sensitivity of ataxia-telangiectasia (A-T) cells to X-ray. We have examined 8 lines of human fibroblasts (including 4 A-T lines) for stimulation of the synthesis of poly(ADP-R) by X-irradiation. Similar amounts of X-ray-stimulated synthesis of poly(ADP-R) were detected in 4 lines of A-T fibroblasts, and in fibrolasts from a xeroderma pigmentosum (XP) patient, a Fanconi's anemia (FA) patient and 2 normal patients. 6 lines of human lymphoblastoid lines were also examined for X-ray-stimulated poly(ADP-R) synthesis. 4 A-T lines displayed an unusually high synthesis of poly(ADP-R) in unirradiated cells compared with 2 normal lines.

  17. File list: Pol.Adp.50.RNA_polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.50.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Adipocyt...e SRX682084,SRX682086,SRX682085,SRX682083 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Adp.50.RNA_polymerase_II.AllCell.bed ...

  18. Modulated anharmonic ADPs are intrinsic to aperiodic crystals: a case study on incommensurate Rb2ZnCl4

    International Nuclear Information System (INIS)

    Li, Liang; Wölfel, Alexander; Schönleber, Andreas; Mondal, Swastik; Schreurs, Antoine M. M.; Kroon-Batenburg, Loes M. J.; Smaalen, Sander van

    2011-01-01

    The superspace maximum entropy method (MEM) density in combination with structure refinements has been used to uncover the modulation in incommensurate Rb 2 ZnCl 4 close to the lock-in transition. Modulated atomic displacement parameters (ADPs) and modulated anharmonic ADPs are found to form an intrinsic part of the modulation. Refined values for the displacement modulation function depend on the presence or absence of modulated ADPs in the model. A combination of structure refinements, analysis of the superspace MEM density and interpretation of difference-Fourier maps has been used to characterize the incommensurate modulation of rubidium tetrachlorozincate, Rb 2 ZnCl 4 , at a temperature of T = 196 K, close to the lock-in transition at T lock-in = 192 K. The modulation is found to consist of a combination of displacement modulation functions, modulated atomic displacement parameters (ADPs) and modulated third-order anharmonic ADPs. Up to fifth-order Fourier coefficients could be refined against diffraction data containing up to fifth-order satellite reflections. The center-of-charge of the atomic basins of the MEM density and the displacive modulation functions of the structure model provide equivalent descriptions of the displacive modulation. Modulations of the ADPs and anharmonic ADPs are visible in the MEM density, but extracting quantitative information about these modulations appears to be difficult. In the structure refinements the modulation parameters of the ADPs form a dependent set, and ad hoc restrictions had to be introduced in the refinements. It is suggested that modulated harmonic ADPs and modulated third-order anharmonic ADPs form an intrinsic part, however small, of incommensurately modulated structures in general. Refinements of alternate models with and without parameters for modulated ADPs lead to significant differences between the parameters of the displacement modulation in these two types of models, thus showing the modulation of ADPs to

  19. File list: His.Adp.10.AllAg.Adipose_progenitor_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: His.Adp.05.AllAg.Adipose_progenitor_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: ALL.Adp.10.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  4. File list: Oth.Adp.20.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: Oth.Adp.05.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: ALL.Adp.50.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: ALL.Adp.20.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: ALL.Adp.05.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: InP.Adp.50.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

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  10. File list: NoD.Adp.20.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

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  11. File list: InP.Adp.20.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: NoD.Adp.05.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

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  13. File list: NoD.Adp.50.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

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  14. File list: InP.Adp.10.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: NoD.Adp.50.AllAg.Adipose_progenitor_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: NoD.Adp.05.AllAg.Adipose_progenitor_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: Pol.Adp.50.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.50.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Adipocyte... SRX800011,SRX800010,SRX341031,SRX341032,SRX341029,SRX800016,SRX800017,SRX341030 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Adp.50.RNA_Polymerase_II.AllCell.bed ...

  19. Prevalence and Characterization of a Binary Toxin (Actin-Specific ADP-Ribosyltransferase) from Clostridium difficile

    Science.gov (United States)

    Gonçalves, Carina; Decré, Dominique; Barbut, Frédéric; Burghoffer, Béatrice; Petit, Jean-Claude

    2004-01-01

    In addition to the two large clostridial cytotoxins (TcdA and TcdB), some strains of Clostridium difficile also produce an actin-specific ADP-ribosyltransferase, called binary toxin CDT. We used a PCR method and Southern blotting for the detection of genes encoding the enzymatic (CDTa) and binding (CDTb) components of the binary toxin in 369 strains isolated from patients with suspected C. difficile-associated diarrhea or colitis. Twenty-two strains (a prevalence of 6%) harbored both genes. When binary toxin production was assessed by Western blotting, 19 of the 22 strains reacted with antisera against the iota toxin of C. perfringens (anti-Ia and anti-Ib). Additionally, binary toxin activity, detected by the ADP-ribosyltransferase assay, was present in only 17 of the 22 strains. Subsequently, all 22 binary toxin-positive strains were tested for the production of toxins TcdA and TcdB, toxinotyped, and characterized by serogrouping, PCR ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis. All binary toxin-positive strains also produced TcdB and/or TcdA. However, they had significant changes in the tcdA and tcdB genes and belonged to variant toxinotypes III, IV, V, VII, IX, and XIII. We could differentiate 16 profiles by using typing methods, indicating that most of the binary toxin-positive strains were unrelated. PMID:15131151

  20. Age-Associated Impairments in Mitochondrial ADP Sensitivity Contribute to Redox Stress in Senescent Human Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Graham P. Holloway

    2018-03-01

    Full Text Available Summary: It remains unknown if mitochondrial bioenergetics are altered with aging in humans. We established an in vitro method to simultaneously determine mitochondrial respiration and H2O2 emission in skeletal muscle tissue across a range of biologically relevant ADP concentrations. Using this approach, we provide evidence that, although the capacity for mitochondrial H2O2 emission is not increased with aging, mitochondrial ADP sensitivity is impaired. This resulted in an increase in mitochondrial H2O2 and the fraction of electron leak to H2O2, in the presence of virtually all ADP concentrations examined. Moreover, although prolonged resistance training in older individuals increased muscle mass, strength, and maximal mitochondrial respiration, exercise training did not alter H2O2 emission rates in the presence of ADP, the fraction of electron leak to H2O2, or the redox state of the muscle. These data establish that a reduction in mitochondrial ADP sensitivity increases mitochondrial H2O2 emission and contributes to age-associated redox stress. : Holloway et al. show that an inability of ADP to decrease mitochondrial reactive oxygen species emission contributes to redox stress in skeletal muscle tissue of older individuals and that this process is not recovered following prolonged resistance-type exercise training, despite the general benefits of resistance training for muscle health. Keywords: mitochondria, aging, muscle, ROS, H2O2, ADP, respiration, bioenergetics, exercise, resistance training

  1. Marine debris accumulation in the Northwestern Hawaiian Islands: an examination of rates and processes.

    Science.gov (United States)

    Dameron, Oliver J; Parke, Michael; Albins, Mark A; Brainard, Russell

    2007-04-01

    Large amounts of derelict fishing gear accumulate and cause damage to shallow coral reefs of the Northwestern Hawaiian Islands (NWHI). To facilitate maintenance of reefs cleaned during 1996-2005 removal efforts, we identify likely high-density debris areas by assessing reef characteristics (depth, benthic habitat type, and energy regime) that influence sub-regional debris accumulation. Previously cleaned backreef and lagoonal reefs at two NWHI locations were resurveyed for accumulated debris using two survey methods. Accumulated debris densities and weights were found to be greater in lagoonal reef areas. Sample weight-based debris densities are extrapolated to similar habitats throughout the NWHI using a spatial 'net habitat' dataset created by generalizing IKONOS satellite derivatives for depth and habitat classification. Prediction accuracy for this dataset is tested using historical debris point data. Annual NWHI debris accumulation is estimated to be 52.0 metric tonnes. For planning purposes, individual NWHI atolls/reefs are allotted a proportion of this total.

  2. Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, J.; Xue, Z.; Du, Z.; Melese, T.; Boyer, P.D.

    1988-07-12

    Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F/sub 1/ ATPase (CF/sub 1/) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. The authors have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg/sup 2 +/ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF/sub 1/ that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF/sub 1/. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (P/sub i/) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with (/sup 32/P)P/sub i/, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. They also report the occurrence of a 1-2-min delay in the onset of the Mg/sup 2 +/-induced inhibition after addition of CF/sub 1/ to solutions containing Mg/sup 2 +/ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of P/sub i/ formation is followed by a much lower, constant steady-state rate. The burst is not observed with GTP as a substrate or with Ca/sup 2 +/ as the activating cation.

  3. File list: InP.Adp.05.AllAg.Adipose_progenitor_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.05.AllAg.Adipose_progenitor_cells mm9 Input control Adipocyte Adipose progeni...tor cells SRX127367,SRX127370 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.05.AllAg.Adipose_progenitor_cells.bed ...

  4. File list: InP.Adp.20.AllAg.Adipose_progenitor_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.20.AllAg.Adipose_progenitor_cells mm9 Input control Adipocyte Adipose progeni...tor cells SRX127370,SRX127367 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.20.AllAg.Adipose_progenitor_cells.bed ...

  5. File list: InP.Adp.50.AllAg.Adipose_progenitor_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.50.AllAg.Adipose_progenitor_cells mm9 Input control Adipocyte Adipose progeni...tor cells SRX127370,SRX127367 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.50.AllAg.Adipose_progenitor_cells.bed ...

  6. Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin.

    Directory of Open Access Journals (Sweden)

    Alexander Belyy

    Full Text Available Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia.

  7. Altered poly(ADP-ribose) metabolism impairs cellular responses to genotoxic stress in a hypomorphic mutant of poly(ADP-ribose) glycohydrolase

    International Nuclear Information System (INIS)

    Gao Hong; Coyle, Donna L.; Meyer-Ficca, Mirella L.; Meyer, Ralph G.; Jacobson, Elaine L.; Wang, Zhao-Qi; Jacobson, Myron K.

    2007-01-01

    Genotoxic stress activates nuclear poly(ADP-ribose) (PAR) metabolism leading to PAR synthesis catalyzed by DNA damage activated poly(ADP-ribose) polymerases (PARPs) and rapid PAR turnover by action of nuclear poly(ADP-ribose) glycohydrolase (PARG). The involvement of PARP-1 and PARP-2 in responses to DNA damage has been well studied but the involvement of nuclear PARG is less well understood. To gain insights into the function of nuclear PARG in DNA damage responses, we have quantitatively studied PAR metabolism in cells derived from a hypomorphic mutant mouse model in which exons 2 and 3 of the PARG gene have been deleted (PARG-Δ2,3 cells), resulting in a nuclear PARG containing a catalytic domain but lacking the N-terminal region (A domain) of the protein. Following DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we found that the activity of both PARG and PARPs in intact cells is increased in PARG-Δ2,3 cells. The increased PARG activity leads to decreased PARP-1 automodification with resulting increased PARP activity. The degree of PARG activation is greater than PARP, resulting in decreased PAR accumulation. Following MNNG treatment, PARG-Δ2,3 cells show reduced formation of XRCC1 foci, delayed H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. Our results show that a precise coordination of PARPs and PARG activities is important for normal cellular responses to DNA damage and that this coordination is defective in the absence of the PARG A domain

  8. The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch.

    Science.gov (United States)

    Doan; Rudi; Olsen

    1999-11-01

    We present the results of studies of an unmodified version of the recombinant major barley (Hordeum vulgare) endosperm ADP-glucose pyrophoshorylase (AGPase) expressed in insect cells, which corroborate previous data that this isoform of the enzyme acts independently of the allosteric regulators 3-phosphoglycerate and inorganic phosphate. We also present a characterization of the individual subunits expressed separately in insect cells, showing that the SS AGPase is active in the presence of 3-phosphoglycerate and is inhibited by inorganic phosphate. As a step toward the elucidation of the role of the two AGPase isoforms in barley, the temporal and spatial expression profile of the four barley AGPase transcripts encoding these isoforms were studied. The results show that the steady-state level of beps and bepl, the transcripts encoding the major endosperm isoform, correlated positively with the rate of endosperm starch accumulation. In contrast, blps and blpl, the transcripts encoding the major leaf isoform, were constitutively expressed at a very low steady-state level throughout the barley plant. The implications of these findings for the evolution of plant AGPases are discussed.

  9. Aero-Propulsion Technology (APT) Task V Low Noise ADP Engine Definition Study

    Science.gov (United States)

    Holcombe, V.

    2003-01-01

    A study was conducted to identify and evaluate noise reduction technologies for advanced ducted prop propulsion systems that would allow increased capacity operation and result in an economically competitive commercial transport. The study investigated the aero/acoustic/structural advancements in fan and nacelle technology required to match or exceed the fuel burned and economic benefits of a constrained diameter large Advanced Ducted Propeller (ADP) compared to an unconstrained ADP propulsion system with a noise goal of 5 to 10 EPNDB reduction relative to FAR 36 Stage 3 at each of the three measuring stations namely, takeoff (cutback), approach and sideline. A second generation ADP was selected to operate within the maximum nacelle diameter constrain of 160 deg to allow installation under the wing. The impact of fan and nacelle technologies of the second generation ADP on fuel burn and direct operating costs for a typical 3000 nm mission was evaluated through use of a large, twin engine commercial airplane simulation model. The major emphasis of this study focused on fan blade aero/acoustic and structural technology evaluations and advanced nacelle designs. Results of this study have identified the testing required to verify the interactive performance of these components, along with noise characteristics, by wind tunnel testing utilizing and advanced interaction rig.

  10. Improved Triacylglycerol Production in Acinetobacter baylyi ADP1 by Metabolic Engineering

    Directory of Open Access Journals (Sweden)

    Karp Matti

    2011-05-01

    Full Text Available Abstract Background Triacylglycerols are used in various purposes including food applications, cosmetics, oleochemicals and biofuels. Currently the main sources for triacylglycerol are vegetable oils, and microbial triacylglycerol has been suggested as an alternative for these. Due to the low production rates and yields of microbial processes, the role of metabolic engineering has become more significant. As a robust model organism for genetic and metabolic studies, and for the natural capability to produce triacylglycerol, Acinetobacter baylyi ADP1 serves as an excellent organism for modelling the effects of metabolic engineering for energy molecule biosynthesis. Results Beneficial gene deletions regarding triacylglycerol production were screened by computational means exploiting the metabolic model of ADP1. Four deletions, acr1, poxB, dgkA, and a triacylglycerol lipase were chosen to be studied experimentally both separately and concurrently by constructing a knock-out strain (MT with three of the deletions. Improvements in triacylglycerol production were observed: the strain MT produced 5.6 fold more triacylglycerol (mg/g cell dry weight compared to the wild type strain, and the proportion of triacylglycerol in total lipids was increased by 8-fold. Conclusions In silico predictions of beneficial gene deletions were verified experimentally. The chosen single and multiple gene deletions affected beneficially the natural triacylglycerol metabolism of A. baylyi ADP1. This study demonstrates the importance of single gene deletions in triacylglycerol metabolism, and proposes Acinetobacter sp. ADP1 as a model system for bioenergetic studies regarding metabolic engineering.

  11. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

    Science.gov (United States)

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-10-28

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

  12. Poly (ADP-ribose polymerase 1 is required for protein localization to Cajal body.

    Directory of Open Access Journals (Sweden)

    Elena Kotova

    2009-02-01

    Full Text Available Recently, the nuclear protein known as Poly (ADP-ribose Polymerase1 (PARP1 was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose, PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

  13. Correlations of serum levels of leptin and other related factor (NPY, ADP) in female children with simple obesity

    International Nuclear Information System (INIS)

    Bai Hua; Wei Chunlei; Qian Mingzhu

    2008-01-01

    Objective: To study the changes of serum levels of leptin, NPY and ADP in female children with simple obesity. Methods: Serum levels of leptin, NPY and ADP were measured with radioimmunoassay (RIA) in 32 female children with simple obesity and 35 controls. Results: The serum levels of leptin, NPY were significantly higher in the obese children than those in controls (P<0.01), while the serum levels of ADP were significantly lower (P<0.01). Serum leptin levels were significantly positively correlated (r=0.6014, P<0.01) with NPY levels but were negatively correlated (r=-0.4786, P<0.01) with adiponectin (ADP) levels. Conclusion: Determination of serum leptin, NPY and ADP levels is of help for judgement of degree of obesity as wen as outcome prediction in female children. (authors)

  14. D.C. electrical conductivity measurements on ADP single crystals ...

    Indian Academy of Sciences (India)

    Unknown

    Impurity added ADP crystals; density; electrical conductivity measurements. 1. Introduction ... determined by the intrinsic defects caused by thermal fluctuations in the ... beaker (corning glass vessel) and allowed to equilibrate at the desired ...

  15. ADP Security Plan, Math Building, Room 1139

    Energy Technology Data Exchange (ETDEWEB)

    Melton, R.

    1985-08-27

    This document provides the draft copy of an updated (ADP) Security Plan for an IBM Personal Computer to be used in the Math Building at PNL for classified data base management. Using the equipment specified in this document and implementing the administrative and physical procedures as outlined will provide the secure environment necessary for this work to proceed.

  16. The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch1

    Science.gov (United States)

    Doan, Danny N.P.; Rudi, Heidi; Olsen, Odd-Arne

    1999-01-01

    We present the results of studies of an unmodified version of the recombinant major barley (Hordeum vulgare) endosperm ADP-glucose pyrophoshorylase (AGPase) expressed in insect cells, which corroborate previous data that this isoform of the enzyme acts independently of the allosteric regulators 3-phosphoglycerate and inorganic phosphate. We also present a characterization of the individual subunits expressed separately in insect cells, showing that the SS AGPase is active in the presence of 3-phosphoglycerate and is inhibited by inorganic phosphate. As a step toward the elucidation of the role of the two AGPase isoforms in barley, the temporal and spatial expression profile of the four barley AGPase transcripts encoding these isoforms were studied. The results show that the steady-state level of beps and bepl, the transcripts encoding the major endosperm isoform, correlated positively with the rate of endosperm starch accumulation. In contrast, blps and blpl, the transcripts encoding the major leaf isoform, were constitutively expressed at a very low steady-state level throughout the barley plant. The implications of these findings for the evolution of plant AGPases are discussed. PMID:10557246

  17. Adrenaline potentiates PI 3-kinase in platelets stimulated with thrombin and SFRLLN: role of secreted ADP.

    Science.gov (United States)

    Selheim, F; Frøyset, A K; Strand, I; Vassbotn, F S; Holmsen, H

    2000-11-17

    Adrenaline significantly potentiated late thrombin- and SFRLLN-induced PtdIns(3,4)P(2) production. Furthermore, the potentiating effect of adrenaline on thrombin-induced PtdIns(3, 4)P(2) production was independent on secreted ADP, whereas, the effect of adrenaline on SFRLLN-induced PtdIns(3,4)P(2) production was completely dependent of secreted ADP. However, the ADP-dependent accumulation of PtdIns(3,4)P(2) was not required for irreversible platelet aggregation induced by SFRLLN in the presence of adrenaline. It is concluded that adrenaline can replace secreted ADP to potentiate PtdIns(3,4)P(2) production in thrombin-stimulated but not in SFRLLN-stimulated platelets, thus demonstrating a qualitative difference between platelet stimulation by thrombin and the thrombin receptor activating peptide SFRLLN.

  18. Nucleolin down-regulation is involved in ADP-induced cell cycle arrest in S phase and cell apoptosis in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Wenmeng Wang

    Full Text Available High concentration of extracellular ADP has been reported to induce cell apoptosis, but the molecular mechanisms remain not fully elucidated. In this study, we found by serendipity that ADP treatment of human umbilical vein endothelial cells (HUVEC and human aortic endothelial cells (HAEC down-regulated the protein level of nucleolin in a dose- and time-dependent manner. ADP treatment did not decrease the transcript level of nucloelin, suggesting that ADP might induce nucleolin protein degradation. HUVEC and HAEC expressed ADP receptor P2Y13 receptor, but did not express P2Y1 or P2Y12 receptors. However, P2Y1, 12, 13 receptor antagonists MRS2179, PSB0739, MRS2211 did not inhibit ADP-induced down-regulation of nucleolin. Moreover, MRS2211 itself down-regulated nucleolin protein level. In addition, 2-MeSADP, an agonist for P2Y1, 12 and 13 receptors, did not down-regulate nucleolin protein. These results suggested that ADP-induced nucleolin down-regulation was not due to the activation of P2Y1, 12, or 13 receptors. We also found that ADP treatment induced cell cycle arrest in S phase, cell apoptosis and cell proliferation inhibition via nucleolin down-regulation. The over-expression of nucleolin by gene transfer partly reversed ADP-induced cell cycle arrest, cell apoptosis and cell proliferation inhibition. Furthermore, ADP sensitized HUVEC to cisplatin-induced cell death by the down-regulation of Bcl-2 expression. Taken together, we found, for the first time to our knowledge, a novel mechanism by which ADP regulates cell proliferation by induction of cell cycle arrest and cell apoptosis via targeting nucelolin.

  19. PARP2 Is the Predominant Poly(ADP-Ribose Polymerase in Arabidopsis DNA Damage and Immune Responses.

    Directory of Open Access Journals (Sweden)

    Junqi Song

    2015-05-01

    Full Text Available Poly (ADP-ribose polymerases (PARPs catalyze the transfer of multiple poly(ADP-ribose units onto target proteins. Poly(ADP-ribosylation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390, rather than PARP1 (At2g31320, makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose glycohydrolase (PARG enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosylation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosylation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation.

  20. 7 CFR 272.10 - ADP/CIS Model Plan.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 4 2010-01-01 2010-01-01 false ADP/CIS Model Plan. 272.10 Section 272.10 Agriculture Regulations of the Department of Agriculture (Continued) FOOD AND NUTRITION SERVICE, DEPARTMENT OF AGRICULTURE... benefit computation (including but not limited to all household members' names, addresses, dates of birth...

  1. ADP stimulation of inositol phosphates in hepatocytes: role of conversion to ATP and stimulation of P2Y2 receptors.

    Science.gov (United States)

    Dixon, C Jane; Hall, John F; Boarder, Michael R

    2003-01-01

    1 Accumulation of inositol (poly)phosphates (InsP(x)) has been studied in rat hepatocytes labelled with [(3)H]inositol. Stimulation with ADP resulted in a significant increase in total [(3)H]InsP(x), whereas 2-MeSADP had only a small effect and ADPbetaS was ineffective. UTP and ITP also stimulated substantial increases in [(3)H]InsP(x). 2 The dose-response curve to ADP was largely unaltered by the presence of the P2Y(1) antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P). Similarly, inclusion of MRS 2179, a more selective P2Y(1) antagonist, had no effect on the dose-response curve to ADP. 3 The inclusion of hexokinase in the assay reduced, but did not abolish, the response to ADP. 4 HPLC analysis revealed that ADP in the medium was rapidly converted to AMP and ATP. The inclusion of hexokinase removed ATP, but exacerbated the decline in ADP concentration, leading to increased levels of AMP. 2-MeSADP was stable in the medium and ATP was largely unaffected. 5 The addition of the adenylate kinase inhibitor, diadenosine pentaphosphate (Ap(5)A) significantly reduced the ADP response. HPLC analysis conducted in parallel demonstrated that this treatment inhibited conversion of ADP to ATP and AMP. 6 Inclusion of the P1 antagonist CGS 15943 had no effect on the dose-response curve to ADP. 7 These observations indicate that hepatocytes respond to ADP with an increase in inositol (poly)phosphates following conversion to ATP. P2Y(1) activation in hepatocytes does not appear to be coupled to inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) production.

  2. Pleiotropic regulatory genes bldA, adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin.

    Science.gov (United States)

    Makitrynskyy, Roman; Ostash, Bohdan; Tsypik, Olga; Rebets, Yuriy; Doud, Emma; Meredith, Timothy; Luzhetskyy, Andriy; Bechthold, Andreas; Walker, Suzanne; Fedorenko, Victor

    2013-10-23

    Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production-bldA, adpA and absB-exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNA(Leu)UAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs-that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.

  3. Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells

    International Nuclear Information System (INIS)

    Fendrick, J.L.; Iglewski, W.J.

    1989-01-01

    Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing [ 32 P]orthophosphate and 105 (vol/vol) fetal bovine serum. A 32 P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The 32 P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The 32 P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as [ 32 P]AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the 32 P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and [ 32 P]NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When 32 P-labeled cultures were incubated in medium containing unlabeled phosphate, the 32 P label was lost from the EF-2 within 30 min

  4. Class I ADP-ribosylation factors are involved in enterovirus 71 replication.

    Directory of Open Access Journals (Sweden)

    Jianmin Wang

    Full Text Available Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies.

  5. Class I ADP-ribosylation factors are involved in enterovirus 71 replication.

    Science.gov (United States)

    Wang, Jianmin; Du, Jiang; Jin, Qi

    2014-01-01

    Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies.

  6. 32 CFR Appendix J to Part 154 - ADP Position Categories and Criteria for Designating Positions

    Science.gov (United States)

    2010-07-01

    ... maintenance of a computer system, and whose work is technically reviewed by a higher authority of the ADP-I... agency computer security programs, and also including direction and control of risk analysis and/or... the activities of the individual are not subject to technical review by higher authority in the ADP-I...

  7. Poly(ADP-ribosyl)ation is recognized by ECT2 during mitosis.

    Science.gov (United States)

    Li, Mo; Bian, Chunjing; Yu, Xiaochun

    2014-01-01

    Poly(ADP-ribosyl)ation is an unique posttranslational modification and required for spindle assembly and function during mitosis. However, the molecular mechanism of poly(ADP-ribose) (PAR) in mitosis remains elusive. Here, we show the evidence that PAR is recognized by ECT2, a key guanine nucleotide exchange factor in mitosis. The BRCT domain of ECT2 directly binds to PAR both in vitro and in vivo. We further found that α-tubulin is PARylated during mitosis. PARylation of α-tubulin is recognized by ECT2 and recruits ECT2 to mitotic spindle for completing mitosis. Taken together, our study reveals a novel mechanism by which PAR regulates mitosis.

  8. Preliminary crystallographic analysis of ADP-glucose pyrophosphorylase from Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Cupp-Vickery, Jill R.; Igarashi, Robert Y.; Meyer, Christopher R.

    2005-01-01

    Crystallization and X-ray diffraction methods for native A. tumefaciens ADP-glucose pyrophosphorylase and its selenomethionyl derivative are described. Two crystal forms are identified, both of which diffract to 2 Å

  9. Third-order nonlinear optical properties of ADP crystal

    Science.gov (United States)

    Wang, Mengxia; Wang, Zhengping; Chai, Xiangxu; Sun, Yuxiang; Sui, Tingting; Sun, Xun; Xu, Xinguang

    2018-05-01

    By using the Z-scan method, we investigated the third-order nonlinear optical (NLO) properties of ADP crystal at different wavelengths (355, 532, and 1064 nm) and different orientations ([001], [100], [110], I and II). The experimental data were fitted by NLO theory, to give out the two photon absorption (TPA) coefficient β 2 and the nonlinear refractive index n 2. When the light source changed from a 40 ps, 1064 nm fundamental laser to a 30 ps, 355 nm third-harmonic-generation (THG) laser, the β 2 value increased about 5 times (0.2 × 10‑2 → 1 × 10‑2 cm GW‑1), and the n 2 value increased about 1.5 times (1.5 × 10‑16 → 2.2 × 10‑16 cm2 W‑1). Among all of the orientations, the [110] sample exhibits the smallest β 2, and the second smallest n 2. It indicates that this orientation and its surroundings will be the preferred directions for high-power laser applications of ADP crystal.

  10. Structure and properties of Al-MIL-53-ADP, a breathing MOF based on the aliphatic linker molecule adipic acid.

    Science.gov (United States)

    Reinsch, Helge; Pillai, Renjith S; Siegel, Renée; Senker, Jürgen; Lieb, Alexandra; Maurin, Guillaume; Stock, Norbert

    2016-03-14

    The new aluminium based metal-organic framework [Al(OH)(O2C-C4H8-CO2)]·H2O denoted as Al-MIL-53-ADP-lp (lp stands for large pore) was synthesised under solvothermal conditions. This solid is an analogue of the archetypical aluminium terephthalate Al-MIL-53 based on the aliphatic single-chain linker molecule adipic acid (H2ADP, hexanedioic acid). In contrast to its aromatic counterparts, Al-MIL-53-ADP exhibits a structural breathing behaviour solely upon dehydration/rehydration. The crystal structure of the anhydrous compound denoted as Al-MIL-53-ADP-np (np stands for narrow pore) was determined by a combination of forcefield-based computations and Rietveld refinement of the powder X-ray diffraction data while the structure of the hydrated form Al-MIL-53-ADP-lp was derived computationally by a combination of force field based methods and Density Functional Theory calculations. Both structures were further supported by (1)H, (13)C and (27)Al high-resolution NMR MAS 1D data coupled again with simulations. Al-MIL-53-ADP was further characterised by means of vibrational spectroscopy, elemental analysis, thermogravimetry and water vapour sorption.

  11. Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells

    Energy Technology Data Exchange (ETDEWEB)

    Fendrick, J.L.; Iglewski, W.J. (Univ. of Rochester, NY (USA))

    1989-01-01

    Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing ({sup 32}P)orthophosphate and 105 (vol/vol) fetal bovine serum. A {sup 32}P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The {sup 32}P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The {sup 32}P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as ({sup 32}P)AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the {sup 32}P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and ({sup 32}P)NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When {sup 32}P-labeled cultures were incubated in medium containing unlabeled phosphate, the {sup 32}P label was lost from the EF-2 within 30 min.

  12. Radiation-induced DNA breaks detected by immuno labelling of poly(ADP-ribose) in CHO cells. Standardization by pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    Varlet, P.; Bidon, N.; Noel, G.; Averbeck, D.; Salamero, J.; DeMurcia, G.

    1998-01-01

    The poly (ADP-ribose) polymerase is an ubiquitous nuclear protein capable of binding specifically to DNA strand breaks. It synthesizes ADP-ribose polymers proportionally to DNA breaks. The actual method of reference to determine DNA double strand breaks is pulsed-field gel electrophoresis, but this requires many cells. It thus appeared of interest to use poly (ADP-ribos)ylation to follow and estimate γ-ray-induced DNA fragmentation at the level of isolated cells after γ-irradiation in chinese hamster ovary cells (CHO-K1). The results obtained by the immuno-labelling technique of ADP-ribose polymers were compared to those obtained by pulsed-field gel electrophoresis. They show that poly (ADP-ribos)ylation reflects the occurrence of radiation-induced DNA strand breaks. A clear relationship exists between the amount of ADP-ribose polymers detected and DNA double strand breaks after γ-irradiation. (authors)

  13. Uridylylation of Herbaspirillum seropedicae GlnB and GlnK proteins is differentially affected by ATP, ADP and 2-oxoglutarate in vitro.

    Science.gov (United States)

    Bonatto, Ana C; Souza, Emanuel M; Oliveira, Marco A S; Monteiro, Rose A; Chubatsu, Leda S; Huergo, Luciano F; Pedrosa, Fábio O

    2012-08-01

    PII are signal-transducing proteins that integrate metabolic signals and transmit this information to a large number of proteins. In proteobacteria, PII are modified by GlnD (uridylyltransferase/uridylyl-removing enzyme) in response to the nitrogen status. The uridylylation/deuridylylation cycle of PII is also regulated by carbon and energy signals such as ATP, ADP and 2-oxoglutarate (2-OG). These molecules bind to PII proteins and alter their tridimensional structure/conformation and activity. In this work, we determined the effects of ATP, ADP and 2-OG levels on the in vitro uridylylation of Herbaspirillum seropedicae PII proteins, GlnB and GlnK. Both proteins were uridylylated by GlnD in the presence of ATP or ADP, although the uridylylation levels were higher in the presence of ATP and under high 2-OG levels. Under excess of 2-OG, the GlnB uridylylation level was higher in the presence of ATP than with ADP, while GlnK uridylylation was similar with ATP or ADP. Moreover, in the presence of ADP/ATP molar ratios varying from 10/1 to 1/10, GlnB uridylylation level decreased as ADP concentration increased, whereas GlnK uridylylation remained constant. The results suggest that uridylylation of both GlnB and GlnK responds to 2-OG levels, but only GlnB responds effectively to variation on ADP/ATP ratio.

  14. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    International Nuclear Information System (INIS)

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-01-01

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  15. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    Energy Technology Data Exchange (ETDEWEB)

    Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Wagner, Silvia [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany); Buerkle, Alexander [Molecular Toxicology Group, Department of Biology, University of Konstanz (Germany); Koenigsrainer, Alfred [Clinic of General, Visceral- and Transplantation Surgery, ZMF, University Hospital Tuebingen (Germany)

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  16. Molecular and biochemical analysis of the plastidic ADP-glucose transporter (ZmBT1) from Zea mays.

    NARCIS (Netherlands)

    Kirchberger, S.; Leroch, M.; Huynen, M.A.; Wahl, M.; Neuhaus, H.E.; Tjaden, J.

    2007-01-01

    Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous

  17. Molecular and biochemical analysis of the plastidic ADP-glucose transporter (ZmBT1) from Zea mays

    NARCIS (Netherlands)

    Kirchberger, S.; Leroch, M.; Huynen, M.A.; Wahl, M.; Neuhaus, H.E.; Tjaden, J.

    2007-01-01

    Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous

  18. Higher cytoplasmic and nuclear poly(ADP-ribose) polymerase expression in familial than in sporadic breast cancer

    NARCIS (Netherlands)

    Klauke, M.L.; Hoogerbrugge-van der Linden, N.; Budczies, J.; Bult, P.; Prinzler, J.; Radke, C.; van Krieken, J.H.; Dietel, M.; Denkert, C.; Muller, B.M.

    2012-01-01

    Poly(ADP-ribose) polymerase 1 (PARP) is a key element of the single-base excision pathway for repair of DNA single-strand breaks. To compare the cytoplasmic and nuclear poly(ADP-ribose) expression between familial (BRCA1, BRCA2, or non BRCA1/2) and sporadic breast cancer, we investigated 39 sporadic

  19. VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt

    Directory of Open Access Journals (Sweden)

    Laura Lafon-Hughes

    2014-10-01

    Full Text Available Poly-ADP-ribose (PAR is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs and degraded by poly-ADP-ribose-glycohydrolase (PARG. Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair. Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt. In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO. PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications.

  20. Poly(ADP-ribose) polymerase-independent potentiation of nitrosourea cytotoxicity by 3-aminobenzamide in human malignant glioma cells.

    Science.gov (United States)

    Winter, S; Weller, M

    2000-06-16

    Poly(ADP-ribose) polymerase is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents and is thought to be involved in DNA repair. Here, we examined the effects of 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, on the chemosensitivity of human malignant glioma cells. 3-Aminobenzamide selectively potentiated the cytotoxicity of the nitrosoureas, nimustine, carmustine and lomustine in 10 of 12 human malignant glioma cell lines. In contrast, 3-aminobenzamide did not modulate the cytotoxic effects of doxorubicine, teniposide, vincristine, camptothecin or cytarabine. The nitrosoureas did not induce poly(ADP-ribose) polymerase activity in the glioma cells. Ectopic expression of truncated poly(ADP-ribose) polymerase containing the poly(ADP-ribose) polymerase DNA-binding domain, which acts as a dominant-negative mutant, in LN-18 or LN-229 cells did not alter the 3-aminobenzamide effect on nitrosourea-mediated cytotoxicity. Thus, 3-aminobenzamide may target another nicotinamide adenine dinucleotide (NAD)-requiring enzyme, but not poly(ADP-ribose) polymerase, when enhancing nitrosourea cytotoxicity in human malignant glioma cells. Carmustine cytotoxicity was associated with a G2/M arrest. Coexposure to carmustine and 3-aminobenzamide overcame this G2/M arrest in T98G cells, which are sensitized to carmustine by 3-aminobenzamide, but not in U251MG cells, which are refractory to 3-aminobenzamide-mediated sensitization to carmustine. Thus, 3-aminobenzamide-mediated sensitization to carmustine cytotoxicity may result from interference with the stable G2/M arrest response to carmustine in human glioma cells.

  1.   Adenosine-diphosphate (ADP) reduces infarct size and improves porcine heart function after myocardial infarction

    DEFF Research Database (Denmark)

    Bune, Laurids Touborg; Larsen, Jens Kjærgaard Rolighed; Thaning, Pia

    2013-01-01

    Acute myocardial infarction continues to be a major cause of morbidity and mortality. Timely reperfusion can substantially improve outcomes and the administration of cardioprotective substances during reperfusion is therefore highly attractive. Adenosine diphosphate (ADP) and uridine-5-triphoshat...... infusion during reperfusion reduces IS by ~20% independently from systemic release of t-PA. ADP-induced reduction in both preload and afterload could account for the beneficial myocardial effect....

  2. Overexpression, purification, and partial characterization of ADP-ribosyltransferases modA and modB of bacteriophage T4.

    Science.gov (United States)

    Tiemann, B; Depping, R; Rüger, W

    1999-01-01

    There is increasing experimental evidence that ADP-ribosylation of host proteins is an important means to regulate gene expression of bacteriophage T4. Surprisingly, this phage codes for three different ADP-ribosyltransferases, gene products Alt, ModA, and ModB, modifying partially overlapping sets of host proteins. While gene product Alt already has been isolated as a recombinant protein and its action on host RNA polymerases and transcription regulation have been studied, the nucleotide sequences of the two mod genes was published only recently. Their mode of action in the course of the infection cycle and the consequences of the ADP-ribosylations catalyzed by these enzymes remain to be investigated. Here we describe the cloning of the genes, the overexpression, purification, and partial characterization of ADP-ribosyltransferases ModA and ModB. Both proteins seem to act independently, and the ADP-ribosyl moieties are transferred to different sets of host proteins. While gene product ModA, similarly to the Alt protein, acts also on the alpha-subunit of host RNA polymerase, the ModB activity serves another set of proteins, one of which was identified as the S1 protein associated with the 30S subunit of the E. coli ribosomes.

  3. Niacin, poly(ADP-ribose) polymerase-1 and genomic stability

    NARCIS (Netherlands)

    Hageman, G.J.; Stierum, R.H.

    2001-01-01

    Nicotinic acid (NA) and nicotinamide (NAM), commonly called niacin, are the dietary precursors for NAD+ (nicotinamide adenine dinucleotide), which is required for DNA synthesis, as well as for the activity of the enzyme poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) for which NAD+ is the sole

  4. A new approach of optimal control for a class of continuous-time chaotic systems by an online ADP algorithm

    Science.gov (United States)

    Song, Rui-Zhuo; Xiao, Wen-Dong; Wei, Qing-Lai

    2014-05-01

    We develop an online adaptive dynamic programming (ADP) based optimal control scheme for continuous-time chaotic systems. The idea is to use the ADP algorithm to obtain the optimal control input that makes the performance index function reach an optimum. The expression of the performance index function for the chaotic system is first presented. The online ADP algorithm is presented to achieve optimal control. In the ADP structure, neural networks are used to construct a critic network and an action network, which can obtain an approximate performance index function and the control input, respectively. It is proven that the critic parameter error dynamics and the closed-loop chaotic systems are uniformly ultimately bounded exponentially. Our simulation results illustrate the performance of the established optimal control method.

  5. Control of ATP hydrolysis by ADP bound at the catalytic site of chloroplast ATP synthase as related to protonmotive force and Mg2+

    International Nuclear Information System (INIS)

    Du, Z.; Boyer, P.D.

    1989-01-01

    The activation of the ATP synthesis and hydrolysis capacity of isolated chloroplast membranes by protonmotive force is known to be associated with the release of tightly bound ADP from the ATP synthase. The data support the view that the activation requires only those structural changes occurring in the steady-state reaction mechanism. The trapping of ADP released during light activation or the chelation of Mg 2+ with EDTA effectively reduces the rate of decay of the ATPase activity. When the release of tightly bound ADP and Mg 2+ is promoted by light activation, followed by immediate dilution and washing to retard the rebinding of the ADP and Mg 2+ released, the ATPase activity remains high in the dark long after the protonmotive force has disappeared. After the addition of ADP and Mg 2+ the decay of the ATPase activity has the same characteristics as those of the unwashed chloroplast membrane. The results are interpreted as indicating that both Mg 2+ and ADP must be present prior to exposure to MgATP for the ATPase to be inhibited. However, in contrast to the isolated chloroplast ATPase, the steady-state activity of the membrane-bound ATPase is not inhibited by excess Mg 2+ . The replacement of [ 3 H]ADP from catalytic sites during hydrolysis of unlabeled ATP or during photophosphorylation with unlabeled ADP occurs as anticipated if Mg 2+ and ADP bound at one catalytic site without P i block catalysis by all three enzyme sites. The inhibited form induced by Mg 2+ and ADP may occur only under laboratory conditions and not have an in vivo role

  6. Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

    Science.gov (United States)

    Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K; Miyazaki, Hiroshi; Lewis, Janina P

    2014-06-01

    Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

  7. Pratt & Whitney Advanced Ducted Propulsor (ADP) Engine Test in 40x80ft w.t.: Engineers Peter

    Science.gov (United States)

    1993-01-01

    Pratt & Whitney Advanced Ducted Propulsor (ADP) Engine Test in 40x80ft w.t.: Engineers Peter Zell (left) and Dr Clifton Horne (right) are shown preparing a laser light sheet for a flow visualization test. Shown standing in the nacelle of the ADP is John Girvin, senior test engineer for Pratt & Whitney.

  8. PARP16/ARTD15 is a novel endoplasmic-reticulum-associated mono-ADP-ribosyltransferase that interacts with, and modifies karyopherin-ß1.

    Directory of Open Access Journals (Sweden)

    Simone Di Paola

    Full Text Available BACKGROUND: Protein mono-ADP-ribosylation is a reversible post-translational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. The latter include members of three different families of proteins: the well characterized arginine-specific ecto-enzymes ARTCs, two sirtuins and, more recently, novel members of the poly(ADP-ribose polymerase (PARP/ARTD family that have been suggested to act as cellular mono-ADP-ribosyltransferases. Here, we report on the characterisation of human ARTD15, the only known ARTD family member with a putative C-terminal transmembrane domain. METHODOLOGY/PRINCIPAL FINDINGS: Immunofluorescence and electron microscopy were performed to characterise the sub-cellular localisation of ARTD15, which was found to be associated with membranes of the nuclear envelope and endoplasmic reticulum. The orientation of ARTD15 was determined using protease protection assay, and is shown to be a tail-anchored protein with a cytosolic catalytic domain. Importantly, by combining immunoprecipitation with mass spectrometry and using cell lysates from cells over-expressing FLAG-ARTD15, we have identified karyopherin-ß1, a component of the nuclear trafficking machinery, as a molecular partner of ARTD15. Finally, we demonstrate that ARTD15 is a mono-ADP-ribosyltransferase able to induce the ADP-ribosylation of karyopherin-ß1, thus defining the first substrate for this enzyme. CONCLUSIONS/SIGNIFICANCE: Our data reveal that ARTD15 is a novel ADP-ribosyltransferase enzyme with a new intracellular location. Finally, the identification of karyopherin-ß1 as a target of ARTD15-mediated ADP-ribosylation, hints at a novel regulatory mechanism of karyopherin-ß1 functions.

  9. Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

    Science.gov (United States)

    Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

    2009-12-15

    Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

  10. Control of ATP hydrolysis by ADP bound at the catalytic site of chloroplast ATP synthase as related to protonmotive force and Mg sup 2+

    Energy Technology Data Exchange (ETDEWEB)

    Du, Z.; Boyer, P.D. (Univ. of California, Los Angeles (USA))

    1989-01-24

    The activation of the ATP synthesis and hydrolysis capacity of isolated chloroplast membranes by protonmotive force is known to be associated with the release of tightly bound ADP from the ATP synthase. The data support the view that the activation requires only those structural changes occurring in the steady-state reaction mechanism. The trapping of ADP released during light activation or the chelation of Mg{sup 2+} with EDTA effectively reduces the rate of decay of the ATPase activity. When the release of tightly bound ADP and Mg{sup 2+} is promoted by light activation, followed by immediate dilution and washing to retard the rebinding of the ADP and Mg{sup 2+} released, the ATPase activity remains high in the dark long after the protonmotive force has disappeared. After the addition of ADP and Mg{sup 2+} the decay of the ATPase activity has the same characteristics as those of the unwashed chloroplast membrane. The results are interpreted as indicating that both Mg{sup 2+} and ADP must be present prior to exposure to MgATP for the ATPase to be inhibited. However, in contrast to the isolated chloroplast ATPase, the steady-state activity of the membrane-bound ATPase is not inhibited by excess Mg{sup 2+}. The replacement of ({sup 3}H)ADP from catalytic sites during hydrolysis of unlabeled ATP or during photophosphorylation with unlabeled ADP occurs as anticipated if Mg{sup 2+} and ADP bound at one catalytic site without P{sub i} block catalysis by all three enzyme sites. The inhibited form induced by Mg{sup 2+} and ADP may occur only under laboratory conditions and not have an in vivo role.

  11. Determinants of rice output among ADP contact farmers in mining ...

    African Journals Online (AJOL)

    The study analyzed factors affecting rice output among Agricultural Development Programme (ADP) contact farmers in the mining and non mining locations of IVO LGA of Ebonyi State, Nigeria. Multistage random sampling technique was used to select agricultural circles and rice farmers. The sample size was 120 rice ...

  12. Multiple origins of hydrogenosomes: functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytrid Neocallimastix sp.

    NARCIS (Netherlands)

    Voncken, F.L.M.; Boxma, B.; Tjaden, J.; Akhmanova, A.S.; Huynen, M.A.; Verbeek, F.; Tielens, A.G.; Haferkamp, I.; Neuhaus, H.E.; Vogels, G.D.; Veenhuis, M.; Hackstein, J.H.P.

    2002-01-01

    A mitochondrial-type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the

  13. Multiple origins of hydrogenosomes : functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytrid Neocallimastix sp.

    NARCIS (Netherlands)

    Voncken, F; Boxma, B; Tjaden, J; Akhmanova, A; Huynen, M; Tielens, AGM; Haferkamp, [No Value; Neuhaus, HE; Vogels, G; Veenhuis, M; Hackstein, JHP; Tielens, Aloysius G.M.; Haferkamp, Ilka; Hackstein, Johannes H.P.

    A mitochondrial-type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the

  14. Poly (ADP-Ribose) Polymerase is Involved in the Repair of DNA Damage Due to Sulfur Mustard by a Mechanism Other Than DNA Ligase I Activation

    National Research Council Canada - National Science Library

    Bhat, K. Ramachandra; Benton, Betty J; Ray, Radharaman

    2004-01-01

    Poly (ADP-ribose) polymerase (PARP) modulates several cellular functional proteins by a mechanism in which the proteins are poly-ADP-ribosylated by transferring the ADP-ribose moieties from the enzyme substrate NAD+ to the proteins...

  15. Human mass balance study and metabolite profiling of 14C-niraparib, a novel poly(ADP-Ribose) polymerase (PARP)-1 and PARP-2 inhibitor, in patients with advanced cancer

    NARCIS (Netherlands)

    van Andel, Lotte; Zhang, Z; Lu, S.; Kansra, V; Agarwal, S.; Hughes, L.; Tibben, M.; Gebretensae, A.; Lucas, L.; Hillebrand, Michel J X; Rosing, H.; Schellens, J H M|info:eu-repo/dai/nl/073926272; Beijnen, J H|info:eu-repo/dai/nl/071919570

    2017-01-01

    Niraparib is an investigational oral, once daily, selective poly(ADP-Ribose) polymerase (PARP)-1 and PARP-2 inhibitor. In the pivotal Phase 3 NOVA/ENGOT/OV16 study, niraparib met its primary endpoint of improving progression-free survival (PFS) for adult patients with recurrent, platinum sensitive,

  16. Interaction of cytochalasin D with actin filaments in the presence of ADP and ATP.

    Science.gov (United States)

    Carlier, M F; Criquet, P; Pantaloni, D; Korn, E D

    1986-02-15

    Cytochalasin D strongly inhibits the faster components in the reactions of actin filament depolymerization and elongation in the presence of 10 mM Tris-Cl-, pH 7.8, 0.2 mM dithiothreitol, 1 mM MgCl2, 0.1 mM CaCl2, and 0.2 mM ATP or ADP. Assuming an exclusive and total capping of the barbed end by the drug, the kinetic parameters derived at saturation by cytochalasin D refer to the pointed end and are 10-15-fold lower than at the barbed end. In ATP, the critical concentration increases with cytochalasin D up to 12-fold its value when both ends are free; as a result of the lowering of the free energy of nucleation by cytochalasin D, short oligomers of F-actin exist just above and below the critical concentration. Cytochalasin D interacts strongly with the barbed ends independently of the ADP-G-actin concentration (K = 0.5 nM-1). In contrast, the affinity of cytochalasin D decreases cooperatively with increasing ATP-G-actin concentration. These data are equally well accounted for by two different models: either cytochalasin D binds very poorly to ATP-capped filament ends whose proportion increases with actin concentration, or cytochalasin D binds equally well to ATP-ends and ADP-ends and also binds to actin dimers in ATP but not in ADP. A linear actin concentration dependence of the rate of growth was found at the pointed end, consistent with the virtual absence of an ATP cap at that end.

  17. On the mechanism of sulfite activation of chloroplast thylakoid ATPase and the relation of ADP tightly bound at a catalytic site to the binding change mechanism

    International Nuclear Information System (INIS)

    Du, Z.; Boyer, P.D.

    1990-01-01

    Washed chloroplast thylakoid membranes upon exposure to [ 3 H]ADP retain in tightly bound [ 3 H]ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg 2+ results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound [ 3 H]ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme. The authors present evidence that this is not the case. The Mg 2+ - and ADP-inhibited enzyme when exposed to MgATP and 20-100 mM sulfite shows a lag of about 1 min at 22 degree C and of about 15 s at 37 degree C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg 2+ and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound [ 3 H]ADP parallels the onset of ATPase activity, although some [ 3 H]ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound [ 3 H]ADP being at a catalytic site and being replaced as this Mg 2+ - and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme. The sulfite activation can be explained by sulfite combination at a P i binding site of the enzyme-ADP-Mg 2+ complex to give a form more readily activated by ATP binding at an alternative site

  18. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure*

    Science.gov (United States)

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O.; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M. Cristina

    2016-01-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1−/− compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  19. Overproduction of the poly(ADP-ribose)polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

    NARCIS (Netherlands)

    M. Molinete; W. Vermeulen (Wim); A. Bürkle; J. Mé nissier-de Murcia; J.H. Küpper; J.H.J. Hoeijmakers (Jan); G. de Murcia

    1993-01-01

    textabstractThe zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during

  20. Correlation between increased platelet ADP aggregability and silent brain infarcts

    International Nuclear Information System (INIS)

    Ono, Kenichiro; Arimoto, Hirohiko; Shirotani, Toshiki

    2012-01-01

    The purpose of this study was to investigate the correlation between platelet aggregability and silent brain infarcts. The study subjects were 445 people (264 men, 181 women; mean age, 53±14 years) with no neurologic signs, history of brain tumor, trauma, cerebrovascular disease, or antiplatelet medications. Adenosine diphosphate (ADP)-induced platelet aggregation was measured by the aggregation-size analytic method. Platelet aggregability was classified into 9 classes. The presence of headache/vertigo, hypertension, diabetes mellitus, hyperlipidemia, or smoking was elicited by questioning or blood sampling. A head MRI scan was performed, and if marked atherosclerosis or obvious stenosis in the intracranial vessels was detected, it was defined as a positive MR angiography (MRA) finding. Silent brain infarcts were detected in 26.3% of subjects. Hyperaggregability defined as that above class 6, 7, and 8 was present in 43.8%, 30.8%, and 15.7% of subjects, respectively. The risk factors for silent brain infarcts by multiple logistic regression analysis were aging, hypertension, positive MRA findings, and hyperaggregability. Platelet ADP hyperaggregability might be a risk factor for silent brain infarcts. (author)

  1. 10 CFR 95.49 - Security of automatic data processing (ADP) systems.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Security of automatic data processing (ADP) systems. 95.49 Section 95.49 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) FACILITY SECURITY CLEARANCE AND SAFEGUARDING OF NATIONAL SECURITY INFORMATION AND RESTRICTED DATA Control of Information § 95.49 Security of...

  2. On the mechanism of sulfite activation of chloroplast thylakoid ATPase and the relation of ADP tightly bound at a catalytic site to the binding change mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Du, Z.; Boyer, P.D. (Univ. of California, Los Angeles (USA))

    1990-01-16

    Washed chloroplast thylakoid membranes upon exposure to ({sup 3}H)ADP retain in tightly bound ({sup 3}H)ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg{sup 2+} results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound ({sup 3}H)ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme. The authors present evidence that this is not the case. The Mg{sup 2+}- and ADP-inhibited enzyme when exposed to MgATP and 20-100 mM sulfite shows a lag of about 1 min at 22{degree}C and of about 15 s at 37{degree}C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg{sup 2+} and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound ({sup 3}H)ADP parallels the onset of ATPase activity, although some ({sup 3}H)ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound ({sup 3}H)ADP being at a catalytic site and being replaced as this Mg{sup 2+}- and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme. The sulfite activation can be explained by sulfite combination at a P{sub i} binding site of the enzyme-ADP-Mg{sup 2+} complex to give a form more readily activated by ATP binding at an alternative site.

  3. Robust/optimal temperature profile control of a high-speed aerospace vehicle using neural networks.

    Science.gov (United States)

    Yadav, Vivek; Padhi, Radhakant; Balakrishnan, S N

    2007-07-01

    An approximate dynamic programming (ADP)-based suboptimal neurocontroller to obtain desired temperature for a high-speed aerospace vehicle is synthesized in this paper. A 1-D distributed parameter model of a fin is developed from basic thermal physics principles. "Snapshot" solutions of the dynamics are generated with a simple dynamic inversion-based feedback controller. Empirical basis functions are designed using the "proper orthogonal decomposition" (POD) technique and the snapshot solutions. A low-order nonlinear lumped parameter system to characterize the infinite dimensional system is obtained by carrying out a Galerkin projection. An ADP-based neurocontroller with a dual heuristic programming (DHP) formulation is obtained with a single-network-adaptive-critic (SNAC) controller for this approximate nonlinear model. Actual control in the original domain is calculated with the same POD basis functions through a reverse mapping. Further contribution of this paper includes development of an online robust neurocontroller to account for unmodeled dynamics and parametric uncertainties inherent in such a complex dynamic system. A neural network (NN) weight update rule that guarantees boundedness of the weights and relaxes the need for persistence of excitation (PE) condition is presented. Simulation studies show that in a fairly extensive but compact domain, any desired temperature profile can be achieved starting from any initial temperature profile. Therefore, the ADP and NN-based controllers appear to have the potential to become controller synthesis tools for nonlinear distributed parameter systems.

  4. MgADP-induced changes in the structure of myosin S1 near the ATPase-related thiol SH1 probed by cross-linking

    International Nuclear Information System (INIS)

    Rajasekharan, K.N.; Mayadevi, M.; Agarwal, R.; Burke, M.

    1990-01-01

    The structural consequence of MgADP binding at the vicinity of the ATPase-related thiol SH1 (Cys-707) have been examined by subjecting myosin subfragment 1, premodified at SH2 (Cys-697) with N-ethylmaleimide (NEM), to reaction with the bifunctional reagent p-phenylenedimaleimide (pPDM) in the presence and absence of MgADP. By monitoring the changes in the Ca 2+ -ATPase activity as a function of reaction time, it appears that the reagent rapidly modifies SH1 irrespective of whether MgADP is present or not. In the absence of nucleotide, only extremely low levels of cross-linking to the 50-kDa middle segment of S1 can be detected, while in the presence of MgADP substantial cross-linking to this segment is observed. A similar cross-link is also formed if MgADP is added subsequent to the reaction of the SH2-NEM-premodified S1 with pPDM in the absence of nucleotide. Isolation of the labeled tryptic peptide from the cross-linked adduct formed with [ 14 C]pPDM, and subsequent partial sequence analyses, indicates that the cross-link is made from SH1 to Cys-522. Moreover, it appears that this cross-link results in the trapping of MgADP in this S1 species. These data suggest that the binding of MgADP results in a change in the structure of S1 in the vicinity of the SH1 thiol relative to the 50-kDa domain which enables Cys-522 to adopt the appropriate configuration to enable it to be cross-linked to SH1 by pPDM

  5. Effects of inorganic phosphate and ADP on calcium handling by the sarcoplasmic reticulum in rat skinned cardiac muscles.

    Science.gov (United States)

    Xiang, J Z; Kentish, J C

    1995-03-01

    The aim was to investigate whether, and how, increases in inorganic phosphate (Pi) and ADP, similar to those occurring intracellularly during early myocardial ischaemia, affect the calcium handling of the sarcoplasmic reticulum. Rat ventricular trabeculae were permeabilised with saponin. The physiological process of calcium induced calcium release (CICR) from the muscle sarcoplasmic reticulum was triggered via flash photolysis of the "caged Ca2+", nitr-5. Alternatively, calcium release was induced by rapid application of caffeine to give an estimate of sarcoplasmic reticular calcium loading. The initial rate of sarcoplasmic reticular calcium pumping was also assessed by photolysis of caged ATP at saturating [Ca2+]. Myoplasmic [Ca2+] (using fluo-3) and isometric force were measured. Pi (2-20 mM) significantly depressed the magnitude of CICR and the associated force transient. Sarcoplasmic reticular calcium loading was inhibited even more than CICR by Pi, suggesting that reduced calcium loading could account for all of the inhibitory effect of Pi on CICR and that Pi may slightly activate the calcium release mechanism. The reduced sarcoplasmic reticular calcium loading seemed to be due to a fall in the free energy of ATP hydrolysis (delta GATP) available for the calcium pump, since equal decreases in delta GATP produced by adding both Pi and ADP in various ratios caused similar falls in the calcium loading of the sarcoplasmic reticulum. The caged ATP experiments indicated that Pi (20 mM) did not affect the rate constant of sarcoplasmic reticular calcium uptake. ADP (10 mM) alone, or with 1 mM Pi, inhibited calcium loading. In spite of this, ADP (10 mM) did not alter CICR and, when 1 mM Pi was added, ADP increased CICR above control. An increase in intracellular Pi reduces sarcoplasmic reticular calcium loading and thus depresses the CICR. This could be an important contributing factor in the hypoxic or ischaemic contractile failure of the myocardium. However the

  6. Profiling of Biomarkers for the Exposure of Polycyclic Aromatic Hydrocarbons: Lamin-A/C Isoform 3, Poly[ADP-ribose] Polymerase 1, and Mitochondria Copy Number Are Identified as Universal Biomarkers

    Directory of Open Access Journals (Sweden)

    Hwan-Young Kim

    2014-01-01

    Full Text Available This study investigated the profiling of polycyclic aromatic hydrocarbon- (PAH- induced genotoxicity in cell lines and zebrafish. Each type of cells displayed different proportionality of apoptosis. Mitochondrial DNA (mtDNA copy number was dramatically elevated after 5-day treatment of fluoranthene and pyrene. The notable deregulated proteins for PAHs exposure were displayed as follows: lamin-A/C isoform 3 and annexin A1 for benzopyrene; lamin-A/C isoform 3 and DNA topoisomerase 2-alpha for pentacene; poly[ADP-ribose] polymerase 1 (PARP-1 for fluoranthene; and talin-1 and DNA topoisomerase 2-alpha for pyrene. Among them, lamin-A/C isoform 3 and PARP-1 were further confirmed using mRNA and protein expression study. Obvious morphological abnormalities including curved backbone and cardiomegaly in zebrafish were observed in the 54 hpf with more than 400 nM of benzopyrene. In conclusion, the change of mitochondrial genome (increased mtDNA copy number was closely associated with PAH exposure in cell lines and mesenchymal stem cells. Lamin-A/C isoform 3, talin-1, and annexin A1 were identified as universal biomarkers for PAHs exposure. Zebrafish, specifically at embryo stage, showed suitable in vivo model for monitoring PAHs exposure to hematopoietic tissue and other organs.

  7. 211At-α-dose dependence of poly-ADP-ribosylation of human glioblastoma cells in vitro. Suitability in cancer therapy?

    International Nuclear Information System (INIS)

    Schneeweiss, F.H.A.

    1999-01-01

    Aim: It was intended to test the biological response (poly-ADP-ribosylation of cellular proteins) of α-particles from extracellular 211 At for enhanced damage to human glioblastoma cells in vitro and to discuss its suitability for potential application in therapy of high-grade gliomas. Materials and Methods: Confluent cultures of human glioblastoma cells were exposed to different doses of α-radiations from homogeneously distributed extracellular 211 At. Cellular poly-ADP-ribosylation of all proteins including histones was monitored since it is an indirect but sensitive indicator of chromatin damage and putative repair in both normal and malignant mammalian cells. Results: A significant diminution (average 85.6%) in poly-ADP-ribosylation of total cellular proteins relative to that for non-irradiated glioblastoma cells was observed following 0.025 to 1.0 Gy α-radiations. In the dose range of 0.0025 to 0.01 Gy there was an increase with a maximum value of approximately 119.0% at 0.0025 Gy. Below 0.0025 Gy no change in poly-ADP-ribosylation was observed. Conclusions: Level of cellular poly-ADP-ribosylation of proteins at 0.025 to 1.0 Gy of α-radiation dose from 211 At appears to cause enhanced damage by creating molecular conditions which are not conductive to repair of DNA damages in human glioblastoma cells in vitro. Therefore, it is assumed that clinical application of 211 At at least in this dose range might enhance clinical efficacy in radiotherapy of cancer. (orig.) [de

  8. Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

    Directory of Open Access Journals (Sweden)

    Benjamin Clémençon

    2012-02-01

    Full Text Available The existence of a mitochondrial interactosome (MI has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp and inorganic phosphate (PiC carriers as well as the VDAC (or mitochondrial porin catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1 under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003.

  9. Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells

    Science.gov (United States)

    Mortusewicz, Oliver; Amé, Jean-Christophe; Leonhardt, Heinrich

    2007-01-01

    Genome integrity is constantly threatened by DNA lesions arising from numerous exogenous and endogenous sources. Survival depends on immediate recognition of these lesions and rapid recruitment of repair factors. Using laser microirradiation and live cell microscopy we found that the DNA-damage dependent poly(ADP-ribose) polymerases (PARP) PARP-1 and PARP-2 are recruited to DNA damage sites, however, with different kinetics and roles. With specific PARP inhibitors and mutations, we could show that the initial recruitment of PARP-1 is mediated by the DNA-binding domain. PARP-1 activation and localized poly(ADP-ribose) synthesis then generates binding sites for a second wave of PARP-1 recruitment and for the rapid accumulation of the loading platform XRCC1 at repair sites. Further PARP-1 poly(ADP-ribosyl)ation eventually initiates the release of PARP-1. We conclude that feedback regulated recruitment of PARP-1 and concomitant local poly(ADP-ribosyl)ation at DNA lesions amplifies a signal for rapid recruitment of repair factors enabling efficient restoration of genome integrity. PMID:17982172

  10. Data-Driven Zero-Sum Neuro-Optimal Control for a Class of Continuous-Time Unknown Nonlinear Systems With Disturbance Using ADP.

    Science.gov (United States)

    Wei, Qinglai; Song, Ruizhuo; Yan, Pengfei

    2016-02-01

    This paper is concerned with a new data-driven zero-sum neuro-optimal control problem for continuous-time unknown nonlinear systems with disturbance. According to the input-output data of the nonlinear system, an effective recurrent neural network is introduced to reconstruct the dynamics of the nonlinear system. Considering the system disturbance as a control input, a two-player zero-sum optimal control problem is established. Adaptive dynamic programming (ADP) is developed to obtain the optimal control under the worst case of the disturbance. Three single-layer neural networks, including one critic and two action networks, are employed to approximate the performance index function, the optimal control law, and the disturbance, respectively, for facilitating the implementation of the ADP method. Convergence properties of the ADP method are developed to show that the system state will converge to a finite neighborhood of the equilibrium. The weight matrices of the critic and the two action networks are also convergent to finite neighborhoods of their optimal ones. Finally, the simulation results will show the effectiveness of the developed data-driven ADP methods.

  11. ADP-ribosylation of nonhistone proteins from metaphase and interphase HeLa cells: factors responsible for differences

    International Nuclear Information System (INIS)

    Adolph, K.W.

    1986-01-01

    A striking reduction was previously detected for HeLa metaphase chromosomes, compared to interphase nuclei, in the number of modified nonhistone species. Several factors which could contribute to this cell cycle change in ADP-ribosylation have therefore been examined. In these experiments, mitotic or interphase cells were incubated with [ 32 P]NAD, chromosomes and nuclei were prepared, and the proteins were resolved by polyacrylamide gel electrophoresis. The level of incorporation of 32 P label was found to be substantially influenced by chromosome expansion, DNA nicking, disruption of chromosomes or nuclei, and the growth activity of cells. The level of ADP-ribosylation was not greatly affected by the presence of inhibitors of RNA, DNA, and protein synthesis. NAD concentration influenced the extent of labelling but not the pattern of labeled species. A similar change in the pattern from interphase to mitosis was observed for whole cells as well as for isolated chromosomes and nuclei. The procedure used to arrest cells in mitosis was not artifactually responsible for the results. The difference in metaphase and interphase ADP-ribosylation is not confined to HeLa cells, since comparable patterns were found for chromosomes and nuclei from Novikoff rat hepatoma cells

  12. Site of ADP-ribosylation and the RNA-binding site are situated in different domains of the elongation factor EF-2

    International Nuclear Information System (INIS)

    Davydova, E.K.

    1987-01-01

    One of the proteins participating in the process of elongation of polypeptide chains - elongation factor 2 (EF-2) - can be ADP-ribosylated at a unique amino acid residue - diphthamide. Since the ADP-ribosylation of EF-2 at dipthamide leads to a loss of affinity of the factor for RNA while the presence of RNA inhibits the ADP-ribosylation reaction, it seemed probable to the authors that diphthamide participated directly in the binding of EF-2 to DNA. The experiments presented in this article showed that this was not the case: diphthamide and the RNA-binding site are situated on different domains of EF-2. Thus, ADP-ribosylation of factor EF-2 in one domain leads to a loss of the ability to bind to RNA in the other. The authors investigated the mutual arrangement of diphthamide and the RNA-binding site on the EF-2 molecule by preparing a factor from rabbit reticulocytes and subjecting it to proteolytic digestion with elastase. The factor was incubated with elastase for 15 min at 37 0 C at an enzyme:substrate ratio of 1:100 in buffer solution containing 20 mM Tris-HCl, pH 7.6, 10 mM KCl, 1 mM MgCl 2 , and 2 mM dithiothreitol. The reaction was stopped by adding para-methylsulfonyl fluoride to 50 micro-M. The authors obtained a preparation as a result of proteolysis and applied it on a column with RNA-Sepharose and separated into two fractions: RNA-binding and without affinity for RNA. The initial preparation and its fractions were subjected to exhaustive ADP-ribosylation in the presence of diphtheria toxin and [U- 14 C] nicotinaide adenine dinucleotide ([ 14 C]NAD) (296 mCi/mmole). The samples were analyzed electrophoretically in a polyacrylamide gel gradient in the presence of sodium dodecyl sulfate. For the detection of [ 14 C] ADP-ribosylated components, the gels were dried and exposed with RM-V x-ray film

  13. Effects of the gelatin plasma substitutes Haemaccel, Plasmagel and Plasmion (Geloplasma) on collagen-, ADP- and adrenaline-induced aggregation of human platelets in vitro.

    Science.gov (United States)

    Stibbe, J; van der Plas, P M; Ong, G L; ten Hoor, F; Nauta, J; de Jong, D S; Krenning-Douma, E; Gomes, M

    1981-01-01

    The effect of some gelatin plasma substitutes (Haemaccel, plasmagel and Plasmion (Geloplasma), which are widely used in Europe) on collagen-, ADP- and adrenaline-induced platelet aggregation in human PRP in vitro was studied under controlled conditions (pH, electrolyte composition). Haemaccel inhibited these aggregations, both in citrated as well as in heparinised PRP, whereas they were enhanced by both Plasmagel and Plasmion as compared to the appropriate control. Increasing teh concentration of the inducer overcame the inhibition by Haemaccel. Haemaccel inhibited, while Plasmion enhanced 14C-serotonin release induced by collagen, ADP or adrenaline. Also in the presence of indomethacin (90 muM) Haemaccel inhibited aggregation induced by high concentrations of collagen and the primary aggregation induced by ADP and adrenaline, while Plasmion enhanced these aggregations induced by ADP and adrenaline, while Plasmion enhanced these aggregations. The inhibition by Haemaccel was not caused by binding of Ca2+ to haemaccel.

  14. Role of reactive oxygen species and poly-ADP-ribose polymerase in the development of AZT-induced cardiomyopathy in rat.

    Science.gov (United States)

    Szabados, E; Fischer, G M; Toth, K; Csete, B; Nemeti, B; Trombitas, K; Habon, T; Endrei, D; Sumegi, B

    1999-02-01

    The short term cardiac side-effects of AZT (3'-azido-3'-deoxythymidine, zidovudine) was studied in rats to understand the biochemical events contributing to the development of AZT-induced cardiomyopathy. Developing rats were treated with AZT (50 mg/kg/day) for 2 wk and the structural and functional changes were monitored in the cardiac muscle. AZT treatment provoked a surprisingly fast appearance of cardiac malfunctions in developing animals characterized by prolonged RR, PR and QT intervals and J point depression. Electron microscopy showed abnormal mitochondrial structure but the cardiomyocyte had normal myofibers. The AZT treatment of rats significantly increased ROS and peroxynitrite formation in heart tissues as determined by the oxidation of nonfluorescent dihydrorhodamine123 and dichlorodihydro-fluorescein diacetate (H2DCFDA) to fluorescent dyes, and induced single-strand DNA breaks. Lipid peroxidation and oxidation of cellular proteins determined from protein carbonyl content were increased as a consequence of AZT treatment. Activation of the nuclear poly-ADP-ribose polymerase and the accelerated NAD+ catabolism were also observed in AZT-treated animals. Western blot analysis showed that mono-ADP-ribosylation of glucose regulated protein (GRP78/BIP) was enhanced by AZT treatment, that process inactivates GRP78. In this way moderate decrease in the activity of respiratory complexes was detected in the heart of AZT-treated animals indicating a damaged mitochondrial energy production. There was a significant decrease in creatine phosphate concentration resulting in a decrease in creatine phosphate/creatine ratio from 2.08 to 0.58. ATP level remained close to normal but the total extractable ADP increased with 45%. The calculated free ATP/ADP ratio decreased from 340 to 94 in the heart of AZT-treated rats as a consequence of increased free ADP concentration. It was assumed that the increased free ADP in AZT-treated cardiomyocyte may help cells to compensate the

  15. Mono(ADP-ribosyl)ation of the N2 amino groups of guanine residues in DNA by pierisin-2, from the cabbage butterfly, Pieris brassicae

    International Nuclear Information System (INIS)

    Takamura-Enya, Takeji; Watanabe, Masahiko; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

    2004-01-01

    Pierisin-2 is a cytotoxic and apoptosis-inducing protein present in Pieris brassicae with a 91% homology in the deduced amino acid sequences to pierisin-1 from Pieris rapae. We earlier showed pierisin-1 to catalyze mono(ADP-ribosyl)ation of 2'-deoxyguanosine (dG) in DNA to form N 2 -(ADP-ribos-1-yl)-2'-deoxyguanosine, this DNA modification appearing linked to its cytotoxicity and ability to induce apoptosis in mammalian cell lines. In this paper, we documented evidence that pierisin-2 also catalyzed ADP-ribosylation of dG in DNA to give the same reaction product as demonstrated for pierisin-1, with similar efficiency. With oligonucleotides as substrates, ADP-ribosylation by pierisin-2 was suggested to occur by one-side attack of the carbon atom at 1 position of the ribose moiety in NAD toward N 2 of dG. The presence of a unique ADP-ribosylation toxin targeting dG in DNA in two distinct species in a Pieris genus could be a quite important finding to better understand biological functions of pierisin-1 and -2 in Pieris butterflies and the generic evolution of these cabbage butterflies

  16. Poly(ADP-ribose) polymerase inhibitors suppress UV-induced human immunodeficiency virus type 1 gene expression at the posttranscriptional level

    International Nuclear Information System (INIS)

    Yamagoe, S.; Kohda, T.; Oishi, M.

    1991-01-01

    Gene expression of human immunodeficiency virus type 1 (HIV-1) is induced not only by trans activation mediated through a gene product (tat) encoded by the virus but also by treatment of virus-carrying cells with DNA-damaging agents such as UV light. Employing an artificially constructed DNA in which the chloramphenicol acetyltransferase gene was placed under the control of the HIV-1 long terminal repeat, we analyzed the induction process in HeLa cells and found that inhibitors of poly(ADP-ribose) polymerase suppressed UV-induced HIV-1 gene expression but not tat-mediated expression. We also found that suppression occurs at the posttranscriptional level. These results indicate that HIV-1 gene expression is activated by at least two different mechanisms, one of which involves poly-ADP ribosylation. A possible new role of poly-ADP ribosylation in the regulation of specific gene expression is also discussed

  17. Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

    Energy Technology Data Exchange (ETDEWEB)

    Jones, J.; Schultz, R.M. (Univ. of Pennsylvania, Philadelphia (USA))

    1990-06-01

    G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with Mr = 38,000 and 39,000 (alpha 38 and alpha 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of alpha 38-39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12-O-tetradecanoyl phorbol 13-acetate), but not an inactive one (4 alpha-phorbol 12,13-didecanoate, 4 alpha-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo. In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or alpha-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of alpha 38-39. Results of immunoblotting experiments using an antibody that is highly specific for alpha il-3 reveal the presence of a cross-reactive species of Mr = 38,000 (alpha 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos.

  18. Levels of adenine nucleotides (ATP, ADP, AMP) and of inorganic phosphate in needles of Picea abies, representing different stages of development and of pollution dependence

    Energy Technology Data Exchange (ETDEWEB)

    Benz, T; Hampp, R; Horsch, F; Filby, G; Fund, N; Gross, S; Hanisch, B; Kilz, E; Seidel, A [comps.

    1986-04-01

    Levels of adenine nucleotides (ATP, ADP, AMP) and of inorganic phosphate in needles of Picea abies, representing different stages of development and of pollution dependence. Lyophilized needles of Picea abies (Kaelbelescheuer, southern Black Forest) were analyzed for their content of adenine nucleotides (ATP, ADP, AMP: AdN) and of inorganic phosphate (Psub(i)). The metabolite levels were related to needle age, vegetation period and degree of damage (chlorophyll content). The results were as follows: 1) With increasing needle age there is a general decrease in the total AdN-pool. This decrease is most pronounced in very young needles and occurs in both healthy and damaged tissue. 2) The ATP/ADP-ratio of damaged needle is significantly higher than that of healthy ones. 3) Both phosphorylation potential (ATP.(ADP.Psub(i))/sup -1/) and adenylate energy charge ((ATP + 0.5.ADP).(AdN)/sup -1/) are significantly reduced in damaged needles. This is due to relatively higher levels of Psub(i) and of AMP. The results, although incomplete and preliminary, indicate metabolic alterations which have been described for other tissues in response to pollution by photooxidants.

  19. Effect of oxalic acid on the optical, thermal, dielectric and mechanical behaviour of ADP crystals

    International Nuclear Information System (INIS)

    Rajesh, P.; Ramasamy, P.

    2009-01-01

    The effect of the addition, over a concentration range from 1 to 5 mol%, of oxalic acid on the growth rate, optical transparency, hardness, dielectric behaviour, and SHG efficiency of ammonium dihydrogen phosphate single crystals grown by slow evaporation method has been investigated. UV-Vis studies show that the transparency of the oxalic acid added crystals decreased gradually. Thermal studies indicate that the decomposition temperatures of the crystal are decreased in oxalic acid added ADP crystals. It is observed from the dielectric measurements that the dielectric constant and dielectric loss increase with increase in temperature for all the crystals. Vicker's microhardness study reveals that the addition of higher concentration of oxalic acid decreases the hardness of the crystal. SHG efficiency of 1 mol% of oxalic acid is higher than the pure ADP.

  20. Differences in the regulation by poly(ADP-ribose) of repair of DNA damage from alkylating agents and ultraviolet light according to cell type

    Energy Technology Data Exchange (ETDEWEB)

    Cleaver, J.E.; Bodell, W.J.; Morgan, W.F.; Zelle, B.

    1983-08-10

    Inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide in various human and hamster cells influenced the responses to DNA damage from methyl methanesulfonate, but not from ultraviolet light. After exposure to methyl methanesulfonate, 3-aminobenzamide increased the strand break frequency in all cell types studied, but only stimulated repair replication in lymphoid and HeLa cells, suggesting these are independent effects. 3-Aminobenzamide also inhibited the pathway for de novo synthesis of DNA purines, suggesting that some of its effects may be due to disturbance of precursor pathways and irrelevant to the role of poly(ADP-ribose) in repair. Previous claims that 3-aminobenzamide stimulates repair synthesis after exposure to UV light are probably artifacts, because the stimulations are only observed in lymphocytes in the presence of a high concentration of hydroxyurea that itself inhibits repair. The initial inhibition of semiconservative DNA synthesis and the excision of the major alkylation products and pyrimidine dimers were unaffected by 3-aminobenzamide. In general poly(ADP-ribose) synthesis appears to be uniquely involved in regulating the ligation stage of repair of alkylation damage but not ultraviolet damage. By regulating the ligation efficiency, poly(ADP-ribosylation) modulates the dynamic balance between incision and ligation, so as to minimize the frequency of DNA breaks. The ligation stage of repair of UV damage appears different and is not regulated by poly(ADP-ribosylation).

  1. Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms

    DEFF Research Database (Denmark)

    Ross, Fiona A; Jensen, Thomas Elbenhardt; Hardie, D Grahame

    2016-01-01

    The g subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different g isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged ve...

  2. Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

    International Nuclear Information System (INIS)

    Kaslow, H.R.; Groppi, V.E.; Abood, M.E.; Bourne, H.R.

    1981-01-01

    Cholera toxin catalyzes transfer of radiolabel from [ 32 P]NAD + to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of M/sub r/ = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (M/sub r/ = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and [ 32 P]NAD + caused radiolabeling of purified microtubule and intermediate filament proteins

  3. Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y12/13 receptors among BV-2 microglia.

    Directory of Open Access Journals (Sweden)

    Pengchong Jiang

    Full Text Available Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as 'find/eat-me' signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme, suramin (broad-spectrum P2 receptor antagonist, 2-APB (IP3 receptor blocker and thapsigargin (endoplasmic reticulum calcium pump inhibitor potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 μM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.

  4. Poly(ADP-ribosyl)ation as a fail-safe, transcription-independent, suicide mechanism in acutely DNA-damaged cells: a hypothesis

    International Nuclear Information System (INIS)

    Nagele, A.

    1995-01-01

    Poly(ADP-ribose) polymerase is an abundant nuclear protein that is higly conserved and consitutively expressed in all higher eukaryotic cells in investigated. Today, after about two decades of intensive research, we have a fairly comprehensive picture of its remarkable enzymatic functions and of its molecular structure. Its physiological role, however, remains controversial. The present hypothesis attempts to reconcile the different findings. By extending and earlier hypothesis, it is proposed that poly(ADP-ribosy)ation is primarily a mechanism to prevent survival of mutated, possibly apoptosis-incompetent, cells after acute DNA-damage. (orig.)

  5. Hydrogen ADPs with Cu Kα data? Invariom and Hirshfeld atom modelling of fluconazole.

    Science.gov (United States)

    Orben, Claudia M; Dittrich, Birger

    2014-06-01

    For the structure of fluconazole [systematic name: 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-ol] monohydrate, C13H12F2N6O·H2O, a case study on different model refinements is reported, based on single-crystal X-ray diffraction data measured at 100 K with Cu Kα radiation to a resolution of sin θ/λ of 0.6 Å(-1). The structure, anisotropic displacement parameters (ADPs) and figures of merit from the independent atom model are compared to `invariom' and `Hirshfeld atom' refinements. Changing from a spherical to an aspherical atom model lowers the figures of merit and improves both the accuracy and the precision of the geometrical parameters. Differences between results from the two aspherical-atom refinements are small. However, a refinement of ADPs for H atoms is only possible with the Hirshfeld atom density model. It gives meaningful results even at a resolution of 0.6 Å(-1), but requires good low-order data.

  6. Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis.

    Science.gov (United States)

    Han, S; Arvai, A S; Clancy, S B; Tainer, J A

    2001-01-05

    Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors

  7. Poly(ADP-ribose) metabolism in X-irradiated Chinese hamster cells: its relation to repair of potentially lethal damage

    International Nuclear Information System (INIS)

    Ben-Hur, E.; Elkind, M.M.

    1984-01-01

    Nicotinamide-adenine dinucleotide (NAD + ) is the substrate used by cells in poly(ADP-ribose) synthesis. X-irradiation of log-phase Chinese hamster cells caused a rapid decrease in NAD + levels which was linearly dependent on radiation dose. The activity of ADP-ribosyl transferase (ADPRT) also increased linearly with radiation dose. The decrease of NAD + was slower, and the increase in ADPRT activity was less pronounced, in a radiation sensitive line, V79-AL162/S-10. An inhibitor of ADPRT, m-aminobenzamide, largely prevented the depletion of cellular NAD + and reduced the rate at which ADPRT activity disappeared during post-irradiation incubation. Post-irradiation treatment with hypertonic buffer or with medium containing D 2 O-which inhibit repair of radiation-induced potentially lethal damage-enhanced the depletion of NAD + and prevented the reduction in ADPRT activity following irradiation. The characteristics of the effects of treatment with hypertonic buffer on NAD + metabolism were qualitatively similar to the effects that such treatment has on radiation-induced cell killing. These results suggest that poly(ADP-ribose) synthesis after irradiation plays a role in the repair of potentially lethal damage. (author)

  8. Pharmacological Inhibition of poly(ADP-ribose) polymerases improves fitness and mitochondrial function in skeletal muscle.

    NARCIS (Netherlands)

    Pirinen, E.; Canto, C.; Jo, Y.S.; Morato, L.; Zhang, H.; Menzies, K.J.; Williams, E.G.; Mouchiroud, L.; Moullan, N.; Hagberg, C.; Li, W.; Timmers, S.; Imhof, R.; Verbeek, J.; Pujol, A.; Loon, B. van; Viscomi, C.; Zeviani, M.; Schrauwen, P.; Sauve, A.A.; Schoonjans, K.; Auwerx, J.

    2014-01-01

    We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show

  9. Acceptors for poly(ADP-ribose) in irradiated Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Xue, L.Y.; Sokany, N.M.; Friedman, L.R.; Oleinick, N.L.

    1985-01-01

    Strand breaks in DNA, as produced by ionizing radiation, stimulate the synthesis of poly(ADP-ribose) (pADPR) by the nuclear enzyme pADPR transferase (ADPRT). The polymer is covalently bound to chromatin-associated proteins and may function in repair of DNA lesions. When total /sup 32/P-pADPR-protein is analyzed by electrophoresis on SDS-polyacrylamide gels, the major radioactive bands correspond to the 116 kD ADPRT and the low molecular weight (histone) region. On two-dimensional gels (isoelectric focusing followed by SDS-PAGE) several ADP-ribosylated species can be detected in each molecular weight range. The intensity of label in each species is greater for proteins isolated from irradiated (10 or 100 Cy) rather than control cells. For detailed analysis of histones, the authors incubated isolated nuclei with /sup 32/P-NAD, extracted histones in acid, and subjected them to electrophoresis in acid-urea gels. Specific radiation-induced increases in pADPR were seen on some nucleosomal core histone bands but not on histone H1. The results suggest that radiation-induced strand breaks stimulate ADPRT to modify core histones; the resultant increase in negative charge could loosen nucleosomal structure, permitting access of repair enzymes to the DNA lesions

  10. Yeast Mitochondrial ADP/ATP Carriers Are Monomeric in Detergents as Demonstrated by Differential Affinity Purification

    NARCIS (Netherlands)

    Bamber, Lisa; Slotboom, Dirk-Jan; Kunji, Edmund R.S.; Barber, L

    2007-01-01

    Most mitochondrial carriers carry out equimolar exchange of substrates and they are believed widely to exist as homo-dimers. Here we show by differential tagging that the yeast mitochondrial ADP/ATP carrier AAC2 is a monomer in mild detergents. Carriers with and without six-histidine or

  11. A Simple and Rapid Determination of ATP, ADP and AMP Concentrations in Pericarp Tissue of Litchi Fruit by High Performance Liquid Chromatography

    Directory of Open Access Journals (Sweden)

    Weibo Jiang

    2006-01-01

    Full Text Available A simple and rapid method using high performance liquid chromatography (HPLC was developed to determine levels of adenosine triphosphate (ATP, adenosine diphosphate (ADP and adenosine monophosphate (AMP in litchi fruit pericarp tissue. This HPLC method used acetonitrile gradient elution and shortened the time required for determinations of adenosine phosphates. This analysis exhibited good repeatability (coefficients of variation 1.28–1.80 % and recovery rate (94.7–97.1 %. The correlation coefficients of ATP, ADP and AMP with their peak areas at a range of 0–80 ng were 0.9946, 0.9994 and 0.9974, respectively. This method was applied to determine levels of adenosine phosphates in pericarp tissue of litchi fruit at harvest. There were 27.4 μg/g of ATP, 35.4 μg/g of ADP and 7.9 μg/g of AMP on a fresh mass basis.

  12. The Impact of Type 2 Diabetes on the Efficacy of ADP Receptor Blockers in Patients with Acute ST Elevation Myocardial Infarction: A Pilot Prospective Study

    Directory of Open Access Journals (Sweden)

    Matej Samoš

    2016-01-01

    Full Text Available Background. The aim of this study was to validate the impact of type 2 diabetes (T2D on the platelet reactivity in patients with acute ST elevation myocardial infarction (STEMI treated with adenosine diphosphate (ADP receptor blockers. Methods. A pilot prospective study was performed. Totally 67 patients were enrolled. 21 patients had T2D. Among all study population, 33 patients received clopidogrel and 34 patients received prasugrel. The efficacy of ADP receptor blocker therapy had been tested in two time intervals using light transmission aggregometry with specific inducer and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P flow cytometry assay. Results. There were no significant differences in platelet aggregability among T2D and nondiabetic (ND group. The platelet reactivity index of VASP-P did not differ significantly between T2D and ND group (59.4±30.9% versus 60.0±25.2% and 33.9±25.3% versus 38.6±29.3% in second testing. The number of ADP receptor blocker nonresponders did not differ significantly between T2D and ND patients. The time interval from ADP receptor blocker loading dosing to the blood sampling was similar in T2D and ND patients in both examinations. Conclusion. This prospective study did not confirm the higher platelet reactivity and higher prevalence of ADP receptor blocker nonresponders in T2D acute STEMI patients.

  13. Poly(ADP-ribose) polymerase inhibition reduces tumor necrosis factor-induced inflammatory response in rheumatoid synovial fibroblasts

    NARCIS (Netherlands)

    García, S.; Bodaño, A.; Pablos, J. L.; Gómez-Reino, J. J.; Conde, C.

    2008-01-01

    To investigate the effect of poly(ADP-ribose) polymerase (PARP) inhibition on the production of inflammatory mediators and proliferation in tumour necrosis factor (TNF)-stimulated fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). Cultured FLS from patients with RA were

  14. Hypo-and hyperthyroidism affect the ATP, ADP and AMP hydrolysis in rat hippocampal and cortical slices.

    Science.gov (United States)

    Bruno, Alessandra Nejar; Diniz, Gabriela Placoná; Ricachenevsky, Felipe Klein; Pochmann, Daniela; Bonan, Carla Denise; Barreto-Chaves, Maria Luiza M; Sarkis, João José Freitas

    2005-05-01

    The presence of severe neurological symptoms in thyroid diseases has highlighted the importance of thyroid hormones in the normal functioning of the mature brain. Since, ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system (CNS), the ectonucleotidase cascade that hydrolyzes ATP to adenosine, is also involved in the control of brain functions. Thus, we investigated the influence of hyper-and hypothyroidism on the ATP, ADP and AMP hydrolysis in hippocampal and cortical slices from adult rats. Hyperthyroidism was induced by daily injections of l-thyroxine (T4) 25 microg/100 g body weight, for 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) added to their drinking water for 14 days. Hypothyroid rats were hormonally replaced by daily injections of T4 (5 microg/100 g body weight, i.p.) for 5 days. Hyperthyroidism significantly inhibited the ATP, ADP and AMP hydrolysis in hippocampal slices. In brain cortical slices, hyperthyroidism inhibited the AMP hydrolysis. In contrast, hypothyroidism increased the ATP, ADP and AMP hydrolysis in both hippocampal and cortical slices and these effects were reverted by T4 replacement. Furthermore, hypothyroidism increased the expression of NTPDase1 and 5'-nucleotidase, whereas hyperthyroidism decreased the expression of 5'-nucleotidase in hippocampus of adult rats. These findings demonstrate that thyroid disorders may influence the enzymes involved in the complete degradation of ATP to adenosine and possibly affects the responses mediated by adenine nucleotides in the CNS of adult rats.

  15. Guidelines for developing NASA (National Aeronautics and Space Administration) ADP security risk management plans

    Science.gov (United States)

    Tompkins, F. G.

    1983-01-01

    This report presents guidance to NASA Computer security officials for developing ADP security risk management plans. The six components of the risk management process are identified and discussed. Guidance is presented on how to manage security risks that have been identified during a risk analysis performed at a data processing facility or during the security evaluation of an application system.

  16. Two distinct genes for ADP/ATP translocase are expressed at the mRNA level in adult human liver

    International Nuclear Information System (INIS)

    Houldsworth, J.; Attardi, G.

    1988-01-01

    Several clones hybridizing with a bovine ADP/ATP translocase cDNA were isolated from an adult human liver cDNA library in the vector pEX1. DNA sequence analysis revealed that these clones encode two distinct forms of translocase. In particular, two clones specifying the COOH-end-proximal five-sixths of the protein exhibit a 9% amino acid sequence divergence and totally dissimilar 3' untranslated regions. One of these cDNAs is nearly identical in sequence to an ADP/ATP translocase clone (hp2F1) recently isolated from a human fibroblast cDNA library with three amino acid changes and a few differences in the 3' untranslated region. Another clone isolated from the pEX1 library contains a reading frame encoding the remaining, NH 2 -end-proximal, 37 amino acids of the translocase. This sequence differs significantly (14% amino acid sequence divergence) from the corresponding segment of hp2F1, and the 5' untranslated regions of the two clones are totally dissimilar. RNA transfer hybridization experiments utilizing the clones isolated from the pEX1 library revealed the presence in HeLa cells of three distinct mRNA species. The pattern of hybridization and the sizes of these mRNAs suggest a greater complexity of organization and expression of the ADP/ATP translocase genes in human cells than indicated by the analysis of the cDNA clones

  17. ModA and ModB, two ADP-ribosyltransferases encoded by bacteriophage T4: catalytic properties and mutation analysis.

    Science.gov (United States)

    Tiemann, Bernd; Depping, Reinhard; Gineikiene, Egle; Kaliniene, Laura; Nivinskas, Rimas; Rüger, Wolfgang

    2004-11-01

    Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Alt- and ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.

  18. PROTEOLYTIC DEGRADATION OF POLY (ADP-RIBOSE POLYMERASE IN RATS WITH CARRAGEENAN-INDUCED GASTROENTEROCOLITIS

    Directory of Open Access Journals (Sweden)

    Tkachenko A. S.

    2017-12-01

    Full Text Available The aim of the research was to study the activity of poly (ADP-ribose polymerase in small intestinal homogenate of rats with chronic carrageenan-induced gastroenterocolitis, as well as mechanisms of regulation of the enzyme in this pathology. Twenty Wistar Albino Glaxo rats were divided into two groups. Animals of group 1 (n = 10 consumed 1 % carrageenan solution for 28 days, which resulted in the development of gastroenterocolitis confirmed morphologically. The control group consisted of intact animals (n = 10. The activity of poly (ADP-ribose polymerase (PARP in the homogenate of small intestine, as well as caspase-3, matrix metalloproteinase-2 (MMP-2 and matrix metalloproteinase-9 (MMP-9 serum levels were determined. Obtained data were statistically processed using the Mann-Whitney U test and calculating median and interquartile range (Me, 25th–75th percentile with the help of the GraphPad Prism 5 application. The development of carrageenan-induced gastroenterocolitis was accompanied by an increase in caspase-3, MMP-2, MMP-9 concentrations in blood serum and a decrease in the activity of PARP in small intestinal homogenates. The reduced activity of PARP in chronic carrageenan-induced gastroenterocolitis may be due to the proteolysis of this enzyme under the action of caspase-3, MMP-2, and MMP-9.

  19. Inhibitors of poly (ADP-ribose) synthesis inhibit the two types of repair of potentially lethal damage

    International Nuclear Information System (INIS)

    Utsumi, Hiroshi; Elkind, M.M.

    1994-01-01

    The purpose of this study was to examine whether 3-amino-benzamide (3ABA), an inhibitor of poly (ADP-ribose) synthesis, inhibits the two types of potentially lethal damage (PLD) repair, termed slow and fast. The fast-type PLD repair was measured by the decrease in survival of V79 Chinese hamster cells by postirradiation treatment with 3ABA. The slow-type PLD repair was measured by the increase in survival by posttreatment with conditioned medium (CM), which became conditioned by growing a crowed culture of cells and supports the slow-type PLD repair. Up to 1 mM 3-ABA inhibited the slow type repair; at doses of 2 mM and above, it inhibited the fast type of PLD repair. There are quantitative differences in cellular effects of 3ABA dependent on concentration. Poly (ADP-ribose) appears to play an important role in the PLD repairs and has little effect on the repair of sublethal damage. 10 refs., 2 figs

  20. Poly(ADP-ribose polymerase (PARP-1 is not involved in DNA double-strand break recovery

    Directory of Open Access Journals (Sweden)

    Fernet Marie

    2003-07-01

    Full Text Available Abstract Background The cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose polymerase (PARP-1 in DNA double-strand break repair. Results PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by γ-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose synthesis following γ-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as γ-rays and H2O2. Conclusions The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to γ-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.

  1. A Poly-ADP-Ribose Trigger Releases the Auto-Inhibition of a Chromatin Remodeling Oncogene.

    Science.gov (United States)

    Singh, Hari R; Nardozza, Aurelio P; Möller, Ingvar R; Knobloch, Gunnar; Kistemaker, Hans A V; Hassler, Markus; Harrer, Nadine; Blessing, Charlotte; Eustermann, Sebastian; Kotthoff, Christiane; Huet, Sébastien; Mueller-Planitz, Felix; Filippov, Dmitri V; Timinszky, Gyula; Rand, Kasper D; Ladurner, Andreas G

    2017-12-07

    DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATPase module mediates auto-inhibition. PARP1 activation suppresses this inhibitory interaction. Crucially, release from auto-inhibition requires a poly-ADP-ribose (PAR) binding macrodomain. We identify tri-ADP-ribose as a potent PAR-mimic and synthetic allosteric effector that abrogates ATPase-macrodomain interactions, promotes an ungated conformation, and activates the remodeler's ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain's interaction with PARP1 under non-DNA damage conditions. Somatic cancer mutants disrupt ALC1's auto-inhibition and activate chromatin remodeling. Our data show that the NAD + -metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Uemura, Sotaro; Ishiwata, Shin'ichi

    2003-04-01

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

  3. Clostridium botulinum C2 toxin--new insights into the cellular up-take of the actin-ADP-ribosylating toxin.

    Science.gov (United States)

    Aktories, Klaus; Barth, Holger

    2004-04-01

    Clostridium botulinum C2 toxin is a member of the family of binary actin-ADP-ribosylating toxins. It consists of the enzyme component C2I, and the separated binding/translocation component C2II. Proteolytically activated C2II forms heptamers and binds to a carbohydrate cell surface receptor. After attachment of C2I, the toxin complex is endocytosed to reach early endosomes. At low pH of endosomes, C2II-heptamers insert into the membrane, form pores and deliver C2I into the cytosol. Here, C2I ADP-ribosylates actin at Arg177 to block actin polymerization and to induce depolymerization of actin filaments. The mini-review describes main properties of C2 toxin and discusses new findings on the involvement of chaperones in the up-take process of the toxin.

  4. Poly(ADP-ribose) synthesis following DNA damage in cells heterozygous or homozygous for the xeroderma pigmentosum genotype

    International Nuclear Information System (INIS)

    McCurry, L.S.; Jacobson, M.K.

    1981-01-01

    Treatment of normal human cells with DNA-damaging agents such as uv light or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) stimulates the conversion of NAD to the chromosomal polymer poly(ADP-ribose) which in turn results in a rapid depletion of the cellular NAD pool. The effect of uv light or MNNG on the NAD pools of seven cell lines of human fibroblasts either homozygous or heterozygous for the xeroderma pigmentosum genotype has been studied. Xeroderma pigmentosum cells of genetic complementation groups A, C, and D are deficient in the excision repair of DNA damage caused by uv light. Following uv treatment, the NAD content of these cells was unchanged or only slightly reduced. All of the cell lines are able to excise DNA damage caused by MNNG and all of the cell lines had a greatly reduced content of NAD following MNNG treatment. The results demonstrate a close relationship between the conversion of NAD to poly(ADP-ribose) and DNA excision repair in human cells

  5. Thrombomodulin Is Silenced in Malignant Mesothelioma by a Poly(ADP-ribose) Polymerase-1-mediated Epigenetic Mechanism

    Czech Academy of Sciences Publication Activity Database

    Nocchi, L.; Tomasetti, M.; Amati, M.; Neužil, Jiří; Santarelli, L.; Saccucci, F.

    2011-01-01

    Roč. 286, č. 22 (2011), s. 19478-19488 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA204/08/0811 Institutional research plan: CEZ:AV0Z50520701 Keywords : Thrombomodulin gene promoter * malignant mesothelioma * poly(ADP-ribose) polymerase-1 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.773, year: 2011

  6. Bioenergetic profile of human coronary artery smooth muscle cells and effect of metabolic intervention.

    Directory of Open Access Journals (Sweden)

    Mingming Yang

    Full Text Available Bioenergetics of artery smooth muscle cells is critical in cardiovascular health and disease. An acute rise in metabolic demand causes vasodilation in systemic circulation while a chronic shift in bioenergetic profile may lead to vascular diseases. A decrease in intracellular ATP level may trigger physiological responses while dedifferentiation of contractile smooth muscle cells to a proliferative and migratory phenotype is often observed during pathological processes. Although it is now possible to dissect multiple building blocks of bioenergetic components quantitatively, detailed cellular bioenergetics of artery smooth muscle cells is still largely unknown. Thus, we profiled cellular bioenergetics of human coronary artery smooth muscle cells and effects of metabolic intervention. Mitochondria and glycolysis stress tests utilizing Seahorse technology revealed that mitochondrial oxidative phosphorylation accounted for 54.5% of ATP production at rest with the remaining 45.5% due to glycolysis. Stress tests also showed that oxidative phosphorylation and glycolysis can increase to a maximum of 3.5 fold and 1.25 fold, respectively, indicating that the former has a high reserve capacity. Analysis of bioenergetic profile indicated that aging cells have lower resting oxidative phosphorylation and reduced reserve capacity. Intracellular ATP level of a single cell was estimated to be over 1.1 mM. Application of metabolic modulators caused significant changes in mitochondria membrane potential, intracellular ATP level and ATP:ADP ratio. The detailed breakdown of cellular bioenergetics showed that proliferating human coronary artery smooth muscle cells rely more or less equally on oxidative phosphorylation and glycolysis at rest. These cells have high respiratory reserve capacity and low glycolysis reserve capacity. Metabolic intervention influences both intracellular ATP concentration and ATP:ADP ratio, where subtler changes may be detected by the latter.

  7. Poly (ADP) ribose polymerase enzyme inhibitor, veliparib, potentiates chemotherapy and radiation in vitro and in vivo in small cell lung cancer

    International Nuclear Information System (INIS)

    Owonikoko, Taofeek K; Zhang, Guojing; Deng, Xingming; Rossi, Michael R; Switchenko, Jeffrey M; Doho, Gregory H; Chen, Zhengjia; Kim, Sungjin; Strychor, Sandy; Christner, Susan M; Beumer, Jan; Li, Chunyang; Yue, Ping; Chen, Alice; Sica, Gabriel L; Ramalingam, Suresh S; Kowalski, Jeanne; Khuri, Fadlo R; Sun, Shi-Yong

    2014-01-01

    Poly (ADP) ribose polymerase (PARP) plays a key role in DNA repair and is highly expressed in small cell lung cancer (SCLC). We investigated the therapeutic impact of PARP inhibition in SCLC. In vitro cytotoxicity of veliparib, cisplatin, carboplatin, and etoposide singly and combined was determined by MTS in 9 SCLC cell lines (H69, H128, H146, H526, H187, H209, DMS53, DMS153, and DMS114). Subcutaneous xenografts in athymic nu/nu mice of H146 and H128 cells with relatively high and low platinum sensitivity, respectively, were employed for in vivo testing. Mechanisms of differential sensitivity of SCLC cell lines to PARP inhibition were investigated by comparing protein and gene expression profiles of the platinum sensitive and the less sensitive cell lines. Veliparib showed limited single-agent cytotoxicity but selectively potentiated (≥50% reduction in IC 50 ) cisplatin, carboplatin, and etoposide in vitro in five of nine SCLC cell lines. Veliparib with cisplatin or etoposide or with both cisplatin and etoposide showed greater delay in tumor growth than chemotherapy alone in H146 but not H128 xenografts. The potentiating effect of veliparib was associated with in vitro cell line sensitivity to cisplatin (CC = 0.672; P = 0.048) and DNA-PKcs protein modulation. Gene expression profiling identified differential expression of a 5-gene panel (GLS, UBEC2, HACL1, MSI2, and LOC100129585) in cell lines with relatively greater sensitivity to platinum and veliparib combination. Veliparib potentiates standard cytotoxic agents against SCLC in a cell-specific manner. This potentiation correlates with platinum sensitivity, DNA-PKcs expression and a 5-gene expression profile

  8. Cloning and characterization of Escherichia coli DUF299: a bifunctional ADP-dependent kinase - Pi-dependent pyrophosphorylase from bacteria

    Directory of Open Access Journals (Sweden)

    Burnell Jim N

    2010-01-01

    Full Text Available Abstract Background Phosphoenolpyruvate synthetase (PEPS; EC 2.7.9.2 catalyzes the synthesis of phosphoenolpyruvate from pyruvate in Escherichia coli when cells are grown on a three carbon source. It also catalyses the anabolic conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis. A bioinformatics search conducted following the successful cloning and expression of maize leaf pyruvate, orthophosphate dikinase regulatory protein (PDRP revealed the presence of PDRP homologs in more than 300 bacterial species; the PDRP homolog was identified as DUF299. Results This paper describes the cloning and expression of both PEPS and DUF299 from E. coli and establishes that E. coli DUF299 catalyzes both the ADP-dependent inactivation and the Pi-dependent activation of PEPS. Conclusion This paper represents the first report of a bifunctional regulatory enzyme catalysing an ADP-dependent phosphorylation and a Pi-dependent pyrophosphorylation reaction in bacteria.

  9. Functional characterisation of an Arabidopsis gene strongly induced by ionising radiation: the gene coding the poly(ADP-ribose)polymerase-1 (AthPARP-1)

    International Nuclear Information System (INIS)

    Doucet-Chabeaud, G.

    2000-01-01

    Arabidopsis thaliana, the model-system in plant genetics, has been used to study the responses to DNA damage, experimentally introduced by γ-irradiation. We have characterised a radiation-induced gene coding a 111 kDa protein, AthPARP-1, homologous to the human poly(ADP-ribose)polymerase-1 (hPARP-1). As hPARP-1 is composed by three functional domain with characteristic motifs, AthPARP-1 binds to DNA bearing single-strand breaks and shows DNA damage-dependent poly(ADP-ribosyl)ation. The preferential expression of AthPARP-1 in mitotically active tissues is in agreement with a potential role in the maintenance of genome integrity during DNA replication, as proposed for its human counterpart. Transcriptional gene activation by ionising radiation of AthPARP-1 and AthPARP-2 genes is to date plant specific activation. Our expression analyses after exposure to various stress indicate that 1) AthPARP-1 and AthPARP-2 play an important role in the response to DNA lesions, particularly they are activated by genotoxic agents implicating the BER DNA repair pathway 2) AthPARP-2 gene seems to play an additional role in the signal transduction induced by oxidative stress 3) the observed expression profile of AthPARP-1 is in favour of the regulation of AthPARP-1 gene expression at the level of transcription and translation. This mode of regulation of AthPARP-1 protein biosynthesis, clearly distinct from that observed in animals, needs the implication of a so far unidentified transcription factor that is activated by the presence of DNA lesions. The major outcome of this work resides in the isolation and characterisation of such new transcription factor, which will provide new insight on the regulation of plant gene expression by genotoxic stress. (author) [fr

  10. Enhancement of radiosensitivity of recombinant Ad-p53 gene on human lung adenocarcinoma cell with different p53 status

    International Nuclear Information System (INIS)

    Pang Dequan; Wang Peiguo; Wang Ping; Zhang Weiming

    2008-01-01

    Objective: To investigate the enhancement of radiosensitivity of recombinant Ad-p53 gene on human lung adenocarcinoma cell lines(A549 and GLC-82) with different p53 status in vitro. Methods: Two human lung adenocarcinoma cell lines of A549 and GLC-82 were examined on their difference in p53 status with immunohistochemistry stain and PCR-SSCP technique. Expand Ad-wtp53 was transfected into tumor cells. Clonogenic assays were performed to evaluate the inhibition effect on cell growth and the degree of sensitization to irradiation. Apoptosis and cell cycle changes were determined using the flow cytometry assay. Results: The A549 cell line presented positive P53 expression while GLC-82 negative. GLC-82 bore mutant p53 on the exon 7. The wtp53 gene could be efficiently expressed in the two cell lines and greatly inhibit the cell growth. Its efficiency didn't depend on the intrinsic p53 genetic status. After irradiation, its function of inducing G 1 arrest and apoptosis on GLC-82 cell line was much stronger than the A549 cell line. In both the A549 and GLC-82 cell lines, the combination of Ad-p53 plus radiation resulted in more apoptosis than the others. There was no significant difference between two groups. Conclusions: Ad-p53 can depress the tumor growth and enhance the radiosensitivity of human lung adenocarcinoma cells. And this effect is independent of endogenous p53 status. (authors)

  11. High similarity of phylogenetic profiles of rate-limiting enzymes with inhibitory relation in Human, Mouse, Rat, budding Yeast and E. coli.

    Science.gov (United States)

    Zhao, Min; Qu, Hong

    2011-11-30

    The phylogenetic profile is widely used to characterize functional linkage and conservation between proteins without amino acid sequence similarity. To survey the conservative regulatory properties of rate-limiting enzymes (RLEs) in metabolic inhibitory network across different species, we define the enzyme inhibiting pair as: where the first enzyme in a pair is the inhibitor provider and the second is the target of the inhibitor. Phylogenetic profiles of enzymes in the inhibiting pairs are further generated to measure the functional linkage of these enzymes during evolutionary history. We find that the RLEs generate, on average, over half of all in vivo inhibitors in each surveyed model organism. And these inhibitors inhibit on average over 85% targets in metabolic inhibitory network and cover the majority of targets of cross-pathway inhibiting relations. Furthermore, we demonstrate that the phylogenetic profiles of the enzymes in inhibiting pairs in which at least one enzyme is rate-limiting often show higher similarities than those in common inhibiting enzyme pairs. In addition, RLEs, compared to common metabolic enzymes, often tend to produce ADP instead of AMP in conservative inhibitory networks. Combined with the conservative roles of RLEs in their efficiency in sensing metabolic signals and transmitting regulatory signals to the rest of the metabolic system, the RLEs may be important molecules in balancing energy homeostasis via maintaining the ratio of ATP to ADP in living cells. Furthermore, our results indicate that similarities of phylogenetic profiles of enzymes in the inhibiting enzyme pairs are not only correlated with enzyme topological importance, but also related with roles of the enzymes in metabolic inhibitory network.

  12. The mechanism of action of poly (ADP-ribose) polymerases inhibitors and its application perspective

    International Nuclear Information System (INIS)

    Huang Xiaofei; Cao Jianping

    2008-01-01

    Poly (ADP-ribose) polymerases (PARP) constitute a family of enzymes involved in the regulation of many cellular processes. It plays a vital role in many physical and physiopathological processes,, In the past ten years scientists have conducted extensive research on PARP and its inhibitors, among which the role of PARP inhihitors in radiosensitization, chemopotentiation and neuroprotection have been placed close attention. There have been several PARP inhibitors entering the clinical trials, which predicts its sound application perspectives. (authors)

  13. Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.; Poland, M.; Meyer, C.R.

    2009-05-14

    ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. The A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.

  14. The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

    International Nuclear Information System (INIS)

    Bolin, Celeste; Boudra, Mohammed-Tayyib; Fernet, Marie; Vaslin, Laurence; Pennaneach, Vincent; Zaremba, Tomasz; Favaudon, Vincent; Megnin-Chanet, Frederique; Hall, Janet; Biard, Denis; Cordelieres, Fabrice P.

    2012-01-01

    Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro irradiated Cdk5KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5- dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels. (authors)

  15. Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

    Science.gov (United States)

    Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

    2017-06-02

    Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

  16. A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles.

    NARCIS (Netherlands)

    Tjaden, J.; Haferkamp, I.; Boxma, B.; Tielens, A.G.; Huynen, M.A.; Hackstein, J.H.P.

    2004-01-01

    The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and

  17. Gamma-ray induced DNA breaks and repair studied by immuno-labelling of poly(ADP-ribose) polymerase (PARP) in chinese hamster ovary cells (CHO)

    International Nuclear Information System (INIS)

    Bidon, N.; Noel, G.; Averbeck, D.; Varlet, P.; Salamero, J.; DeMurcia, G.

    1998-01-01

    The poly(ADP-ribose)polymerase is a nuclear ubiquitous enzyme capable of binding to DNA breaks. Chinese hamster ovary cells were (CHO-K1) cultured on slides and γ-irradiated ( 137 Cs) at a high (12.8 Gy/min) or medium dose rate (5 Gy/min), and immuno-labelling against (ADP-ribose) polymers immediately or three hours after irradiation. Quantification and localisation of γ-ray induced breaks was performed by confocal microscopy. The results show a dose effect relationship, a dose-rate effect and the signal disappearance after 3 hours at 37 deg.C. The presence of PARP activity appears to reflect γ-rays induced DNA fragmentation. (authors)

  18. Genes regulation encoding ADP/ATP carrier in yeasts Saccharomyces cerevisiae and Candida parapsilosis

    International Nuclear Information System (INIS)

    Nebohacova, M.

    2000-01-01

    Genes encoding a mitochondrial ADP/ATP carrier (AAC) in yeast Saccharomyces cerevisiae and Candida parapsilosis were investigated. AAC2 is coding for the major AAC isoform in S. cerevisiae. We suggest that AAC2 is a member of a syn-expression group of genes encoding oxidative phosphorylation proteins. Within our previous studies on the regulation of the AAC2 transcription an UAS (-393/-268) was identified that is essential for the expression of this gene. Two functional regulatory cis-elements are located within this UAS -binding sites for an ABFl factor and for HAP2/3/4/5 heteromeric complex. We examined relative contributions and mutual interactions of the ABFl and HAP2/3/4/5 factors in the activation of transcription from the UAS of the AAC2 gene. The whole UAS was dissected into smaller sub-fragments and tested for (i) the ability to form DNA-protein complexes with cellular proteins in vitro, (ii) the ability to confer heterologous expression using AAC3 gene lacking its own promoter, and (iii) the expression of AAC3-lacZ fusion instead of intact AAC3 gene. The obtained results demonstrated that: a) The whole UAS as well as sub-fragment containing only ABF1-binding site are able to form DNA-protein complexes with cellular proteins in oxygen- and heme- dependent manner. The experiments with antibody against the ABF1 showed that the ABF1 factor is one of the proteins binding to AAC2 promoter. We have been unsuccessful to prove the binding of cellular proteins to the HAP2/3/4/5-binding site. However, the presence of HAP2/3/4/5-binding site is necessary to drive a binding of cellular proteins to the ABF1-binding site in carbon source-dependent manner. b) The presence of both ABF1- and HAP2/3/4/5-binding sites and original spacing between them is necessary to confer the growth of Aaac2 mutant strain on non- fermentable carbon source when put in front of AAC3 gene introduced on centromeric vector to Aaac2 mutant strain. c) For the activation of AAC3-lacZ expression on

  19. Metabolic consequences of DNA damage: The role of poly (ADP-ribose) polymerase as mediator of the suicide response

    International Nuclear Information System (INIS)

    Berger, N.A.; Berger, S.J.

    1986-01-01

    Recent studies show that DNA damage can produce rapid alterations in steady state levels of deoxynucleoside triphosphate pools, for example, MNNG or uv-irradiation cause rapid increases in dATP and dTTP pools without significant changes in dGTP or dCTP pools. In vitro, studies with purified eukaryotic DNA polymerases show that the frequency of nucleotide misincorporation was affected by alterations in relative concentrations of the deoxynucleoside triphosphates. Thus the alterations in dNTP pool sizes that occur consequent to DNA damage may contribute to an increased mutagenic frequency. Poly(ADP-ribose) polymerase mediated suicide mechanism may participate in the toxicity of adenosine deaminase deficiency and severe combined immune deficiency disease in humans. Individuals with this disease suffer severe lymphopenia due to the toxic effects of deoxyadenosine. The lymphocytotoxic effect of adenosine deaminase deficiency can be simulated in lymphocyte cell lines from normal individuals by incubating them with the adenosine deaminase inhibitor, deoxycoformycin. Incubation of such leukocytes with deoxycoformycin and deoxyadenosine results in the gradual accumulation of DNA strand breaks and the depletion of NAD + leading to cell death over a period of several days. This depletion of NAD and loss of cell viability were effectively blocked by nicotinamide or 3-amino benzamide. Thus, persistent activation of poly(ADP-ribose) polymerase by unrepaired or recurrent DNA strand breaks may activate the suicide mechanism of cell death. This study provides a basis for the interesting suggestion that treatment with nicotinamide could block the persistent activity of poly(ADP-ribose) polymerase and may help preserve lymphocyte function in patients with adenosine deaminase deficiency. 16 refs., 3 figs., 2 tabs

  20. Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

    OpenAIRE

    Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

    2008-01-01

    ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotat...

  1. Model study of ATP and ADP buffering, transport of Ca(2+) and Mg(2+), and regulation of ion pumps in ventricular myocyte

    Science.gov (United States)

    Michailova, A.; McCulloch, A.

    2001-01-01

    We extended the model of the ventricular myocyte by Winslow et al. (Circ. Res 1999, 84:571-586) by incorporating equations for Ca(2+) and Mg(2+) buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na(+)-K(+) pump, sarcolemmal and sarcoplasmic Ca(2+) pumps). The results indicate that, under normal conditions, Ca(2+) binding by low-affinity ATP and diffusion of CaATP may affect the amplitude and time course of intracellular Ca(2+) signals. The model also suggests that a fall in ATP/ADP ratio significantly reduces sarcoplasmic Ca(2+) content, increases diastolic Ca(2+), lowers systolic Ca(2+), increases Ca(2+) influx through L-type channels, and decreases the efficiency of the Na(+)/Ca(2+) exchanger in extruding Ca(2+) during periodic voltage-clamp stimulation. The analysis suggests that the most important reason for these changes during metabolic inhibition is the down-regulation of the sarcoplasmic Ca(2+)-ATPase pump by reduced diastolic MgATP levels. High Ca(2+) concentrations developed near the membrane might have a greater influence on Mg(2+), ATP, and ADP concentrations than that of the lower Ca(2+) concentrations in the bulk myoplasm. The model predictions are in general agreement with experimental observations measured under normal and pathological conditions.

  2. 31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg2+ in human erythrocytes.

    Science.gov (United States)

    Petersen, A; Kristensen, S R; Jacobsen, J P; Hørder, M

    1990-08-17

    Absolute 31P-NMR measurements of ATP, ADP and 2,3-diphosphoglycerate (2,3-DPG) in oxygenated and partly deoxygenated human erythrocytes, compared to measurements by standard assays after acid extraction, show that ATP is only 65% NMR visible, ADP measured by NMR is unexpectedly 400% higher than the enzymatic measurement and 2,3-DPG is fully NMR visible, regardless of the degree of oxygenation. These results show that binding to hemoglobin is unlikely to cause the decreased visibility of ATP in human erythrocytes as deoxyhemoglobin binds the phosphorylated metabolites more tightly than oxyhemoglobin. The high ADP visibility is unexplained. The levels of free Mg2+ [( Mg2+]free) in human erythrocytes are 225 mumol/l at an oxygen saturation of 98.6% and instead of the expected increase, the level decreased to 196 mumol/l at an oxygen saturation of 38.1% based on the separation between the alpha- and beta-ATP peaks. [Mg2+]free in the erythrocytes decreased to 104 mumol/l at a high 2,3-DPG concentration of 25.4 mmol/l red blood cells (RBC) and a normal ATP concentration of 2.05 mmol/l RBC. By increasing the ATP concentration to 3.57 mmol/l RBC, and with a high 2,3-DPG concentration of 24.7 mmol/l RBC, the 31P-NMR measured [Mg2+]free decreased to 61 mumol/l. These results indicate, that the 31P-NMR determined [Mg2+]free in human erythrocytes, based solely on the separation of the alpha- and beta-ATP peaks, does not give a true measure of intracellular free Mg2+ changes with different oxygen saturation levels. Furthermore the measurement is influenced by the concentration of the Mg2+ binding metabolites ATP and 2,3-DPG. Failure to take these factors into account when interpreting 31P-NMR data from human erythrocytes may explain some discrepancies in the literature regarding [Mg2+]free.

  3. Activation of ADP-ribosyltransferase in polyamine-depleted mammalian cells.

    Science.gov (United States)

    Wallace, H M; Gordon, A M; Keir, H M; Pearson, C K

    1984-01-01

    Mammalian fibroblasts were cultured in the presence of alpha-methylornithine and/or methylglyoxal bis(guanylhydrazone), which inhibit the synthesis of polyamines. This led to a decrease in the cellular content of the polyamines spermine and spermidine by up to 60% when the cells were grown in the presence of both drugs together. The activity of the chromatin-associated enzyme ADP-ribosyltransferase was enhanced 2-3-fold in the drug-treated cells when measured in cells subsequently rendered permeable to exogenous NAD+, the substrate for the transferase. This is a novel and surprising observation, since the transferase is invariably activated by the addition of polyamines to a suitable incubation system such as permeabilized cells, isolated nuclei or the purified enzyme. We found no evidence that the activation was due to the appearance of DNA strand breaks, by using a variety of procedures including both neutral [the 'nucleoid' technique of Cook & Brazell [(1975) J. Cell Sci. 19, 261-279; (1976) J. Cell Sci. 22, 287-302

  4. Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamashita, Sachiko [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Tanaka, Masakazu [Department of Microbiology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata City, Osaka 573-1010 (Japan); Sato, Teruaki [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Ida, Chieri [Department of Applied Life Studies, College of Nagoya Women’s University, 3-40 Shioji-cho, Mizuho-ku, Nagoya-shi, Aichi 467-8610 (Japan); Ohta, Narumi; Hamada, Takashi; Uetsuki, Taichi; Nishi, Yoshisuke [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Moss, Joel [Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1590 (United States); Miwa, Masanao, E-mail: m_miwa@nagahama-i-bio.ac.jp [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan)

    2016-08-05

    Poly (ADP-ribose) (PAR) is rapidly synthesized by PAR polymerases (PARPs) upon activation by DNA single- and double-strand breaks. In this study, we examined the quantitative amount of PAR in HeLa cells cultured within the physiological temperatures below 41 °C for verification of the effect of shifting-up or -down the temperature from 37.0 °C on the DNA breaks, whether the temperature-shift caused breaks that could be monitored by the level of PAR. While PAR level did not change significantly when HeLa cells were cultured at 33.5 °C or 37.0 °C, it was significantly increased 2- and 3-fold when cells were cultured for 12 h and 24 h, respectively, at 40.5 °C as compared to 37.0 °C. Similar to the results with HeLa cells, PAR level was increased 2-fold in CHO-K1 cells cultured at 40.5 °C for 24 h as compared to 37.0 °C. As the cellular levels of PAR polymerase1 (PARP1) and PAR glycohydrolase (PARG), a major degradation enzyme for PAR, did not seem to change significantly, this increase could be caused by activation of PARP1 by DNA strand breaks. In fact, γH2AX, claimed to be a marker of DNA double-strand breaks, was found in cell extracts of HeLa cells and CHO-K1 cells at elevated temperature vs. 37.0 °C, and these γH2AX signals were intensified in the presence of 3-aminobenzamide, a PARP inhibitor. The γH2AX immunohistochemistry results in HeLa cells were consistent with Western blot analyses. In HeLa cells, proliferation was significantly suppressed at 40.5 °C in 72 h-continuous cultures and decreased viabilities were also observed after 24–72 h at 40.5 °C. Flow cytometric analyses showed that the HeLa cells were arrested at G2/M after temperature shift-up to 40.5 °C. These physiological changes were potentiated in the presence of 3-aminobenzamide. Decrease in growth rates, increased cytotoxicity and G2/M arrest, were associated with the temperature-shift to 40.5 °C and are indirect evidence of DNA breaks. In addition to γH2AX

  5. Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells

    International Nuclear Information System (INIS)

    Yamashita, Sachiko; Tanaka, Masakazu; Sato, Teruaki; Ida, Chieri; Ohta, Narumi; Hamada, Takashi; Uetsuki, Taichi; Nishi, Yoshisuke; Moss, Joel; Miwa, Masanao

    2016-01-01

    Poly (ADP-ribose) (PAR) is rapidly synthesized by PAR polymerases (PARPs) upon activation by DNA single- and double-strand breaks. In this study, we examined the quantitative amount of PAR in HeLa cells cultured within the physiological temperatures below 41 °C for verification of the effect of shifting-up or -down the temperature from 37.0 °C on the DNA breaks, whether the temperature-shift caused breaks that could be monitored by the level of PAR. While PAR level did not change significantly when HeLa cells were cultured at 33.5 °C or 37.0 °C, it was significantly increased 2- and 3-fold when cells were cultured for 12 h and 24 h, respectively, at 40.5 °C as compared to 37.0 °C. Similar to the results with HeLa cells, PAR level was increased 2-fold in CHO-K1 cells cultured at 40.5 °C for 24 h as compared to 37.0 °C. As the cellular levels of PAR polymerase1 (PARP1) and PAR glycohydrolase (PARG), a major degradation enzyme for PAR, did not seem to change significantly, this increase could be caused by activation of PARP1 by DNA strand breaks. In fact, γH2AX, claimed to be a marker of DNA double-strand breaks, was found in cell extracts of HeLa cells and CHO-K1 cells at elevated temperature vs. 37.0 °C, and these γH2AX signals were intensified in the presence of 3-aminobenzamide, a PARP inhibitor. The γH2AX immunohistochemistry results in HeLa cells were consistent with Western blot analyses. In HeLa cells, proliferation was significantly suppressed at 40.5 °C in 72 h-continuous cultures and decreased viabilities were also observed after 24–72 h at 40.5 °C. Flow cytometric analyses showed that the HeLa cells were arrested at G2/M after temperature shift-up to 40.5 °C. These physiological changes were potentiated in the presence of 3-aminobenzamide. Decrease in growth rates, increased cytotoxicity and G2/M arrest, were associated with the temperature-shift to 40.5 °C and are indirect evidence of DNA breaks. In addition to γH2AX

  6. Measurement of O-GlcNAcylated endothelial nitric oxide synthase by using 2',5'-ADP-Sepharose pull-down assay.

    Science.gov (United States)

    Long, Yang; Yan, Jianghong; Luo, Suxin; Liu, Zhenguo; Xia, Yong

    2017-11-15

    Endothelial nitric oxide synthase (eNOS) plays central roles in cardiovascular regulation and disease. eNOS function is critically affected by O-linked N-acetylglucosamine (O-GlcNAc) modification. The present method for measuring O-GlcNAcylated eNOS relies on immunoprecipitation. Such method exhibits low detection efficiency and is also costly. We here report a simplified assay by employing the high binding affinity of eNOS with the 2',5'-ADP-Sepharose resins. Together with the O-GlcNAc antibody, this assay readily allows the detection of O-GlcNAcylated eNOS in both cultured endothelial cells and rat vascular tissues. By using this assay, we demonstrate that eNOS O-GlcNAcylation is markedly elevated in the vessels of diabetic rats. Thus, a 2',5'-ADP-Sepharose-based pull-down assay is developed to measure O-GlcNAcylated eNOS. This assay is simple and efficient in detecting O-GlcNAcylated eNOS in cultured cells and animal tissues under both normal and disease conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Self-assembled copper(II) metallacycles derived from asymmetric Schiff base ligands: efficient hosts for ADP/ATP in phosphate buffer.

    Science.gov (United States)

    Kumar, Amit; Pandey, Rampal; Kumar, Ashish; Gupta, Rakesh Kumar; Dubey, Mrigendra; Mohammed, Akbar; Mobin, Shaikh M; Pandey, Daya Shankar

    2015-10-21

    Novel asymmetric Schiff base ligands 2-{[3-(3-hydroxy-1-methyl-but-2-enylideneamino)-2,4,6-trimethylphenylimino]-methyl}-phenol (H2L(1)) and 1-{[3-(3-hydroxy-1-methyl-but-2-enylideneamino)-2,4,6-trimethylphenylimino]-methyl}-naphthalen-2-ol (H2L(2)) possessing dissimilar N,O-chelating sites and copper(ii) metallacycles (CuL(1))4 (1) and (CuL(2))4 (2) based on these ligands have been described. The ligands and complexes have been thoroughly characterized by satisfactory elemental analyses, and spectral (IR, (1)H, (13)C NMR, ESI-MS, UV/vis) and electrochemical studies. Structures of H2L(2) and 1 have been unambiguously determined by X-ray single crystal analyses. The crystal structure of H2L(2) revealed the presence of two distinct N,O-chelating sites on dissimilar cores (naphthalene and β-ketoaminato groups) offering a diverse coordination environment. Metallacycles 1 and 2 having a cavity created by four Cu(ii) centres coordinated in a homo- and heteroleptic fashion with respective ligands act as efficient hosts for adenosine-5'-diphosphate (ADP) and adenosine-5'-triphosphate (ATP) respectively, over other nucleoside polyphosphates (NPPs). The disparate sensitivity of these metallacycles toward ADP and ATP has been attributed to the size of the ligands assuming diverse dimensions and spatial orientations. These are attuned for π-π stacking and electrostatic interactions suitable for different guest molecules under analogous conditions, metallacycle 1 offers better orientation for ADP, while 2 for ATP. The mechanism of the host-guest interaction has been investigated by spectral and electrochemical studies and supported by molecular docking studies.

  8. The Role of Poly(ADP-ribose Polymerase-1 in Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Samuel García

    2015-01-01

    Full Text Available Poly(ADP-ribose polymerase-1 (PARP-1 is a nuclear enzyme with a crucial role in the maintenance of genomic stability. In addition to the role of PARP-1 in DNA repair, multiple studies have also demonstrated its involvement in several inflammatory diseases, such as septic shock, asthma, atherosclerosis, and stroke, as well as in cancer. In these diseases, the pharmacological inhibition of PARP-1 has shown a beneficial effect, suggesting that PARP-1 regulates their inflammatory processes. In recent years, we have studied the role of PARP-1 in rheumatoid arthritis, as have other researchers, and the results have shown that PARP-1 has an important function in the development of this disease. This review summarizes current knowledge on the effects of PARP-1 in rheumatoid arthritis.

  9. LEGACY - EOP Marine Debris

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — These data contains towed diver surveys of and weights of marine debris removed from the near shore environments of the NWHI.

  10. Influence of sulphate on effects of ADP, 3′,5′-cyclic amp and citrate on human platelet phosphofructokinase activity

    NARCIS (Netherlands)

    Akkerman, Jan Willem N.; Gorter, G.; Sixma, J.J.; Staal, Gerard E.J.

    1974-01-01

    1. 1. Sulphate ions activate partially-purified human platelet phosphofructokinase. This is caused by suppression of the cooperativity of the enzyme with respect to fructose 6-phosphate. 2. 2. Sulphate therefore markedly affects the influences of allosteric modifiers such as ADP, 3′,5′-cyclic AMP

  11. 2-Azido-( sup 32 P)NAD+, a photoactivatable probe for G-protein structure: Evidence for holotransducin oligomers in which the ADP-ribosylated carboxyl terminus of alpha interacts with both alpha and gamma subunits

    Energy Technology Data Exchange (ETDEWEB)

    Vaillancourt, R.R.; Dhanasekaran, N.; Johnson, G.L.; Ruoho, A.E. (Univ. of Wisconsin Medical School, Madison (USA))

    1990-05-01

    A radioactive and photoactivatable derivative of NAD+, 2-azido-(adenylate-32P)NAD+, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys347 of the alpha subunit (alpha T) of GT, the retinal guanine nucleotide-binding protein. ADP-ribosylation of alpha T followed by light activation of the azide moiety of 2-azido-(adenylate-32P)ADP-ribose produced four crosslinked species involving the alpha and gamma subunits of the GT heterotrimer: an alpha trimer (alpha-alpha-alpha), and alpha-alpha-gamma crosslink, an alpha dimer (alpha-alpha), and an alpha-gamma crosslink. The alpha trimer, alpha-alpha-gamma complex, alpha dimer, and alpha-gamma complexes were immunoreactive with alpha T antibodies. The alpha-alpha-gamma and the alpha-gamma complexes were immunoreactive with antisera recognizing gamma subunits. No evidence was found for crosslinking of alpha T to beta T subunits. Hydrolysis of the thioglycosidic bond between Cys347 and 2-azido-(adenylate-32P)ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys347 of alpha T in the crosslinked oligomers to alpha monomers, indicative of intermolecular photocrosslinking, and to gamma monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that GT exists as an oligomer and that ADP-ribosylated Cys347, which is four residues from the alpha T-carboxyl terminus, is oriented toward and in close proximity to the gamma subunit.

  12. Developing CNS mitochondria oxidative phosphorylation P/O/ADP/O index for rats

    International Nuclear Information System (INIS)

    Egana, E.; Diaz, G.

    1975-01-01

    The effect of whole-body-gamma irradiation on developing CNS mitochondria oxidative phosphorylation was studied through the P/O/ADP/O index; three irradiation doses (5, 50 and 500 R) were employed at neonatal stage and both 'prompt' (10 min approx,) and 'delayed' (7 days for 500 R exposure, 21 days for 5 and 50 R) effects were observed. In the 'prompt' effects investigated after 500 R exposure, the oxidative phosphorylation diminished; the same occurred at 7 days with this dose ('delayed' effect). With doses of 5 and 50 R there was no alteration of oxidative phosphorylation as a 'prompt' effect, but it diminished at 21 days post irradiation. The uncoupling between respiration and oxidative phosphorylation should explain - at least, in part -these results. (author)

  13. Developing CNS mitochondria oxidative phosphorylation P/O/ADP/O index for rats

    Energy Technology Data Exchange (ETDEWEB)

    Egana, E; Diaz, G [Institute of Experimental Medicine, Santiago (Chile). Lab. of Neurochemistry

    1975-11-01

    The effect of whole-body-gamma irradiation on developing CNS mitochondria oxidative phosphorylation was studied through the P/O/ADP/O index; three irradiation doses (5, 50 and 500 R) were employed at neonatal stage and both 'prompt' (10 min approx,) and 'delayed' (7 days for 500 R exposure, 21 days for 5 and 50 R) effects were observed. In the 'prompt' effects investigated after 500 R exposure, the oxidative phosphorylation diminished; the same occurred at 7 days with this dose ('delayed' effect). With doses of 5 and 50 R there was no alteration of oxidative phosphorylation as a 'prompt' effect, but it diminished at 21 days post irradiation. The uncoupling between respiration and oxidative phosphorylation should explain - at least, in part -these results.

  14. Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria.

    Science.gov (United States)

    Nocek, Boguslaw; Kochinyan, Samvel; Proudfoot, Michael; Brown, Greg; Evdokimova, Elena; Osipiuk, Jerzy; Edwards, Aled M; Savchenko, Alexei; Joachimiak, Andrzej; Yakunin, Alexander F

    2008-11-18

    Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of phosphate residues linked by high-energy bonds. It is found in all organisms and has been proposed to serve as an energy source in a pre-ATP world. This ubiquitous and abundant biopolymer plays numerous and vital roles in metabolism and regulation in prokaryotes and eukaryotes, but the underlying molecular mechanisms for most activities of polyP remain unknown. In prokaryotes, the synthesis and utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 and PPK2, and polyphosphatases. Here, we present structural and functional characterization of the PPK2 family. Proteins with a single PPK2 domain catalyze polyP-dependent phosphorylation of ADP to ATP, whereas proteins containing 2 fused PPK2 domains phosphorylate AMP to ADP. Crystal structures of 2 representative proteins, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa, revealed a 3-layer alpha/beta/alpha sandwich fold with an alpha-helical lid similar to the structures of microbial thymidylate kinases, suggesting that these proteins share a common evolutionary origin and catalytic mechanism. Alanine replacement mutagenesis identified 9 conserved residues, which are required for activity and include the residues from both Walker A and B motifs and the lid. Thus, the PPK2s represent a molecular mechanism, which potentially allow bacteria to use polyP as an intracellular energy reserve for the generation of ATP and survival.

  15. Down-regulation of NTPDase2 and ADP-sensitive P2 Purinoceptors Correlate with Severity of Symptoms during Experimental Autoimmune Encephalomyelitis

    Directory of Open Access Journals (Sweden)

    Marija Jakovljevic

    2017-10-01

    Full Text Available The present study explores tissue and cellular distribution of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2 and the gene and protein expression in rat spinal cord during the course of experimental autoimmune encephalomyelitis (EAE. Given that NTPDase2 hydrolyzes ATP with a transient accumulation of ADP, the expression of ADP-sensitive P2 purinoceptors was analyzed as well. The autoimmune disease was actively induced in Dark Agouti female rats and the changes were analyzed 10, 15 and 29 days after the induction. These selected time points correspond to the onset (Eo, peak (Ep and recovery (Er from EAE. In control animals, NTPDase2 was confined in the white matter, in most of the glial fibrillary acidic protein (GFAP-immunoreactive (ir astrocytes and in a considerable number of nestin-ir cells, while the other cell types were immunonegative. Immunoreactivity corresponding to NTPDase2 decreased significantly at Eo and Ep and then returned to the baseline levels at Er. The preservation of the proportion of GFAP single-labeled and GFAP/NTPDase2 double-labeled elements along the course of EAE indicated that changes in NTPDase2-ir occurred at fibrous astrocytes that typically express NTPDase2 in normal conditions. Significant downregulation of P2Y1 and P2Y12 receptor proteins at Eo and several-fold induction of P2Y12 and P2Y13 receptor proteins at Ep and/or Er were observed implying that the pathophysiological process in EAE may be linked to ADP signaling. Cell-surface expression of NTPDase2, NTPDase1/CD39 and ecto-5′-nucleotidase (eN/CD73 was analyzed in CD4+ T cells of a draining lymph node by fluorescence-activated cell sorting. The induction of EAE was associated with a transient decrease in a number of CD4+ NTPDase2+ T cells in a draining lymph node, whereas the recovery was characterized by an increase in NTPDase2+ cells in both CD4+ and CD4− cell populations. The opposite was found for NTPDase1/CD39+ and eN/CD73+ cells, which

  16. PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

    International Nuclear Information System (INIS)

    Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li; Yang, Wen-Ming

    2013-01-01

    Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression

  17. Deficiency in Poly(ADP-ribose Polymerase-1 (PARP-1 Accelerates Aging and Spontaneous Carcinogenesis in Mice

    Directory of Open Access Journals (Sweden)

    Tatiana S. Piskunova

    2008-01-01

    Full Text Available Genetic and biochemical studies have shown that PARP-1 and poly(ADP-ribosylation play an important role in DNA repair, genomic stability, cell death, inflammation, telomere maintenance, and suppressing tumorigenesis, suggesting that the homeostasis of poly(ADP-ribosylation and PARP-1 may also play an important role in aging. Here we show that PARP-1−/− mice exhibit a reduction of life span and a significant increase of population aging rate. Analysis of noninvasive parameters, including body weight gain, body temperature, estrous function, behavior, and a number of biochemical indices suggests the acceleration of biological aging in PARP-1−/− mice. The incidence of spontaneous tumors in both PARP-1−/− and PARP-1+/+ groups is similar; however, malignant tumors including uterine tumors, lung adenocarcinomas and hepatocellular carcinomas, develop at a significantly higher frequency in PARP-1−/− mice than PARP-1+/+ mice (72% and 49%, resp.; < .05. In addition, spontaneous tumors appear earlier in PARP-1−/− mice compared to the wild type group. Histopathological studies revealed a wide spectrum of tumors in uterus, ovaries, liver, lungs, mammary gland, soft tissues, and lymphoid organs in both groups of the mice. These results demonstrate that inactivation of DNA repair gene PARP-1 in mice leads to acceleration of aging, shortened life span, and increased spontaneous carcinogenesis.

  18. Metabolome and proteome profiling of complex I deficiency induced by rotenone.

    Science.gov (United States)

    Gielisch, Ina; Meierhofer, David

    2015-01-02

    Complex I (CI; NADH dehydrogenase) deficiency causes mitochondrial diseases, including Leigh syndrome. A variety of clinical symptoms of CI deficiency are known, including neurodegeneration. Here, we report an integrative study combining liquid chromatography-mass spectrometry (LC-MS)-based metabolome and proteome profiling in CI deficient HeLa cells. We report a rapid LC-MS-based method for the relative quantification of targeted metabolome profiling with an additional layer of confidence by applying multiple reaction monitoring (MRM) ion ratios for further identity confirmation and robustness. The proteome was analyzed by label-free quantification (LFQ). More than 6000 protein groups were identified. Pathway and network analyses revealed that the respiratory chain was highly deregulated, with metabolites such as FMN, FAD, NAD(+), and ADP, direct players of the OXPHOS system, and metabolites of the TCA cycle decreased up to 100-fold. Synthesis of functional iron-sulfur clusters, which are of central importance for the electron transfer chain, and degradation products like bilirubin were also significantly reduced. Glutathione metabolism on the pathway level, as well as individual metabolite components such as NADPH, glutathione (GSH), and oxidized glutathione (GSSG), was downregulated. Overall, metabolome and proteome profiles in CI deficient cells correlated well, supporting our integrated approach.

  19. Okeanos Explorer (EX1504L1): CAPSTONE NWHI Exploration Mapping

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Multibeam and singlebeam mapping operations will be conducted 24 hours a day throughout the cruise. Sub-bottom profile mapping will be conducted 24 hours a day at...

  20. Lobster Logbook Data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NWHI lobster trap fishery, which commenced in the mid-1970s, is a multispecies fishery and primarily targets the Hawaiian spiny lobster (Panuliris marginatus)...

  1. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Morotomi-Yano, Keiko; Akiyama, Hidenori [Institute of Pulsed Power Science, Kumamoto University, Kumamoto 860-8555 (Japan); Yano, Ken-ichi, E-mail: yanoken@kumamoto-u.ac.jp [Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto 860-8555 (Japan)

    2013-08-30

    Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.

  2. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

    International Nuclear Information System (INIS)

    Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi

    2013-01-01

    Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs

  3. Characterization of Danio rerio Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase, the structural prototype of the ADPRibase-Mn-like protein family.

    Directory of Open Access Journals (Sweden)

    Joaquim Rui Rodrigues

    Full Text Available The ADPRibase-Mn-like protein family, that belongs to the metallo-dependent phosphatase superfamily, has different functional and structural prototypes. The functional one is the Mn(2+-dependent ADP-ribose/CDP-alcohol diphosphatase from Rattus norvegicus, which is essentially inactive with Mg(2+ and active with low micromolar Mn(2+ in the hydrolysis of the phosphoanhydride linkages of ADP-ribose, CDP-alcohols and cyclic ADP-ribose (cADPR in order of decreasing efficiency. The structural prototype of the family is a Danio rerio protein with a known crystallographic structure but functionally uncharacterized. To estimate the structure-function correlation with the same protein, the activities of zebrafish ADPRibase-Mn were studied. Differences between zebrafish and rat enzymes are highlighted. The former showed a complex activity dependence on Mn(2+, significant (≈25% Mg(2+-dependent activity, but was almost inactive on cADPR (150-fold less efficient than the rat counterpart. The low cADPR hydrolase activity agreed with the zebrafish genome lacking genes coding for proteins with significant homology with cADPR-forming enzymes. Substrate-docking to zebrafish wild-type protein, and characterization of the ADPRibase-Mn H97A mutant pointed to a role of His-97 in catalysis by orientation, and to a bidentate water bridging the dinuclear metal center as the potential nucleophile. Finally, three structural elements that delimit the active site entrance in the zebrafish protein were identified as unique to the ADPRibase-Mn-like family within the metallo-dependent phosphatase superfamily.

  4. Poly(ADP-ribose polymerase 1 is indispensable for transforming growth factor-β Induced Smad3 activation in vascular smooth muscle cell.

    Directory of Open Access Journals (Sweden)

    Dan Huang

    Full Text Available BACKGROUND: Transforming growth factor type-β (TGF-β/Smad pathway plays an essential role in vascular fibrosis. Reactive oxygen species (ROS generation also mediates TGF-β signaling-induced vascular fibrosis, suggesting that some sort of interaction exists between Smad and redox pathways. However, the underlying molecular mechanism is largely unknown. This study aims to investigate the influence of poly(ADP-ribose polymerase 1 (PARP1, a downstream effector of ROS, on TGF-β signaling transduction through Smad3 pathway in rat vascular smooth muscle cells (VSMCs. METHODS AND RESULTS: TGF-β1 treatment promoted PARP1 activation through induction of ROS generation in rat VSMCs. TGF-β1-induced phosphorylation and nuclear accumulation of Smad3 was prevented by treatment of cells with PARP inhibitor, 3-aminobenzamide (3AB or N-(6-oxo-5,6-dihydrophenanthridin-2-yl-2-(N,N-dimethylaminoacetami (PJ34, or PARP1 siRNA. TGF-β1 treatment promoted poly(ADP-ribosylation of Smad3 via activation of PARP1 in the nucleus. Poly(ADP-ribosylation enhanced Smad-Smad binding element (SBE complex formation in nuclear extracts and increased DNA binding activity of Smad3. Pretreatment with 3AB, PJ34, or PARP1 siRNA prevented TGF-β1-induced Smad3 transactivation and expression of Smad3 target genes, including collagen Iα1, collagen IIIα1 and tissue inhibitor of metalloproteinase 1, in rat VSMCs. CONCLUSIONS: PARP1 is indispensable for TGF-β1 induced Smad3 activation in rat VSMCs. Targeting PARP1 may be a promising therapeutic approach against vascular diseases induced by dysregulation of TGF-β/Smad3 pathway.

  5. HMSRP Hawaiian Monk Seal Survey Data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set contains records of Hawaiian monk seal and green turtle sightings in the Northwestern Hawaiian Islands (NWHI) since 1982 at Lisianski Island, and since...

  6. A key role for poly(ADP-ribose polymerase 3 in ectodermal specification and neural crest development.

    Directory of Open Access Journals (Sweden)

    Michèle Rouleau

    2011-01-01

    Full Text Available The PARP family member poly(ADP-ribose polymerase 3 (PARP3 is structurally related to the well characterized PARP1 that orchestrates cellular responses to DNA strand breaks and cell death by the synthesis of poly(ADP-ribose. In contrast to PARP1 and PARP2, the functions of PARP3 are undefined. Here, we reveal critical functions for PARP3 during vertebrate development.We have used several in vitro and in vivo approaches to examine the possible functions of PARP3 as a transcriptional regulator, a function suggested from its previously reported association with several Polycomb group (PcG proteins. We demonstrate that PARP3 gene occupancy in the human neuroblastoma cell line SK-N-SH occurs preferentially with developmental genes regulating cell fate specification, tissue patterning, craniofacial development and neurogenesis. Addressing the significance of this association during zebrafish development, we show that morpholino oligonucleotide-directed inhibition of parp3 expression in zebrafish impairs the expression of the neural crest cell specifier sox9a and of dlx3b/dlx4b, the formation of cranial sensory placodes, inner ears and pectoral fins. It delays pigmentation and severely impedes the development of the median fin fold and tail bud.Our findings demonstrate that Parp3 is crucial in the early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions as early as during the specification of the neural plate border.

  7. Role of water in the enzymatic catalysis: study of ATP + AMP → 2ADP conversion by adenylate kinase.

    Science.gov (United States)

    Adkar, Bharat V; Jana, Biman; Bagchi, Biman

    2011-04-28

    The catalytic conversion ATP + AMP → 2ADP by the enzyme adenylate kinase (ADK) involves the binding of one ATP molecule to the LID domain and one AMP molecule to the NMP domain. The latter is followed by a phosphate transfer and then the release of two ADP molecules. We have computed a novel two-dimensional configurational free energy surface (2DCFES), with one reaction coordinate each for the LID and the NMP domain motions, while considering explicit water interactions. Our computed 2DCFES clearly reveals the existence of a stable half-open half-closed (HOHC) intermediate state of the enzyme. Cycling of the enzyme through the HOHC state reduces the conformational free energy barrier for the reaction by about 20 kJ/mol. We find that the stability of the HOHC state (missed in all earlier studies with implicit solvent model) is largely because of the increase of specific interactions of the polar amino acid side chains with water, particularly with the arginine and the histidine residues. Free energy surface of the LID domain is rather rugged, which can conveniently slow down LID's conformational motion, thus facilitating a new substrate capture after the product release in the catalytic cycle.

  8. Influence of ADP on the course of radiation sickness and some metabolic processes in rats after irradiation of abdomen and parenteral feeding

    International Nuclear Information System (INIS)

    Grozdov, S.P.; Fedorovskij, L.L.; Protserova, T.K.

    1985-01-01

    The intrapertitoneal administration of ADP (150-75 mg/kg/day) to rats on days 1-3 after exposure of abdomen to X-rays (13.5 Gy) enhanced the assimilation of glucose, amino acids and electrolytes introduced with the parenteral feeding and increased considerably the survival rate of animals

  9. Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

    Science.gov (United States)

    Ludtmann, Marthe H R; Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Preza, Elisavet; Carro, Eva; Houlden, Henry; Gandhi, Sonia; Wray, Selina; Abramov, Andrey Y

    2017-05-26

    Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Increased transcript level of poly(ADP-ribose) polymerase (PARP-1) in human tricuspid compared with bicuspid aortic valves correlates with the stenosis severity

    International Nuclear Information System (INIS)

    Nagy, Edit; Caidahl, Kenneth; Franco-Cereceda, Anders; Bäck, Magnus

    2012-01-01

    Highlights: ► Oxidative stress has been implicated in the pathomechanism of calcific aortic valve stenosis. ► We assessed the transcript levels for PARP-1 (poly(ADP-ribose) polymerase), acts as a DNA damage nick sensor in stenotic valves. ► Early stage of diseased tricuspid valves exhibited higher mRNA levels for PARP-1 compared to bicuspid valves. ► The mRNA levels for PARP-1 inversely correlated with the clinical stenosis severity in tricuspid valves. ► Our data demonstrated that DNA damage pathways might be associated with stenosis severity only in tricuspid valves. -- Abstract: Oxidative stress may contribute to the hemodynamic progression of aortic valve stenosis, and is associated with activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) 1. The aim of the present study was to assess the transcriptional profile and the topological distribution of PARP-1 in human aortic valves, and its relation to the stenosis severity. Human stenotic aortic valves were obtained from 46 patients undergoing aortic valve replacement surgery and used for mRNA extraction followed by quantitative real-time PCR to correlate the PARP-1 expression levels with the non invasive hemodynamic parameters quantifying the stenosis severity. Primary isolated valvular interstitial cells (VICs) were used to explore the effects of cytokines and leukotriene C 4 (LTC 4 ) on valvular PARP-1 expression. The thickened areas of stenotic valves with tricuspid morphology expressed significantly higher levels of PARP-1 mRNA compared with the corresponding part of bicuspid valves (0.501 vs 0.243, P = 0.01). Furthermore, the quantitative gene expression levels of PARP-1 were inversely correlated with the aortic valve area (AVA) (r = −0.46, P = 0.0469) and AVA indexed for body surface area (BSA) (r = −0.498; P = 0.0298) only in tricuspid aortic valves. LTC 4 (1 nM) significantly elevated the mRNA levels of PARP-1 by 2.38-fold in VICs. Taken together, these data suggest that

  11. Predicting sea-level rise vulnerability of terrestrial habitat and wildlife of the Northwestern Hawaiian Islands

    Science.gov (United States)

    Reynolds, Michelle H.; Berkowitz, Paul; Courtot, Karen N.; Krause, Crystal M.; Reynolds, Michelle H.; Berkowitz, Paul; Courtot, Karen N.; Krause, Crystal M.

    2012-01-01

    If current climate change trends continue, rising sea levels may inundate low-lying islands across the globe, placing island biodiversity at risk. Recent models predict a rise of approximately one meter (1 m) in global sea level by 2100, with larger increases possible in areas of the Pacific Ocean. Pacific Islands are unique ecosystems home to many endangered endemic plant and animal species. The Northwestern Hawaiian Islands (NWHI), which extend 1,930 kilometers (km) beyond the main Hawaiian Islands, are a World Heritage Site and part of the Papahanaumokuakea Marine National Monument. These NWHI support the largest tropical seabird rookery in the world, providing breeding habitat for 21 species of seabirds, 4 endemic land bird species and essential foraging, breeding, or haul-out habitat for other resident and migratory wildlife. In recent years, concern has grown about the increasing vulnerability of the NWHI and their wildlife populations to changing climatic patterns, particularly the uncertainty associated with potential impacts from global sea-level rise (SLR) and storms. In response to the need by managers to adapt future resource protection strategies to climate change variability and dynamic island ecosystems, we have synthesized and down scaled analyses for this important region. This report describes a 2-year study of a remote northwestern Pacific atoll ecosystem and identifies wildlife and habitat vulnerable to rising sea levels and changing climate conditions. A lack of high-resolution topographic data for low-lying islands of the NWHI had previously precluded an extensive quantitative model of the potential impacts of SLR on wildlife habitat. The first chapter (chapter 1) describes the vegetation and topography of 20 islands of Papahanaumokuakea Marine National Monument, the distribution and status of wildlife populations, and the predicted impacts for a range of SLR scenarios. Furthermore, this chapter explores the potential effects of SLR on

  12. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein

    International Nuclear Information System (INIS)

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

    1988-01-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5'-[α- 32 P]triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an α subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera

  13. Combined blockade of ADP receptors and PI3-kinase p110β fully prevents platelet and leukocyte activation during hypothermic extracorporeal circulation.

    Directory of Open Access Journals (Sweden)

    Stefanie Krajewski

    Full Text Available Extracorporeal circulation (ECC and hypothermia are used to maintain stable circulatory parameters and improve the ischemia tolerance of patients in cardiac surgery. However, ECC and hypothermia induce activation mechanisms in platelets and leukocytes, which are mediated by the platelet agonist ADP and the phosphoinositide-3-kinase (PI3K p110β. Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation. This study analyzes effects of ADP receptor P(2Y(12 and P(2Y(1 blockade and PI3K p110β inhibition on platelets and granulocytes during hypothermic ECC. Human blood was treated with the P(2Y(12 antagonist 2-MeSAMP, the P(2Y(1 antagonist MRS2179, the PI3K p110β inhibitor TGX-221, combinations thereof, or PBS and propylene glycol (controls. Under static in vitro conditions a concentration-dependent effect regarding the inhibition of ADP-induced platelet activation was found using 2-MeSAMP or TGX-221. Further inhibition of ADP-mediated effects was achieved with MRS2179. Next, blood was circulated in an ex vivo ECC model at 28°C for 30 minutes and various platelet and granulocyte markers were investigated using flow cytometry, ELISA and platelet count analysis. GPIIb/IIIa activation induced by hypothermic ECC was inhibited using TGX-221 alone or in combination with P(2Y blockers (p<0.05, while no effect of hypothermic ECC or antiplatelet agents on GPIIb/IIIa and GPIbα expression and von Willebrand factor binding was observed. Sole P(2Y and PI3K blockade or a combination thereof inhibited P-selectin expression on platelets and platelet-derived microparticles during hypothermic ECC (p<0.05. P(2Y blockade alone or combined with TGX-221 prevented ECC-induced platelet-granulocyte aggregate formation (p<0.05. Platelet adhesion to the ECC surface, platelet loss and Mac-1 expression on granulocytes were inhibited by combined P(2Y and PI3K blockade (p<0.05. Combined blockade of P

  14. 75 FR 1597 - Western Pacific Crustacean Fisheries; 2010 Northwestern Hawaiian Islands Lobster Harvest Guideline

    Science.gov (United States)

    2010-01-12

    ... Pacific Crustacean Fisheries; 2010 Northwestern Hawaiian Islands Lobster Harvest Guideline AGENCY.... ACTION: Notification of lobster harvest guideline. SUMMARY: NMFS announces that the annual harvest guideline for the commercial lobster fishery in the Northwestern Hawaiian Islands (NWHI) for calendar year...

  15. Arsenite-induced ROS/RNS generation causes zinc loss and inhibits the activity of poly (ADP-ribose) polymerase-1

    OpenAIRE

    Wang, Feng; Zhou, Xixi; Liu, Wenlan; Sun, Xi; Chen, Chen; Hudson, Laurie G.; Liu, Ke Jian

    2013-01-01

    Arsenic enhances genotoxicity of other carcinogenic agents such as ultraviolet radiation and benzo[a]pyrene. Recent reports suggest that inhibition of DNA repair is an important aspect of arsenic co-carcinogenesis, and DNA repair proteins such as poly (ADP ribose) polymerase (PARP)-1 are direct molecular targets of arsenic. Although arsenic has been shown to generate reactive oxygen/nitrogen species (ROS/RNS), little is known about the role of arsenic-induced ROS/RNS in the mechanism underlyi...

  16. Molecular mechanism of the short-term cardiotoxicity caused by 2',3'-dideoxycytidine (ddC): modulation of reactive oxygen species levels and ADP-ribosylation reactions.

    Science.gov (United States)

    Skuta, G; Fischer, G M; Janaky, T; Kele, Z; Szabo, P; Tozser, J; Sumegi, B

    1999-12-15

    The short-term cardiac side effects of 2',3'-dideoxycytidine (ddC, zalcitabine) were studied in rats in order to understand the biochemical events contributing to the development of ddC-induced cardiomyopathy. In developing animals, ddC treatment provoked a surprisingly rapid appearance of cardiac malfunctions characterized by prolonged RR, PR, and QT intervals and J point depression. The energy metabolism in the heart was compromised, characterized by a decreased creatine phosphate/creatine ratio (from 2.05 normal value to 0.75) and a decreased free ATP/ADP ratio (from 332 normal value to 121). The activity of respiratory complexes (NADH: cytochrome c oxidoreductase and cytochrome oxidase) also decreased significantly. Southern blot and polymerase chain reaction analysis did not show deletions or a decrease in the quantity of mitochondrial DNA (mtDNA) deriving from ddC-treated rat hearts, indicating that under our experimental conditions, ddC-induced heart abnormalities were not the direct consequence of mtDNA-related damage. The ddC treatment of rats significantly increased the formation of reactive oxygen species (ROS) in heart and skeletal muscle as determined by the oxidation of non-fluorescent dihydrorhodamine123 to fluorescent rhodamine123 and the oxidation of cellular proteins determined from protein carbonyl content. An activation of the nuclear poly-(ADP-ribose) polymerase (EC 2.4.2.30) and an increase in the mono-ADP-ribosylation of glucose-regulated protein and desmin were observed in the cardiac tissue from ddC-treated animals. A decrease in the quantity of heat shock protein (HSP)70s was also detected, while the level of HSP25 and HSP60 remained unchanged. Surprisingly, ddC treatment induced a skeletal muscle-specific decrease in the quantity of three proteins, one of which was identified by N-terminal sequencing as myoglobin, and another by tandem mass spectrometer sequencing as triosephosphate isomerase (EC 5.3.1.1). These data show that the short

  17. THRIPS SPECIES (INSECTA: THYSANOPTERA OF ORNAMENTAL PLANTS FROM THE PARKS AND GREENHOUSES OF ADP PITESTI

    Directory of Open Access Journals (Sweden)

    Daniela Bărbuceanu

    2012-04-01

    Full Text Available The observations carried-out in 2008/2010 to ornamental plants from parks and greenhouses of ADP Pitesti relieve 12 species of thrips. One species of them, Frankliniella occidentalis was identified in greenhouses on Rosa sp., Dianthus sp. and Zantedeschia sp. In parks, the thrips species belong to 12 species, dominated by Frankliniella intonsa. All of them are polypfagous and divided in two throphic levels: primary and secondary consumers. The thrips species are mentioned for the first time in Romania on this host plant. In greenhouses are necessary intensive chemical treatments and methods of cultural hygiene to limit the F. occidentalis populations.

  18. Targeting poly (ADP-ribose polymerase partially contributes to bufalin-induced cell death in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    He Huang

    Full Text Available Despite recent pharmaceutical advancements in therapeutic drugs, multiple myeloma (MM remains an incurable disease. Recently, ploy(ADP-ribose polymerase 1 (PARP1 has been shown as a potentially promising target for MM therapy. A previous report suggested bufalin, a component of traditional Chinese medicine ("Chan Su", might target PARP1. However, this hypothesis has not been verified. We here showed that bufalin could inhibit PARP1 activity in vitro and reduce DNA-damage-induced poly(ADP-ribosylation in MM cells. Molecular docking analysis revealed that the active site of bufalin interaction is within the catalytic domain of PAPR1. Thus, PARP1 is a putative target of bufalin. Furthermore, we showed, for the first time that the proliferation of MM cell lines (NCI-H929, U266, RPMI8226 and MM.1S and primary CD138(+ MM cells could be inhibited by bufalin, mainly via apoptosis and G2-M phase cell cycle arrest. MM cell apoptosis was confirmed by apoptotic cell morphology, Annexin-V positive cells, and the caspase3 activation. We further evaluated the role of PARP1 in bufalin-induced apoptosis, discovering that PARP1 overexpression partially suppressed bufalin-induced cell death. Moreover, bufalin can act as chemosensitizer to enhance the cell growth-inhibitory effects of topotecan, camptothecin, etoposide and vorinostat in MM cells. Collectively, our data suggest that bufalin is a novel PARP1 inhibitor and a potentially promising therapeutic agent against MM alone or in combination with other drugs.

  19. Cholera toxin-induced ADP-ribosylation of a 46 kDa protein is decreased in brains of ethanol-fed mice

    International Nuclear Information System (INIS)

    Nhamburo, P.T.; Hoffman, P.L.; Tabakoff, B.

    1988-01-01

    The acute in vitro effects of ethanol on cerebral cortical adenylate cyclase activity and beta-adrenergic receptor characteristics suggested a site of action of ethanol at Gs, the stimulatory guanine nucleotide binding protein. After chronic ethanol ingestion, the beta-adrenergic receptor appeared to be uncoupled (i.e., the form of the receptor with high affinity for agonist was undetectable), and stimulation of adenylate cyclase activity by isoproterenol or guanine nucleotides was reduced, suggesting an alteration in the properties of Gs. To further characterize this change, cholera and pertussis toxin-mediated 32 P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingested ethanol in a liquid diet. 32 P-labeled proteins were separated by SDS-PAGE and quantitated by autoradiography. There was a selective 30-50% decrease in cholera toxin-induced labeling of 46 kDa protein band in membranes of ethanol-fed mice, with no apparent change in pertussis toxin-induced labeling. The 46 kDa protein has a molecular weight similar to that of the alpha subunit of Gs, suggesting a reduced amount of this protein or a change in its characteristics as a substrate for cholera toxin-induced ADP-ribosylation in cortical membranes of ethanol-fed mice

  20. 76 FR 77214 - Hawaii Crustacean Fisheries; 2012 Northwestern Hawaiian Islands Lobster Harvest Guideline

    Science.gov (United States)

    2011-12-12

    ... Crustacean Fisheries; 2012 Northwestern Hawaiian Islands Lobster Harvest Guideline AGENCY: National Marine...: Notification of lobster harvest guideline. SUMMARY: NMFS establishes the annual harvest guideline for the commercial lobster fishery in the Northwestern Hawaiian Islands (NWHI) for calendar year 2012 at zero...

  1. Differentiation-Associated Downregulation of Poly(ADP-Ribose Polymerase-1 Expression in Myoblasts Serves to Increase Their Resistance to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Gábor Oláh

    Full Text Available Poly(ADP-ribose polymerase 1 (PARP-1, the major isoform of the poly (ADP-ribose polymerase family, is a constitutive nuclear and mitochondrial protein with well-recognized roles in various essential cellular functions such as DNA repair, signal transduction, apoptosis, as well as in a variety of pathophysiological conditions including sepsis, diabetes and cancer. Activation of PARP-1 in response to oxidative stress catalyzes the covalent attachment of the poly (ADP-ribose (PAR groups on itself and other acceptor proteins, utilizing NAD+ as a substrate. Overactivation of PARP-1 depletes intracellular NAD+ influencing mitochondrial electron transport, cellular ATP generation and, if persistent, can result in necrotic cell death. Due to their high metabolic activity, skeletal muscle cells are particularly exposed to constant oxidative stress insults. In this study, we investigated the role of PARP-1 in a well-defined model of murine skeletal muscle differentiation (C2C12 and compare the responses to oxidative stress of undifferentiated myoblasts and differentiated myotubes. We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes. This alteration correlated with an increased resistance to oxidative stress of the myotubes, as measured by MTT and LDH assays. Mitochondrial function, assessed by measuring mitochondrial membrane potential, was preserved under oxidative stress in myotubes compared to myoblasts. Moreover, basal respiration, ATP synthesis, and the maximal respiratory capacity of mitochondria were higher in myotubes than in myoblasts. Inhibition of the catalytic activity of PARP-1 by PJ34 (a phenanthridinone PARP inhibitor exerted greater protective effects in undifferentiated myoblasts than in differentiated myotubes. The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6. Forced overexpression of PARP1 in C2C12 myotubes sensitized the cells to oxidant

  2. Single-step selection of drug resistant Acinetobacter baylyi ADP1 mutants reveals a functional redundancy in the recruitment of multidrug efflux systems.

    Directory of Open Access Journals (Sweden)

    Anthony J Brzoska

    Full Text Available Members of the genus Acinetobacter have been the focus recent attention due to both their clinical significance and application to molecular biology. The soil commensal bacterium Acinetobacter baylyi ADP1 has been proposed as a model system for molecular and genetic studies, whereas in a clinical environment, Acinetobacter spp. are of increasing importance due to their propensity to cause serious and intractable systemic infections. Clinically, a major factor in the success of Acinetobacter spp. as opportunistic pathogens can be attributed to their ability to rapidly evolve resistance to common antimicrobial compounds. Whole genome sequencing of clinical and environmental Acinetobacter spp. isolates has revealed the presence of numerous multidrug transporters within the core and accessory genomes, suggesting that efflux is an important host defense response in this genus. In this work, we used the drug-susceptible organism A. baylyi ADP1 as a model for studies into the evolution of efflux mediated resistance in genus Acinetobacter, due to the high level of conservation of efflux determinants across four diverse Acinetobacter strains, including clinical isolates. A single exposure of therapeutic concentrations of chloramphenicol to populations of A. baylyi ADP1 cells produced five individual colonies displaying multidrug resistance. The major facilitator superfamily pump craA was upregulated in one mutant strain, whereas the resistance nodulation division pump adeJ was upregulated in the remaining four. Within the adeJ upregulated population, two different levels of adeJ mRNA transcription were observed, suggesting at least three separate mutations were selected after single-step exposure to chloramphenicol. In the craA upregulated strain, a T to G substitution 12 nt upstream of the craA translation initiation codon was observed. Subsequent mRNA stability analyses using this strain revealed that the half-life of mutant craA mRNA was significantly

  3. The influence of inhibitors of poly (ADP-ribose) polymerase on X-ray induced potentially lethal damage repair

    International Nuclear Information System (INIS)

    Brown, D.M.; Evans, J.W.; Brown, J.M.

    1984-01-01

    Inhibition of repair of X-ray-induced potentially lethal damage (PLD) could enhance the curability of radioresistant tumours. We have studied the effect of inhibitors of the enzyme poly (ADP-ribose) polymerase on X-ray PLD repair. Four classes of inhibitors are known: aromatic amides (e.g., 3-aminobenzamide), thymidine, nicotinamides and methyl xanthines (e.g., caffeine). Plateau-phase Chinese hamster ovary (HA-1) cultures were exposed to 10 mM concentrations of thymidine, nicotinamide, 3-aminobenzamide (3-ABA) and caffeine prior to irradiation to 12 Gy in air, and then incubated with drug at 37 0 C for varying times (0-6 h) prior to subculture. Irradiated cells without drug exhibited a 5-6 fold increase in survival over the 6 h period compared to cultures plated immediately after irradiation. Although none of the compounds proved cytotoxic to unirradiated controls over the 6.5 h exposure, all of the compounds except thymidine reduced the capacity of the cells to repair PLD. The order of the inhibitory effect was caffeine > 3-ABA > nicotinamide, and the inhibition was concentration dependent for nicotinamide and 3-ABA. We also studied the effect of 3-ABA on the radiation response of exponentially growing cells. 5 mM 3-ABA for 2h post-irradiation resulted in a dose-multiplicative sensitization reducing the D 0 from 0.88 Gy to 0.69 Gy, indicating an involvement of poly (ADP-ribose) polymerase in the radiosensitivity of exponentially growing as well as plateau-phase cells. (author)

  4. Use of Mycobacterium smegmatis deficient in ADP-ribosyltransferase as surrogate for Mycobacterium tuberculosis in drug testing and mutation analysis.

    Science.gov (United States)

    Agrawal, Priyanka; Miryala, Sandeep; Varshney, Umesh

    2015-01-01

    Rifampicin (Rif) is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr) in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs). Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase) causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr). The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC) for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil) strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G). Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.

  5. Use of Mycobacterium smegmatis deficient in ADP-ribosyltransferase as surrogate for Mycobacterium tuberculosis in drug testing and mutation analysis.

    Directory of Open Access Journals (Sweden)

    Priyanka Agrawal

    Full Text Available Rifampicin (Rif is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs. Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr. The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G. Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.

  6. Derelict fishing gear in the Northwestern Hawaiian Islands: diving surveys and debris removal in 1999 confirm threat to coral reef ecosystems

    Energy Technology Data Exchange (ETDEWEB)

    Donohue, Mary J.; Sramek, Carolyn M.; Antonelis, George A. [National Oceanic and Atmospheric Administration Commissioned Corps, National Marine Fisheries Service Honolulu Lab., Honolulu, HI (United States); Boland, Raymond C. [Hawaii Univ. Research Corp., Joint Inst. for Marine and Atmospheric Research, Honolulu, HI (United States)

    2001-07-01

    Marine debris threatens Northwestern Hawaiian Islands' (NWHI) coral reef ecosystems. Debris, a contaminant, entangles and kills endangered Hawaiian monk seals (Monachus schauinslandi), coral, and other wildlife. We describe a novel multi-agency effort using divers to systematically survey and remove derelict fishing gear from two NWHI in 1999. 14 t of derelict fishing gear were removed and debris distribution, density, type and fouling level documented at Lisianski Island and Pearl and Hermes Atoll. Reef debris density ranged from 3.4 to 62.2 items/km{sup 2}. Trawl netting was the most frequent debris type encountered (88%) and represented the greatest debris component recovered by weight (35%), followed by monofilament gillnet (34%), and maritime line (23%). Most debris recovered, 72%, had light or no fouling, suggesting debris may have short oceanic circulation histories. Our study demonstrates that derelict fishing gear poses a persistent threat to the coral reef ecosystems of the Hawaiian Archipelago. (Author)

  7. Derelict fishing gear in the northwestern Hawaiian Islands: diving surveys and debris removal in 1999 confirm threat to coral reef ecosystems.

    Science.gov (United States)

    Donohue, M J; Boland, R C; Sramek, C M; Antonelis, G A

    2001-12-01

    Marine debris threatens Northwestern Hawaiian Islands' (NWHI) coral reef ecosystems. Debris, a contaminant, entangles and kills endangered Hawaiian monk seals (Monachus schauinslandi), coral, and other wildlife. We describe a novel multi-agency effort using divers to systematically survey and remove derelict fishing gear from two NWHI in 1999. 14 t of derelict fishing gear were removed and debris distribution, density, type and fouling level documented at Lisianski Island and Pearl and Hermes Atoll. Reef debris density ranged from 3.4 to 62.2 items/km2. Trawl netting was the most frequent debris type encountered (88%) and represented the greatest debris component recovered by weight (35%), followed by monofilament gillnet (34%), and maritime line (23%). Most debris recovered, 72%, had light or no fouling, suggesting debris may have short oceanic circulation histories. Our study demonstrates that derelict fishing gear poses a persistent threat to the coral reef ecosystems of the Hawaiian Archipelago.

  8. ADP-ribosylation by cholera toxin: functional analysis of a cellular system that stimulates the enzymic activity of cholera toxin fragment A1

    International Nuclear Information System (INIS)

    Gill, D.M.; Coburn, J.

    1987-01-01

    The authors have clarified relationships between cholera toxin, cholera toxin substrates, a membrane protein S that is required for toxin activity, and a soluble protein CF that is needed for the function of S. The toxin has little intrinsic ability to catalyze ADP-ribosylations unless it encounters the active form of the S protein, which is S liganded to GTP or to a GTP analogue. In the presence of CF, S x GTP forms readily, though reversibly, but a more permanent active species, S-guanosine 5'-O-(3-thiotriphosphate) (S x GTPγS), forms over a period of 10-15 min at 37 0 C. Both guanosine 5'-O-(2-thiodiphosphate) and GTP block this quasi-permanent activation. Some S x GTPγS forms in membranes that are exposed to CF alone and then to GTPγS, with a wash in between, and it is possible that CF facilitates a G nucleotide exchange. S x GTPγS dissolved by nonionic detergents persists in solution and can be used to support the ADP-ribosylation of nucleotide-free substrates. In this circumstance, added guanyl nucleotides have no further effect. This active form of S is unstable, especially when heated, but the thermal inactivation above 45 0 C is decreased by GTPγS. Active S is required equally for the ADP-ribosylation of all of cholera toxin's protein substrates, regardless of whether they bind GTP or not. They suggest that active S interacts directly with the enzymic A 1 fragments of cholera toxin and not with any toxin substrate. The activation and activity of S are independent of the state, or even the presence, of adenylate cyclase and seem to be involved with the cyclase system only via cholera toxin. S is apparently not related by function to certain other GTP binding proteins, including p21/sup ras/, and appears to be a new GTP binding protein whose physiologic role remains to be identified

  9. Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

    Science.gov (United States)

    Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

    2017-09-22

    One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells.

    Science.gov (United States)

    Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi

    2013-08-30

    Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Phylogenetic analysis of the thylakoid ATP/ADP carrier reveals new insights into its function restricted to green plants

    Directory of Open Access Journals (Sweden)

    Cornelia eSpetea

    2012-01-01

    Full Text Available ATP is the common energy currency of cellular metabolism in all living organisms. Most of them synthesize ATP in the cytosol or on the mitochondrial inner membrane, whereas land plants, algae and cyanobacteria also produce it on the thylakoid membrane during the light-dependent reactions of photosynthesis. From the site of synthesis, ATP is transported to the site of utilization via intracellular membranes transporters. One major type of ATP transporter is represented by the mitochondrial ADP/ATP carrier family. Here we review a recently characterized member, namely the thylakoid ATP/ADP carrier from Arabidopsis thaliana (AtTAAC. Thus far, no orthologues of this carrier have been characterized in other organisms, although similar sequences can be recognized in many sequenced genomes. Protein Sequence database searches and phylogenetic analyses indicate the absence of TAAC in cyanobacteria and its appearance early in the evolution of photosynthetic eukaryotes. The TAAC clade is composed of carriers found in land plants and some green algae, but no proteins from other photosynthetic taxa, such as red algae, brown algae and diatoms. This implies that TAAC-like sequences arose only once before the divergence of green algae and land plants. Based on these findings, it is proposed that TAAC may have evolved in response to the need of a new activity in higher photosynthetic eukaryotes. This activity may provide the energy to drive reactions during biogenesis and turnover of photosynthetic complexes, which are heterogenously distributed in a thylakoid membrane system composed of appressed and non-appressed regions.

  12. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint

    Energy Technology Data Exchange (ETDEWEB)

    Nakadate, Yusuke [Shien-Lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Department of Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585 (Japan); Kodera, Yasuo; Kitamura, Yuka [Shien-Lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Tachibana, Taro [Department of Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585 (Japan); Tamura, Tomohide [Division of Thoracic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Koizumi, Fumiaki, E-mail: fkoizumi@ncc.go.jp [Division of Thoracic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2013-11-29

    Highlights: •Radiosensitization by PARG silencing was observed in multiple lung cancer cells. •PAR accumulation was enhanced by PARG silencing after DNA damage. •Radiation-induced G2/M arrest and checkpoint activation were impaired by PARG siRNA. -- Abstract: Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy.

  13. Discriminating silt-and-clay from suspended-sand in rivers using side-looking acoustic profilers

    Science.gov (United States)

    Wright, Scott A.; Topping, David J.; Williams, Cory A.

    2010-01-01

    techniques rely on measurements of ancillary properties that correlate with suspended-sediment concentration and particle size and thus require the collection of traditional samples for calibration. Through in situ deployments, these methods can provide the high temporal resolution that cannot be achieved through traditional sampling. Here we focus on the evaluation of acoustic profiling techniques (e.g. acoustic-Doppler sideways-looking profilers, or ADPs). One major advantage of acoustic profiling is the ability to concurrently measure water velocity (using Doppler-shift methods) and suspended-sediment concentration such that suspended-sediment flux can be directly computed using data from a single instrument. Acoustic-Doppler profilers have become popular for measuring water velocity and discharge in rivers, through both moving-boat operations and from fixed deployments such as bank-mounted sideways-looking instruments (Hirsch and Costa, 2004, Muste et al., 2007). The method presented herein is most suited to sideways-looking applications as a complement to the "index velocity" technique, whereby an index velocity from a sideways-looking instrument is related to the cross-section average velocity (determined from moving-boat discharge measurements) as a means for developing a continuous water-discharge record (Ruhl and Simpson, 2005). Topping et al. (2007) presented a method for discriminating silt-and-clay from suspended sand, using single frequency ADPs. This method takes advantage of the relations among acoustic backscatter, sediment-induced acoustic attenuation, suspended-sediment concentration (SSC), and particle size distribution (PSD). Backscatter is the amount of sound scattered back and received at the transducer while sediment-induced attenuation is the amount of sound scattered in other directions and absorbed by the sediment particles. Both of these parameters can be measured with an ADP, and their different dependencies on SSC and PSD allow for the

  14. 76 FR 32025 - Endangered and Threatened Wildlife and Plants: Proposed Rulemaking To Revise Critical Habitat for...

    Science.gov (United States)

    2011-06-02

    ... extending seaward to the 500-m depth contour around: Kaula Island, Niihau, Kauai, Oahu, Maui Nui (including... coast of Niihau; Pacific Missile Range Facility Main Base at Barking Sands, Kauai; Pacific Missile Range... NWHI as well as utilize some habitat in the MHI, including Kauai and Niihau, which were forming as...

  15. Effect of Vaccinia virus infection on poly(ADP-ribose)synthesis and DNA metabolism in different cells

    Energy Technology Data Exchange (ETDEWEB)

    Topaloglou, A.; Ott, E.; Altmann, H. (Oesterreichisches Forschungszentrum Seibersdorf G.m.b.H. Inst. fuer Biologie); Zashukhina, G.D.; Sinelschikova, T.A. (AN SSSR, Moscow. Inst. Obshchej Genetiki)

    1983-07-14

    In Chang liver cells and rat spleen cells infected with Vaccinia virus, DNA synthesis, repair replication after UV irradiation and poly(ADP-ribose)(PAR) synthesis were determined. In the time post infection semiconservative DNA synthesis showed only a slight reduction. DNA repair replication was not very different from controls 4 hours p.i. but was enhanced 24 hours after infection compared to noninfected cells. PAR synthesis was also not changed very much 4 hours p.i. but was decreased significantly after 24 hours. The determination of radioactivity resulting from /sup 3/H-NAD, showed a marked reduction of PAR in the spacer region of chromatin 24 hours p.i., but in addition, PAR located in the core region, was reduced, too.

  16. 31P NMR measurements of the ADP concentration in yeast cells genetically modified to express creatine kinase

    International Nuclear Information System (INIS)

    Brindle, K.; Braddock, P.; Fulton, S.

    1990-01-01

    Rabbit muscle creatine kinase has been introduced into the yeast Saccharomyces cerevisiae by transforming cells with a multicopy plasmid containing the coding sequence for the enzyme under the control of the yeast phosphoglycerate kinase promoter. The transformed cells showed creating kinase activities similar to those found in mammalian heart muscle. 31 P NMR measurements of the near-equilibrium concentrations of phosphocreatine and cellular pH together with measurements of the total extractable concentrations of phosphocreatine and creatine allowed calculation of the free ADP/ATP ratio in the cell. The calculated ratio of approximately 2 was considerably higher than the ratio of between 0.06 and 0.1 measured directly in cell extracts

  17. Structures of Mycobacterium Tuberculosis Folylpolyglutamate Synthase Complexed With ADP And AMPPCD

    Energy Technology Data Exchange (ETDEWEB)

    Young, P.G.; Smith, C.A.; Metcalf, P.; Baker, E.N.

    2009-05-28

    Folate derivatives are essential vitamins for cell growth and replication, primarily because of their central role in reactions of one-carbon metabolism. Folates require polyglutamation to be efficiently retained within the cell and folate-dependent enzymes have a higher affinity for the polyglutamylated forms of this cofactor. Polyglutamylation is dependent on the enzyme folylpolyglutamate synthetase (FPGS), which catalyzes the sequential addition of several glutamates to folate. FPGS is essential for the growth and survival of important bacterial species, including Mycobacterium tuberculosis, and is a potential drug target. Here, the crystal structures of M. tuberculosis FPGS in complex with ADP and AMPPCP are reported at 2.0 and 2.3 angstroms resolution, respectively. The structures reveal a deeply buried nucleotide-binding site, as in the Escherichia coli and Lactobacillus casei FPGS structures, and a long extended groove for the binding of folate substrates. Differences from the E. coli and L. casei FPGS structures are seen in the binding of a key divalent cation, the carbamylation state of an essential lysine side chain and the adoption of an 'open' position by the active-site beta5-alpha6 loop. These changes point to coordinated events that are associated with dihydropteroate/folate binding and the catalysis of the new amide bond with an incoming glutamate residue.

  18. ADP-ribosylation of actins in fibroblasts and myofibroblasts by botulinum C2 toxin: Influence on microfilament morphology and migratory behavior

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Petersen, Ole William

    1996-01-01

    botulinum C2 toxin. The substrate for C2 toxin is globular actin, which upon ribosylation cannot incorporate into microfilaments. The pattern of actin ADP-ribosylation in (myo)fibroblasts in the presence of [32P]NAD was analyzed by isoelectric focusing, fluorography and immunoblotting. The influence of C2...... toxin on microfilaments in intact cells was further assessed by immunofluorescence, and motility was measured in a mass migration assay and by computerized video time-lapse microscopy. We show here that C2 toxin specifically ribosylates - and -actin in both fibroblasts and myofibroblasts. Whereas...

  19. Porphyrins profile by high performance liquid chromatography ...

    African Journals Online (AJOL)

    E. Fateen

    2008-09-07

    Sep 7, 2008 ... acid dehydratase porphyria (ADP), acute intermittent por- phyria (AIP) ..... affecting motor neurons, and can lead to respiratory and bul- bar paralysis and ... variable degrees of fatty damage and chronic inflammatory changes [1]. ..... ppix influences the hematologic status and iron metabolism in patients with ...

  20. Neonatal diabetes and congenital hyperinsulinism caused by mutations in ABCC8/SUR1 are associated with altered and opposite affinities for ATP and ADP

    Directory of Open Access Journals (Sweden)

    Joseph eBryan

    2015-04-01

    Full Text Available ATP-sensitive K+ (KATP channels composed of potassium inward-rectifier type 6.2 and sulfonylurea receptor type 1 subunits (Kir6.2/SUR14 are expressed in various cells in the brain and endocrine pancreas where they couple metabolic status to membrane potential. In β-cells, increases in cytosolic [ATP/ADP]c inhibit KATP channel activity, leading to membrane depolarization and exocytosis of insulin granules. Mutations in ABCC8 (SUR1 or KCNJ11 (Kir6.2 can result in gain or loss of channel activity and cause neonatal diabetes (ND or congenital hyperinsulinism (CHI, respectively. SUR1 is reported to be a Mg2+-dependent ATPase. A prevailing model posits that ATP hydrolysis at SUR1 is required to stimulate openings of the pore. However, recent work shows nucleotide binding, without hydrolysis, is sufficient to switch SUR1 to stimulatory conformations. The actions of nucleotides, ATP and ADP, on ND (SUR1E1506D and CHI (SUR1E1506K mutants, without Kir6.2, were compared to assess both models. Both substitutions significantly impair hydrolysis in SUR1 homologues. SUR1E1506D has greater affinity for MgATP than wildtype; SUR1E1506K has reduced affinity. Without Mg2+, SUR1E1506K has a greater affinity for ATP4- consistent with electrostatic attraction between ATP4-, unshielded by Mg2+, and the basic lysine. Further analysis of ND and CHI ABCC8 mutants in the second transmembrane and nucleotide binding domains (TMD2 & NBD2, found a relation between their affinities for ATP (± Mg2+ and their clinical phenotype. Increased affinity for ATP is associated with ND; decreased affinity with CHI. In contrast, MgADP showed a weaker relationship. Diazoxide, known to reduce insulin release in some CHI cases, potentiates switching of CHI mutants from non-stimulatory to stimulatory states consistent with diazoxide stabilizing a nucleotide-bound conformation. The results emphasize the greater importance of nucleotide binding vs hydrolysis in the regulation of KATP channels

  1. Functional interaction between the two halves of the photoreceptor-specific ATP binding cassette protein ABCR (ABCA4). Evidence for a non-exchangeable ADP in the first nucleotide binding domain.

    Science.gov (United States)

    Ahn, Jinhi; Beharry, Seelochan; Molday, Laurie L; Molday, Robert S

    2003-10-10

    ABCR, also known as ABCA4, is a member of the superfamily of ATP binding cassette transporters that is believed to transport retinal or retinylidene-phosphatidylethanolamine across photoreceptor disk membranes. Mutations in the ABCR gene are responsible for Stargardt macular dystrophy and related retinal dystrophies that cause severe loss in vision. ABCR consists of two tandemly arranged halves each containing a membrane spanning segment followed by a large extracellular/lumen domain, a multi-spanning membrane domain, and a nucleotide binding domain (NBD). To define the role of each NBD, we examined the nucleotide binding and ATPase activities of the N and C halves of ABCR individually and co-expressed in COS-1 cells and derived from trypsin-cleaved ABCR in disk membranes. When disk membranes or membranes from co-transfected cells were photoaffinity labeled with 8-azido-ATP and 8-azido-ADP, only the NBD2 in the C-half bound and trapped the nucleotide. Co-expressed half-molecules displayed basal and retinal-stimulated ATPase activity similar to full-length ABCR. The individually expressed N-half displayed weak 8-azido-ATP labeling and low basal ATPase activity that was not stimulated by retinal, whereas the C-half did not bind ATP and exhibited little if any ATPase activity. Purified ABCR contained one tightly bound ADP, presumably in NBD1. Our results indicate that only NBD2 of ABCR binds and hydrolyzes ATP in the presence or absence of retinal. NBD1, containing a bound ADP, associates with NBD2 to play a crucial, non-catalytic role in ABCR function.

  2. Poly(ADP-ribose) polymerase-1 inhibits ATM kinase activity in DNA damage response

    International Nuclear Information System (INIS)

    Watanabe, Fumiaki; Fukazawa, Hidesuke; Masutani, Mitsuko; Suzuki, Hiroshi; Teraoka, Hirobumi; Mizutani, Shuki; Uehara, Yoshimasa

    2004-01-01

    DNA double-strand breaks (DSB) mobilize DNA-repair machinery and cell cycle checkpoint by activating the ataxia-telangiectasia (A-T) mutated (ATM). Here we show that ATM kinase activity is inhibited by poly(ADP-ribose) polymerase-1 (PARP-1) in vitro. It was shown by biochemical fractionation procedure that PARP-1 as well as ATM increases at chromatin level after induction of DSB with neocarzinostatin (NCS). Phosphorylation of histone H2AX on serine 139 and p53 on serine 15 in Parp-1 knockout (Parp-1 -/- ) mouse embryonic fibroblasts (MEF) was significantly induced by NCS treatment compared with MEF derived from wild-type (Parp-1 +/+ ) mouse. NCS-induced phosphorylation of histone H2AX on serine 139 in Parp-1 -/- embryonic stem cell (ES) clones was also higher than that in Parp-1 +/+ ES clone. Furthermore, in vitro, PARP-1 inhibited phosphorylation of p53 on serine 15 and 32 P-incorporation into p53 by ATM in a DNA-dependent manner. These results suggest that PARP-1 negatively regulates ATM kinase activity in response to DSB

  3. Analysis of poly(ADP-Ribose polymerases in Arabidopsis telomere biology.

    Directory of Open Access Journals (Sweden)

    Kara A Boltz

    Full Text Available Maintaining the length of the telomere tract at chromosome ends is a complex process vital to normal cell division. Telomere length is controlled through the action of telomerase as well as a cadre of telomere-associated proteins that facilitate replication of the chromosome end and protect it from eliciting a DNA damage response. In vertebrates, multiple poly(ADP-ribose polymerases (PARPs have been implicated in the regulation of telomere length, telomerase activity and chromosome end protection. Here we investigate the role of PARPs in plant telomere biology. We analyzed Arabidopsis thaliana mutants null for PARP1 and PARP2 as well as plants treated with the PARP competitive inhibitor 3-AB. Plants deficient in PARP were hypersensitive to genotoxic stress, and expression of PARP1 and PARP2 mRNA was elevated in response to MMS or zeocin treatment or by the loss of telomerase. Additionally, PARP1 mRNA was induced in parp2 mutants, and conversely, PARP2 mRNA was induced in parp1 mutants. PARP3 mRNA, by contrast, was elevated in both parp1 and parp2 mutants, but not in seedlings treated with 3-AB or zeocin. PARP mutants and 3-AB treated plants displayed robust telomerase activity, no significant changes in telomere length, and no end-to-end chromosome fusions. Although there remains a possibility that PARPs play a role in Arabidopsis telomere biology, these findings argue that the contribution is a minor one.

  4. Analysis of Poly(ADP-Ribose) Polymerases in Arabidopsis Telomere Biology

    Science.gov (United States)

    Townley, Jennifer M.; Shippen, Dorothy E.

    2014-01-01

    Maintaining the length of the telomere tract at chromosome ends is a complex process vital to normal cell division. Telomere length is controlled through the action of telomerase as well as a cadre of telomere-associated proteins that facilitate replication of the chromosome end and protect it from eliciting a DNA damage response. In vertebrates, multiple poly(ADP-ribose) polymerases (PARPs) have been implicated in the regulation of telomere length, telomerase activity and chromosome end protection. Here we investigate the role of PARPs in plant telomere biology. We analyzed Arabidopsis thaliana mutants null for PARP1 and PARP2 as well as plants treated with the PARP competitive inhibitor 3-AB. Plants deficient in PARP were hypersensitive to genotoxic stress, and expression of PARP1 and PARP2 mRNA was elevated in response to MMS or zeocin treatment or by the loss of telomerase. Additionally, PARP1 mRNA was induced in parp2 mutants, and conversely, PARP2 mRNA was induced in parp1 mutants. PARP3 mRNA, by contrast, was elevated in both parp1 and parp2 mutants, but not in seedlings treated with 3-AB or zeocin. PARP mutants and 3-AB treated plants displayed robust telomerase activity, no significant changes in telomere length, and no end-to-end chromosome fusions. Although there remains a possibility that PARPs play a role in Arabidopsis telomere biology, these findings argue that the contribution is a minor one. PMID:24551184

  5. Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin

    Energy Technology Data Exchange (ETDEWEB)

    Chassé, Maggie H.; Muthurajan, Uma M.; Clark, Nicholas J.; Kramer, Michael A.; Chakravarthy, Srinivas; Irving, Thomas; Luger, Karolin [Children; (IIT); (Colorado); (Amgen)

    2018-01-18

    Poly (ADP-Ribose) Polymerase I (PARP-1) is a first responder to DNA damage and participates in the regulation of gene expression. The interaction of PARP-1 with chromatin and DNA is complex and involves at least two different modes of interaction. In its enzymatically inactive state, PARP-1 binds native chromatin with similar affinity as it binds free DNA ends. Automodification of PARP-1 affects interaction with chromatin and DNA to different extents. Here we describe a series of biochemical and biophysical techniques to quantify and dissect the different binding modes of PARP-1 with its various substrates. The techniques listed here allow for high throughput and quantitative measurements of the interaction of different PARP-1 constructs (inactive and automodified) with chromatin and DNA damage models.

  6. Electrophoretic characterization of the Mammalian nuclear matrix proteome, nuclear envelope, nucleoli and covalently bound ADP-ribose polymers: potential applications to cancer.

    Science.gov (United States)

    Aranda, Xavier G; Racho, Ronald G; Pacheco-Rodríguez, Gustavo; Alvarez-González, Rafael

    2014-01-01

    Nucleic acid metabolism is biochemically compartmentalized to the nucleus. Thus, it is necessary to define the proteome of the various macromolecular structures within this organelle. We isolated the nuclear matrix (NM) fraction from rat liver by sequential centrifugation steps at 13,000 rpm, staggered between endogenous nuclease treatment for 2 h at 37°C, followed by high-salt (H.S.; 2.0 M NaCl) and non-ionic detergent extractions (0.1%- or 1.0% Triton X-100) to eliminate the bulk of chromosomal DNA/RNA, histone proteins and the nuclear envelope (NE). Integrity of the NM and NE structures was confirmed by electron microscopy. Next, we analyzed the NM proteome on a 20% polyacrylamide gel using the PhastSystem. We observed the absence of histone proteins and the characteristic presence of the lamins by Coomassie blue staining. By contrast, upon silver staining, following electrophoretic separation with a Tris-Borate-EDTA buffer, we observed the NM-associated nucleic RNA and protein-free ADP-ribose polymers. While polymers are found in much lower concentration than RNA in NM, they were purified by affinity chromatography on boronate resin prior to electrophoresis. We observed the electrophoretic resolution of free ADP-ribose chains (5-25 units) by silver staining. The significance of our observations to cancer studies and carcinogenesis is discussed. Copyright© 2014, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

  7. Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP- and thrombin-induced human platelet activation

    Science.gov (United States)

    Nylander, Sven; Mattsson, Christer; Ramström, Sofia; Lindahl, Tomas L

    2004-01-01

    The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y12 and P2Y1 inhibition and P2Y12 and thrombin inhibition, on ADP- and thrombin-induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both Gαq and Gαi signalling.Blood from healthy volunteers was preincubated with AR-C69931MX, a reversible P2Y12 antagonist; MRS2179, a reversible P2Y1 antagonist; or melagatran, a direct reversible thrombin inhibitor; alone or in various combinations prior to activation with ADP or thrombin. Platelet function in whole blood was assessed by flow cytometry using the antibody PAC-1 to estimate the expression of active αIIbβ3 (the fibrinogen receptor GPIIb/IIIa). A synergistic effect was evaluated by comparing the concentrations in the different combinations with those of corresponding equipotent concentrations of each single inhibitor alone. The equipotent single concentrations were experimentally obtained from concentration response curves performed in parallel.A synergistic effect regarding inhibition of ADP-induced platelet activation (10 μM) was obtained with different combinations of AR-C69931MX and MRS2179.Inhibition of thrombin-induced platelet activation (2 nM) with combinations of AR-C69931MX and the thrombin inhibitor melagatran did also result in a strong synergistic effect.To our knowledge, this is the first time that data supporting a synergistic effect has been published for the inhibitor combinations described.Whether this synergistic effect in vitro also results in an improved antithrombotic effect in vivo with or without an increased risk of bleeding remains to be studied in well-conducted clinical studies. PMID:15265806

  8. Triplicate genes for mitochondrial ADP/ATP carriers in the aerobic yeast Yarrowia lipolytica are regulated differentially in the absence of oxygen

    DEFF Research Database (Denmark)

    Mentel, M.; Piskur, Jure; Neuveglise, C.

    2005-01-01

    Yarrowia lipolytica is a strictly aerobic fungus, which differs from the extensively studied model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe with respect to its physiology, genetics and dimorphic growth habit. We isolated and sequenced cDNA and genomic clones (YlAAC1) from Y....... lipolytica that encode a mitochondrial ADP/ATP carrier. The YlAAC1 gene can complement the S. cerevisiae Delta aac2 deletion mutant. Southern hybridization, analysis of Yarrowia clones obtained in the course of the Genolevures project, and further sequencing revealed the existence of two paralogs of the Yl...

  9. Analysis of the influence of nucleotidases on the apparent activity of exogenous ATP and ADP at P2Y1 receptors

    OpenAIRE

    Vigne, Paul; Philippe Breittmayer, Jean; Frelin, Christian

    1998-01-01

    ADP is a potent agonist of rat and human P2Y1 purinoceptors. ATP is a weak competitive antagonist. This study analyses the situation in which P2Y1 receptors are exposed to ATP in the presence of exogenous ecto-nucleotidases (apyrases) that have high or low ATPase/ADPase activity ratio.Rat brain capillary endothelial cells of the B10 clone express P2Y1 receptors that couple to intracellular Ca2+ mobilization. They have low endogenous ecto-ATPase and ecto-ADPase activities.ATP did not raise int...

  10. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

    Science.gov (United States)

    Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Gomibuchi, Yuki [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Uyeda, Taro Q.P. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Department of Judo Therapy, Faculty of Medical Technology, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan)

    2013-11-29

    ) of the replaced amino acid molecule. Because 1/K{sub app} reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.

  12. Inhibitors of poly (ADP-ribose) polymerase and their enhancement of alkylating agent cytotoxicity in vivo

    International Nuclear Information System (INIS)

    Horsman, M.R.; Brown, D.M.; Hirst, D.G.; Brown, J.M.

    1984-01-01

    The chromosomal enzyme poly (ADP-ribose) polymerase (ADPRP) is involved in the repair of DNA damage caused by both ionizing radiation and alkylating agents. The authors have shown that certain inhibitors of this enzyme decrease potentially lethal damage repair after X-rays. The aim of the present study was to investigate the possible enhancement of alkylating agent damage in vivo by several of these ADPRP inhibitors. 3-aminobenzamide (200 mg/kg), caffeine (200 mg/kg), or nicotinamide (1000 mg/kg) given to RIF-1-tumor-bearing mice immediately before a dose of melphalan (L-PAM) (8 mg/kg) produced enhancement of tumor response as demonstrated by an in vivo in vitro tumor excision assay. Caffeine and nicotinamide provided the greatest enhancement of L-PAM cytotoxicity with at least a 100-fold increase in killing. Data are presented on the mechanism by which these drugs and other more potent inhibitors enhance the tumor cell killing by L-PAM and other alkylating agents

  13. The in vitro screening of aromatic amides as potential inhibitors of poly (ADP-ribose) polymerase

    International Nuclear Information System (INIS)

    Brown, D.M.; Horsman, M.R.; Lee, W.W.; Brown, J.M.

    1984-01-01

    It is now well established that the chromosomal enzyme poly (ADP-ribose) polymerase (ADPRP) is involved in the repair of DNA damage caused by ionizing radiation and alkylating agents, although the mechanisms involved are still not clear. ADPRP inhibitors include thymidine, nicotinamides, benzamides and methyl xanthines. The authors have demonstrated that these compounds are effective inhibitors of X-ray-induced potentially lethal damage repair (PLDR). More recently, they have shown that the cytotoxicity of the bifunctional alkylating L-phenylalanine mustard (L-PAM) was enhanced in vitro and in vivo by 3-aminobenzamide, nicotinamide and caffeine, although in the latter case pharmacokinetic changes could have contributed to the enhanced killing. The authors have examined a series of substituted carbocyclic and heterocyclic aromatic amides as potential inhibitors of ADPRP. The effect of these compounds on ADPRP activity in vitro as well as their effect on the repair of X-ray and alkylation damage in vitro are presented

  14. Differential Role of Poly(ADP-ribose polymerase in D. discoideum growth and development

    Directory of Open Access Journals (Sweden)

    Begum Rasheedunnisa

    2011-03-01

    Full Text Available Abstract Background Poly(ADP-ribose polymerase is evolutionarily conserved as a responder to various forms of stress. Though PARP's role in cell death is well addressed, its role in development and multicellularity is still an enigma. We have previously reported the role of PARP in oxidative stress induced delayed development of D. discoideum. Results In the current study we highlight the involvement of PARP during D. discoideum development. Oxidative stress affects expression of aca and cAR1 thus affecting aggregation. Although parp expression is not affected during oxidative stress but it is involved during normal development as confirmed by our PARP down-regulation studies. Constitutive PARP down-regulation resulted in blocked development while no effect was observed on D. discoideum growth. Interestingly, stage specific PARP down-regulation arrested development at the slug stage. Conclusion These results emphasize that PARP is essential for complex differentiation and its function may be linked to multicellularity. This is the first report where the involvement of PARP during normal multicellular development in D. discoideum, an ancient eukaryote, is established which could be of evolutionary significance. Thus our study adds one more role to the multitasking function of PARP.

  15. Properties of the manganese(II) binding site in ternary complexes of Mnter dot ADP and Mnter dot ATP with chloroplast coupling factor 1: Magnetic field dependence of solvent sup 1 H and sup 2 H NMR relaxation rates

    Energy Technology Data Exchange (ETDEWEB)

    Haddy, A.E.; Frasch, W.D.; Sharp, R.R. (Univ. of Michigan, Ann Arbor (USA))

    1989-05-02

    The influence of the binding of ADP and ATP on the high-affinity Mn(II) binding site of chloroplast coupling factor 1 (CF{sub 1}) was studied by analysis of field-dependent solvent proton and deuteron spin-lattice relaxation data. In order to characterize metal-nucleotide complexes of CF{sub 1} under conditions similar to those of the NMR experiments, the enzyme was analyzed for bound nucleotides and Mn(II) after incubation with AdN and MnCl{sub 2} and removal of labile ligands by extensive gel filtration chromatography. In the field-dependent NMR experiments, the Mn(II) binding site of CF{sub 1} was studied for three mole ratios of added Mn(II) to CF{sub 1}, 0.5, 1.0, and 1.5, in the presence of an excess of either ADP or ATP. The results were extrapolated to zero Mn(II) concentration to characterize the environment of the first Mn(II) binding site of Cf{sub 1}. In the presence of both adenine nucleotides, pronounced changes in the Mn(II) environment relative to that in Mn(II)-CF{sub 1} were evident; the local relaxation rate maxima were more pronounced and shifted to higher field strengths, and the relaxation rate per bound Mn(II) increased at all field strengths. Analysis of the data revealed that the number of exchangeable water molecules liganded to bound Mn(II) increased from one in the binary Mn(II)-CF{sub 1} complex to three and two in the ternary Mn(II)-ADP-CF{sub 1} and Mn(II)-ATP-CF{sub 1} complexes, respectively; these results suggest that a water ligand to bound Mn(II) in the Mn(II)-ADP-CF{sub 1} complex is replaced by the {gamma}-phosphate of ATP in the Mn(II)-ATP-CF{sub 1} complex. A binding model is presented to account for these observations.

  16. Poly(ADP-ribose polymerase inhibition reveals a potential mechanism to promote neuroprotection and treat neuropathic pain

    Directory of Open Access Journals (Sweden)

    Prashanth Komirishetty

    2016-01-01

    Full Text Available Neuropathic pain is triggered by the lesions to peripheral nerves which alter their structure and function. Neuroprotective approaches that limit the pathological changes and improve the behavioral outcome have been well explained in different experimental models of neuropathy but translation of such strategies to clinics has been disappointing. Experimental evidences revealed the role of free radicals, especially peroxynitrite after the nerve injury. They provoke oxidative DNA damage and consequent over-activation of the poly(ADP-ribose polymerase (PARP upregulates pro-inflammatory pathways, causing bioenergetic crisis and neuronal death. Along with these changes, it causes mitochondrial dysfunction leading to neuronal apoptosis. In related preclinical studies agents that neutralize the free radicals and pharmacological inhibitors of PARP have shown benefits in treating experimental neuropathy. This article reviews the involvement of PARP over-activation in trauma induced neuropathy and therapeutic significance of PARP inhibitors in the experimental neuropathy and neuropathic pain.

  17. Poly(ADP-ribose) polymerase inhibition reveals a potential mechanism to promote neuroprotection and treat neuropathic pain.

    Science.gov (United States)

    Komirishetty, Prashanth; Areti, Aparna; Gogoi, Ranadeep; Sistla, Ramakrishna; Kumar, Ashutosh

    2016-10-01

    Neuropathic pain is triggered by the lesions to peripheral nerves which alter their structure and function. Neuroprotective approaches that limit the pathological changes and improve the behavioral outcome have been well explained in different experimental models of neuropathy but translation of such strategies to clinics has been disappointing. Experimental evidences revealed the role of free radicals, especially peroxynitrite after the nerve injury. They provoke oxidative DNA damage and consequent over-activation of the poly(ADP-ribose) polymerase (PARP) upregulates pro-inflammatory pathways, causing bioenergetic crisis and neuronal death. Along with these changes, it causes mitochondrial dysfunction leading to neuronal apoptosis. In related preclinical studies agents that neutralize the free radicals and pharmacological inhibitors of PARP have shown benefits in treating experimental neuropathy. This article reviews the involvement of PARP over-activation in trauma induced neuropathy and therapeutic significance of PARP inhibitors in the experimental neuropathy and neuropathic pain.

  18. 78 FR 2955 - Marine Mammals; File Nos. 14451, 14353, and 13846

    Science.gov (United States)

    2013-01-15

    ..., Oakland, CA 94611; and Jim Darling, Ph.D., Whale Trust, P.O. Box 384, Tofino, BC V0R2Z0, Canada, have... area throughout the Hawaiian Islands, including the Northwestern Hawaiian Islands (NWHI) chain. Permit...., Whale Trust, P.O. Box 384, Tofino, BC V0R2Z0, Canada, to study humpback whales in Hawaii (primarily off...

  19. Poly(ADP-RibosePolymerase-1 in Lung Inflammatory Disorders: A Review

    Directory of Open Access Journals (Sweden)

    Gurupreet S. Sethi

    2017-09-01

    Full Text Available Asthma, acute lung injury (ALI, and chronic obstructive pulmonary disease (COPD are lung inflammatory disorders with a common outcome, that is, difficulty in breathing. Corticosteroids, a class of potent anti-inflammatory drugs, have shown less success in the treatment/management of these disorders, particularly ALI and COPD; thus, alternative therapies are needed. Poly(ADP-ribosepolymerases (PARPs are the post-translational modifying enzymes with a primary role in DNA repair. During the last two decades, several studies have reported the critical role played by PARPs in a good of inflammatory disorders. In the current review, the studies that address the role of PARPs in asthma, ALI, and COPD have been discussed. Among the different members of the family, PARP-1 emerges as a key player in the orchestration of lung inflammation in asthma and ALI. In addition, PARP activation seems to be associated with the progression of COPD. Furthermore, PARP-14 seems to play a crucial role in asthma. STAT-6 and GATA-3 are reported to be central players in PARP-1-mediated eosinophilic inflammation in asthma. Interestingly, oxidative stress–PARP-1–NF-κB axis appears to be tightly linked with inflammatory response in all three-lung diseases despite their distinct pathophysiologies. The present review sheds light on PARP-1-regulated factors, which may be common or differential players in asthma/ALI/COPD and put forward our prospective for future studies.

  20. Poly(ADP-Ribose) Polymerase-1: A Novel Therapeutic Target in Necrotizing Enterocolitis

    Science.gov (United States)

    Giannone, Peter J.; Alcamo, Alicia A.; Schanbacher, Brandon L.; Nankervis, Craig A.; Besner, Gail E.; Bauer, John A.

    2011-01-01

    Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of infancy, afflicting 11% of infants born 22–28 weeks gestational age. Both inflammation and oxidation may be involved in NEC pathogenesis through reactive nitrogen species production, protein oxidation and DNA damage. Poly(ADP-ribose) polymerase-1 (PARP-1) is a critical enzyme activated to facilitate DNA repair using nicotinamide adenine dinucleotide (NAD+) as a substrate. However, in the presence of severe oxidative stress and DNA damage, PARP-1 over-activation may ensue, depleting cells of NAD+ and ATP, killing them by metabolic catastrophe. Here we tested the hypothesis that NO dysregulation in intestinal epithelial cells during NEC leads to marked PARP-1 expression and that administration of a PARP-1 inhibitor (nicotinamide) attenuates intestinal injury in a newborn rat model of NEC. In this model, 56% of control pups developed NEC (any stage), versus 14% of pups receiving nicotinamide. Forty-four percent of control pups developed high-grade NEC (grades 3–4), whereas only 7% of pups receiving nicotinamide developed high-grade NEC. Nicotinamide treatment protects pups against intestinal injury incurred in the newborn rat NEC model. We speculate that PARP-1 over-activation in NEC may drive mucosal cell death in this disease and that PARP-1 may be a novel therapeutic target in NEC. PMID:21399558

  1. Reduced estradiol-induced vasodilation and poly-(ADP-ribose) polymerase (PARP) activity in the aortas of rats with experimental polycystic ovary syndrome (PCOS).

    Science.gov (United States)

    Masszi, Gabriella; Horvath, Eszter Maria; Tarszabo, Robert; Benko, Rita; Novak, Agnes; Buday, Anna; Tokes, Anna-Maria; Nadasy, Gyorgy L; Hamar, Peter; Benyó, Zoltán; Varbiro, Szabolcs

    2013-01-01

    Polycystic ovary syndrome (PCOS) is a complex endocrine disorder characterized by hyperandrogenism and insulin resistance, both of which have been connected to atherosclerosis. Indeed, an increased risk of clinical manifestations of arterial vascular diseases has been described in PCOS. On the other hand endothelial dysfunction can be detected early on, before atherosclerosis develops. Thus we assumed that vascular dysfunction is also related directly to the hormonal imbalance rather than to its metabolic consequences. To detect early functional changes, we applied a novel rodent model of PCOS: rats were either sham operated or hyperandrogenism was achieved by implanting subcutaneous pellets of dihydrotestosterone (DHT). After ten weeks, myograph measurements were performed on isolated aortic rings. Previously we described an increased contractility to norepinephrine (NE). Here we found a reduced immediate relaxation to estradiol treatment in pre-contracted aortic rings from hyperandrogenic rats. Although the administration of vitamin D3 along with DHT reduced responsiveness to NE, it did not restore relaxation to estradiol. Poly-(ADP-ribose) polymerase (PARP) activity was assessed by poly-ADP-ribose immunostaining. Increased PAR staining in ovaries and circulating leukocytes from DHT rats showed enhanced DNA damage, which was reduced by concomitant vitamin D3 treatment. Surprisingly, PAR staining was reduced in both the endothelium and vascular smooth muscle cells of the aorta rings from hyperandrogenic rats. Thus in the early phase of PCOS, vascular tone is already shifted towards vasoconstriction, characterized by reduced vasorelaxation and vascular dysfunction is concomitant with altered PARP activity. Based on our findings, PARP inhibitors might have a future perspective in restoring metabolic disorders in PCOS.

  2. The involvement of poly(ADP-ribose) polymerase in the degradation of NAD caused by γ-radiation and N-methyl-N-nitrosourea

    International Nuclear Information System (INIS)

    Skidmore, C.J.; Davies, M.I.; Goodwin, P.M.; Halldorsson, H.; Lewis, P.J.; Shall, S.; Zia'ee, A.

    1979-01-01

    Both N-methyl-N-nitrosourea and γ-radiation lower cellular NAD in mouse leukaemia cells (L1210) in a dose-dependent way. The minimum NAD level is reached 2 h after a brief exposure to N-methyl-N-nitrosourea, but within 15 min of γ-irradiation. The cells remain metabolically active; they are able to recover their control NAD levels and are impermeable to trypan blue. Several inhibitors of poly(ADP-ribose) polymerase inhibit the drop in cellular NAD caused by these two agents: 2 mM 5-methylnicotinamide, 1 mM theophylline or 1 mM theobromine inhibit the effect of N-methyl-N-nitrosourea on cellular NAD level; 200 μM thymidine, 500 μM 5-methylnicotinaminde, 500 μM thephylline and 500 μM theobromine prevent the lowering of cellular NAD by γ-irradiation. The extent to which the drop in cellular NAD is inhibited is dependent on both the concentration of cytotoxic agent and of polymerase inhibitor. Caffeine will inhibit the drop in NAD but only at 10 mM, while nicotonic acid is ineffictive even at this dose. The activity of poly(ADP-ribose) polymerase is permeabilized cells immediately after γ-radiation increases with dose up to 12 krad, giving a maximal 3.4-fold stimulation of the enzyme activity, whereas the degradation of NAD under conditions optimal for NAD glycohydrolase does not change. The activity of the polymerase shows a close temporal correlation with the NAD drop following both γ-radiation and N-methyl-N-nitrosourea. The enzyme activity is maximal when the NAD content. (orig./AJ) 891 AJ/orig.- 892 HIS [de

  3. Involvement of poly(ADP-ribose polymerase-1 in development of spinal cord injury in Chinese individuals: a Chinese clinical study

    Directory of Open Access Journals (Sweden)

    Meng Q

    2017-12-01

    Full Text Available Qing-Tao Meng,* Guang Yang,* Ren-Bo Li, Jing-Xin Nie, Wei Zhou, Hong-De Yu, Bo Chen, Li Jiang, Jing-Bo Shang Department of Spine Surgery, The Third People’s Hospital of Dalian, Dalian, People’s Republic of China *These authors contributed equally to this work Objective: We aimed to evaluate whether the polymorphism of poly(ADP-ribose polymerase-1 (PARP-1 is involved as potential risk factor in the development of spinal cord injury (SCI among Chinese individuals.Patients and methods: Patients with a confirmed diagnosis of SCI (other than traumatic injury and healthy individuals with no clinical symptoms of SCI were enrolled at Spinal Cord Injury Care Center, The Third People’s Hospital of Dalian, China. Genetic polymorphisms were studied in plasma samples by polymerase chain reaction-restriction fragment length polymorphism assay.Results: A total of 130 Chinese patients with SCI and 130 healthy Chinese individuals were included. We found that patients with the GG genotype (odds ratio [OR]: 4.09, 95% confidence interval [CI] 2.42–6.90, P<0.001 and carriers of the G allele (OR 3.96, 95% CI 2.33–6.74, P<0.0001 were at high risk of developing SCI. A del/ins polymorphism of the NF-κB1 gene (OR 3.32, 95% CI 1.96–5.61, P<0.001 was also found to be associated with SCI.Conclusion: Our study suggests that PARP-1 polymorphisms are involved in the development of SCI in Chinese individuals. Thus, PARP-1 polymorphisms can be considered as one of the potential risk factors for developing SCI. Keywords: spinal cord injury, poly(ADP-ribose polymerase-1, polymorphism 

  4. Interplay between Ubiquitin, SUMO, and Poly(ADP-Ribose) in the Cellular Response to Genotoxic Stress

    Science.gov (United States)

    Pellegrino, Stefania; Altmeyer, Matthias

    2016-01-01

    Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. PMID:27148359

  5. Poly ADP-ribose polymerase-1 as a potential therapeutic target in Merkel cell carcinoma.

    Science.gov (United States)

    Ferrarotto, Renata; Cardnell, Robert; Su, Shirley; Diao, Lixia; Eterovic, A Karina; Prieto, Victor; Morrisson, William H; Wang, Jing; Kies, Merrill S; Glisson, Bonnie S; Byers, Lauren Averett; Bell, Diana

    2018-03-23

    Patients with metastatic Merkel cell carcinoma are treated similarly to small cell lung cancer (SCLC). Poly ADP-ribose polymerase-1 (PARP1) is overexpressed in SCLC and response to PARP inhibitors have been reported in patients with SCLC. Our study explores PARP as a therapeutic target in Merkel cell carcinoma. We evaluated PARP1 expression and Merkel cell polyomavirus (MCPyV) in 19 patients with Merkel cell carcinoma. Target exome-sequencing was performed in 14 samples. Sensitivity to olaparib was tested in 4 Merkel cell carcinoma cell lines. Most Merkel cell carcinomas (74%) express PARP1 at high levels. Mutations in DNA-damage repair genes were identified in 9 samples (64%), occurred exclusively in head neck primaries, and correlated with TP53/RB1 mutations. The TP53/RB1 mutations were more frequent in MCPyV-negative tumors. Sensitivity to olaparib was seen in the Merkel cell carcinoma line with highest PARP1 expression. Based on PARP1 overexpression, DNA-damage repair gene mutations, platinum sensitivity, and activity of olaparib in a Merkel cell carcinoma line, clinical trials with PARP inhibitors are warranted in Merkel cell carcinoma. © 2018 Wiley Periodicals, Inc.

  6. Accounting for subgroup structure in line-transect abundance estimates of false killer whales (Pseudorca crassidens in Hawaiian waters.

    Directory of Open Access Journals (Sweden)

    Amanda L Bradford

    Full Text Available For biological populations that form aggregations (or clusters of individuals, cluster size is an important parameter in line-transect abundance estimation and should be accurately measured. Cluster size in cetaceans has traditionally been represented as the total number of individuals in a group, but group size may be underestimated if group members are spatially diffuse. Groups of false killer whales (Pseudorca crassidens can comprise numerous subgroups that are dispersed over tens of kilometers, leading to a spatial mismatch between a detected group and the theoretical framework of line-transect analysis. Three stocks of false killer whales are found within the U.S. Exclusive Economic Zone of the Hawaiian Islands (Hawaiian EEZ: an insular main Hawaiian Islands stock, a pelagic stock, and a Northwestern Hawaiian Islands (NWHI stock. A ship-based line-transect survey of the Hawaiian EEZ was conducted in the summer and fall of 2010, resulting in six systematic-effort visual sightings of pelagic (n = 5 and NWHI (n = 1 false killer whale groups. The maximum number and spatial extent of subgroups per sighting was 18 subgroups and 35 km, respectively. These sightings were combined with data from similar previous surveys and analyzed within the conventional line-transect estimation framework. The detection function, mean cluster size, and encounter rate were estimated separately to appropriately incorporate data collected using different methods. Unlike previous line-transect analyses of cetaceans, subgroups were treated as the analytical cluster instead of groups because subgroups better conform to the specifications of line-transect theory. Bootstrap values (n = 5,000 of the line-transect parameters were randomly combined to estimate the variance of stock-specific abundance estimates. Hawai'i pelagic and NWHI false killer whales were estimated to number 1,552 (CV = 0.66; 95% CI = 479-5,030 and 552 (CV = 1.09; 95% CI = 97

  7. Formation of PI 3-kinase products in platelets by thrombin, but not collagen, is dependent on synergistic autocrine stimulation, particularly through secreted ADP.

    Science.gov (United States)

    Selheim, F; Idsøe, R; Fukami, M H; Holmsen, H; Vassbotn, F S

    1999-10-05

    Platelet activation by thrombin or collagen results in secretion and synthesis of several platelet agonists that enhance the responses to the primary agonists (autocrine stimulation). To disclose the effects of thrombin and collagen on the phosphorylation of 3-phosphoinositides per se we incubated platelets with five inhibitors of platelet autocrine stimulation (IAS) that act extracellularly. We found that IAS almost totally blocked thrombin-induced production of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. In contrast, collagen induced massive production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) in the presence of IAS. When testing the effect of each inhibitor individually we found the strongest inhibition of thrombin-induced PtdIns(3,4)P(2) production with the ADP scavenger system CP/CPK. Furthermore, we found a strong synergistic effect between exogenously added ADP and thrombin on production of PtdIns(3,4)P(2). In contrast to the results from 3-phosphorylated phosphoinositides, CP/CPK had little effect on thrombin-induced protein tyrosine phosphorylation. Our results show the importance of autocrine stimulation in thrombin-induced accumulation of 3-phosphorylated phosphoinositides and raise the question as to whether thrombin by itself is capable of inducing PI 3-K activation. In marked contrast to thrombin, collagen per se appears to be able to trigger increased production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). Copyright 1999 Academic Press.

  8. Poly(ADP-ribose) polymerase-1 and its cleavage products differentially modulate cellular protection through NF-kB-dependent signaling

    Science.gov (United States)

    Castri, Paola; Lee, Yang-ja; Ponzio, Todd; Maric, Dragan; Spatz, Maria; Bembry, Joliet; Hallenbeck, John

    2014-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) and its cleavage products regulate cell viability and NF-kB activity when expressed in neurons. PARP-1 cleavage generates a 24kDa (PARP-124) and an 89kDa fragment (PARP-189). Compared to WT (PARP-1WT), the expression of an uncleavable PARP-1 (PARP-1UNCL) or of PARP-124 conferred protection from oxygen/glucose deprivation (OGD) or OGD/restoration of oxygen and glucose (ROG) damage in vitro, whereas expression of PARP-189 was cytotoxic. Viability experiments were performed in SH-SY5Y, a human neuroblastoma cell line, as well as in rat primary cortical neurons. Following OGD, the higher viability in the presence of PARP-1UNCL or PARP-124 was not accompanied with decreased formation of poly(ADP-riboses) or higher NAD levels. PARP-1 is a known cofactor for NF-kB, hence we investigated whether PARP-1 cleavage influences the inflammatory response. All PARP-1 constructs mimicked PARP-1WT in regards to induction of NF-kB translocation into the nucleus and its increased activation during ischemic challenge. However, expression of PARP-189 construct induced significantly higher NF-kB activity than PARP-1WT; and the same was true for NF-kB-dependent iNOS promoter binding activity. At a protein level, PARP-1UNCL and PARP-124 decreased iNOS (and lower levels of iNOS transcript) and COX-2, and increased Bcl-xL. The increased levels of NF-kB and iNOS transcriptional activities, seen with cytotoxic PARP-189, were accompanied by higher protein expression of COX-2 and iNOS (and higher levels of iNOS transcript) and lower protein expression of Bcl-xL. Taken together, these findings suggest that PARP-1 cleavage products may regulate cellular viability and inflammatory responses in opposing ways during in vitro models of “ischemia”. PMID:24333653

  9. Reduced estradiol-induced vasodilation and poly-(ADP-ribose polymerase (PARP activity in the aortas of rats with experimental polycystic ovary syndrome (PCOS.

    Directory of Open Access Journals (Sweden)

    Gabriella Masszi

    Full Text Available Polycystic ovary syndrome (PCOS is a complex endocrine disorder characterized by hyperandrogenism and insulin resistance, both of which have been connected to atherosclerosis. Indeed, an increased risk of clinical manifestations of arterial vascular diseases has been described in PCOS. On the other hand endothelial dysfunction can be detected early on, before atherosclerosis develops. Thus we assumed that vascular dysfunction is also related directly to the hormonal imbalance rather than to its metabolic consequences. To detect early functional changes, we applied a novel rodent model of PCOS: rats were either sham operated or hyperandrogenism was achieved by implanting subcutaneous pellets of dihydrotestosterone (DHT. After ten weeks, myograph measurements were performed on isolated aortic rings. Previously we described an increased contractility to norepinephrine (NE. Here we found a reduced immediate relaxation to estradiol treatment in pre-contracted aortic rings from hyperandrogenic rats. Although the administration of vitamin D3 along with DHT reduced responsiveness to NE, it did not restore relaxation to estradiol. Poly-(ADP-ribose polymerase (PARP activity was assessed by poly-ADP-ribose immunostaining. Increased PAR staining in ovaries and circulating leukocytes from DHT rats showed enhanced DNA damage, which was reduced by concomitant vitamin D3 treatment. Surprisingly, PAR staining was reduced in both the endothelium and vascular smooth muscle cells of the aorta rings from hyperandrogenic rats. Thus in the early phase of PCOS, vascular tone is already shifted towards vasoconstriction, characterized by reduced vasorelaxation and vascular dysfunction is concomitant with altered PARP activity. Based on our findings, PARP inhibitors might have a future perspective in restoring metabolic disorders in PCOS.

  10. Diphtheria toxin- and Pseudomonas A toxin-mediated apoptosis. ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumor necrosis factor-alpha.

    Science.gov (United States)

    Morimoto, H; Bonavida, B

    1992-09-15

    We have reported that diphtheria toxin (DTX) mediates target cell lysis and intranucleosomal DNA fragmentation (apoptosis) and also synergizes with TNF-alpha. In this paper, we examined which step in the pathway of DTX-mediated inhibition of protein synthesis was important for induction of cytolytic activity and for synergy. Using a DTX-sensitive tumor cell line, we first examined the activity of the mutant CRM 197, which does not catalyze the ADP ribosylation of elongation factor-2 (EF-2). CRM 197 was not cytolytic for target cells and did not mediate intranucleosomal DNA fragmentation of viable cells. The failure of CRM 197 to mediate target cell lysis suggested that the catalytic activity of DTX is prerequisite for target cell lysis. This was corroborated by demonstrating that MeSAdo, which blocks the biosynthesis of diphthamide, inhibited DTX-mediated protein synthesis inhibition and also blocked target cell lysis. Furthermore, the addition of nicotinamide, which competes with NAD+ on the DTX action site of EF-2, also blocked DTX-mediated lysis. These findings suggest that ADP-ribosylation of EF-2 may be a necessary step in the pathway leading to target cell lysis. In contrast to the sensitive line, the SKOV-3 tumor cell line is sensitive to protein synthesis inhibition by DTX but is not susceptible to cytolysis and apoptosis by DTX. Thus, protein synthesis inhibition by DTX is not sufficient to mediate target cell lysis. The synergy in cytotoxicity obtained with the combination of DTX and TNF-alpha was examined in order to determine the pathway mediated by DTX in synergy. Like the direct lysis by DTX, synergy was significantly reduced by MeSAdo and by nicotinamide. Furthermore, synergy was not observed with combination of CRM 197 and TNF-alpha. These results demonstrate that, in synergy, DTX may utilize the same pathway required for its cytolytic activity. Pseudomonas aeruginosa exotoxin shared most the properties shown for DTX. Altogether, these findings

  11. A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin

    Science.gov (United States)

    Ernst, Katharina; Liebscher, Markus; Mathea, Sebastian; Granzhan, Anton; Schmid, Johannes; Popoff, Michel R.; Ihmels, Heiko; Barth, Holger; Schiene-Fischer, Cordelia

    2016-01-01

    Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins. PMID:26839186

  12. Oxidative stress, metabolomics profiling, and mechanism of local anesthetic induced cell death in yeast

    Directory of Open Access Journals (Sweden)

    Cory H.T. Boone

    2017-08-01

    Full Text Available The World Health Organization designates lidocaine as an essential medicine in healthcare, greatly increasing the probability of human exposure. Its use has been associated with ROS generation and neurotoxicity. Physiological and metabolomic alterations, and genetics leading to the clinically observed adverse effects have not been temporally characterized. To study alterations that may lead to these undesirable effects, Saccharomyces cerevisiae grown on aerobic carbon sources to stationary phase was assessed over 6 h. Exposure of an LC50 dose of lidocaine, increased mitochondrial depolarization and ROS/RNS generation assessed using JC-1, ROS/RNS specific probes, and FACS. Intracellular calcium also increased, assessed by ICP-MS. Measurement of the relative ATP and ADP concentrations indicates an initial 3-fold depletion of ATP suggesting an alteration in the ATP:ADP ratio. At the 6 h time point the lidocaine exposed population contained ATP concentrations roughly 85% that of the negative control suggesting the surviving population adapted its metabolic pathways to, at least partially restore cellular bioenergetics. Metabolite analysis indicates an increase of intermediates in the pentose phosphate pathway, the preparatory phase of glycolysis, and NADPH. Oxidative stress produced by lidocaine exposure targets aconitase decreasing its activity with an observed decrease in isocitrate and an increase citrate. Similarly, increases in α-ketoglutarate, malate, and oxaloacetate imply activation of anaplerotic reactions. Antioxidant molecule glutathione and its precursor amino acids, cysteine and glutamate were greatly increased at later time points. Phosphatidylserine externalization suggestive of early phase apoptosis was also observed. Genetic studies using metacaspase null strains showed resistance to lidocaine induced cell death. These data suggest lidocaine induces perpetual mitochondrial depolarization, ROS/RNS generation along with increased

  13. Activation of Adiponectin Receptor Regulates Proprotein Convertase Subtilisin/Kexin Type 9 Expression and Inhibits Lesions in ApoE-Deficient Mice.

    Science.gov (United States)

    Sun, Lei; Yang, Xiaoxiao; Li, Qi; Zeng, Peng; Liu, Ying; Liu, Lipei; Chen, Yuanli; Yu, Miao; Ma, Chuanrui; Li, Xiaoju; Li, Yan; Zhang, Rongxin; Zhu, Yan; Miao, Qing Robert; Han, Jihong; Duan, Yajun

    2017-07-01

    The reduced adiponectin levels are associated with atherosclerosis. Adiponectin exerts its functions by activating adiponectin receptor (AdipoR). Proprotein convertase subtilisin kexin type 9 (PCSK9) degrades LDLR protein (low-density lipoprotein receptor) to increase serum LDL-cholesterol levels. PCSK9 expression can be regulated by PPARγ (peroxisome proliferator-activated receptor γ) or SREBP2 (sterol regulatory element-binding protein 2). The effects of AdipoR agonists on PCSK9 and LDLR expression, serum lipid profiles, and atherosclerosis remain unknown. At cellular levels, AdipoR agonists (ADP355 and AdipoRon) induced PCSK9 transcription/expression that solely depended on activation of PPAR-responsive element in the PCSK9 promoter. AdipoR agonists induced PPARγ expression; thus, the AdipoR agonist-activated PCSK9 expression/production was impaired in PPARγ deficient hepatocytes. Meanwhile, AdipoR agonists transcriptionally activated LDLR expression by activating SRE in the LDLR promoter. Moreover, AMP-activated protein kinase α (AMPKα) was involved in AdipoR agonist-activated PCSK9 expression. In wild-type mice, ADP355 increased PCSK9 and LDLR expression and serum PCSK9 levels, which was associated with activation of PPARγ, AMPKα and SREBP2 and reduction of LDL-cholesterol levels. In contrast, ADP355 reduced PCSK9 expression/secretion in apoE-deficient (apoE -/- ) mice, but it still activated hepatic LDLR, PPARγ, AMPKα, and SREBP2. More importantly, ADP355 inhibited lesions in en face aortas and sinus lesions in aortic root in apoE -/- mice with amelioration of lipid profiles. Our study demonstrates that AdipoR activation by agonists regulated PCSK9 expression differently in wild-type and apoE -/- mice. However, ADP355 activated hepatic LDLR expression and ameliorated lipid metabolism in both types of mice and inhibited atherosclerosis in apoE -/- mice. © 2017 American Heart Association, Inc.

  14. Effect of Tomato Industrial Processing on Phenolic Profile and Antiplatelet Activity

    Directory of Open Access Journals (Sweden)

    Iván Palomo

    2013-09-01

    Full Text Available Background: Regular consumption of fruits and vegetables (e.g., tomatoes has been shown to be beneficial in terms of reducing the incidence of cardiovascular diseases. The industrial processing of tomatoes into tomato-based products includes several thermal treatments. Very little is known on the effect of tomato industrial processing on antiaggregatory activity and phenolic profile. Methods: It was assessed the effect of tomato and by-products extracts on platelet aggregation induced by ADP, collagen, TRAP-6 and arachidonic acid. These in vitro antithrombotic properties were further supported in an in vivo model of thrombosis. A set of antiplatelet compounds has been selected for HPLC analysis in the different extracts. Results: Some natural compounds such as chlorogenic, caffeic, ferulic and p-coumaric acids were identified by HPLC in tomatoes and its products may inhibit platelet activation. Red tomatoes, tomato products (sauce, ketchup and juice and by-products extracts inhibited platelet aggregation induced adenosine 5'-diphosphate, collagen, thrombin receptor activator peptide-6 and arachidonic acid, but to a different extent. Also, pomace extract presents antithrombotic activity. Conclusions: Processed tomatoes may have a higher content of health-benefiting compounds than fresh ones. Pomace even presents the best antiplatelet activity. Finally, tomato products may be used as a functional ingredient adding antiplatelet activities to processed foods.

  15. pH-tuneable binding of 2′-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study

    Energy Technology Data Exchange (ETDEWEB)

    Ciulli, Alessio [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Lobley, Carina M. C. [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); Tuck, Kellie L. [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Smith, Alison G. [Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA (United Kingdom); Blundell, Tom L. [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); Abell, Chris, E-mail: ca26@cam.ac.uk [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom)

    2007-02-01

    A combined crystallographic, calorimetric and mutagenic study has been used to show how changes in pH give rise to two distinct binding modes of 2′-phospho-ADP-ribose to ketopantoate reductase. The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP{sup +} that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP{sup +}, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ‘reversed binding mode’ observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

  16. pH-tuneable binding of 2′-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study

    International Nuclear Information System (INIS)

    Ciulli, Alessio; Lobley, Carina M. C.; Tuck, Kellie L.; Smith, Alison G.; Blundell, Tom L.; Abell, Chris

    2007-01-01

    A combined crystallographic, calorimetric and mutagenic study has been used to show how changes in pH give rise to two distinct binding modes of 2′-phospho-ADP-ribose to ketopantoate reductase. The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP + that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP + , with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ‘reversed binding mode’ observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes

  17. Suppression of breast cancer metastasis through the inactivation of ADP-ribosylation factor 1.

    Science.gov (United States)

    Xie, Xiayang; Tang, Shou-Ching; Cai, Yafei; Pi, Wenhu; Deng, Libin; Wu, Guangyu; Chavanieu, Alain; Teng, Yong

    2016-09-06

    Metastasis is the major cause of cancer-related death in breast cancer patients, which is controlled by specific sets of genes. Targeting these genes may provide a means to delay cancer progression and allow local treatment to be more effective. We report for the first time that ADP-ribosylation factor 1 (ARF1) is the most amplified gene in ARF gene family in breast cancer, and high-level amplification of ARF1 is associated with increased mRNA expression and poor outcomes of patients with breast cancer. Knockdown of ARF1 leads to significant suppression of migration and invasion in breast cancer cells. Using the orthotopic xenograft model in NSG mice, we demonstrate that loss of ARF1 expression in breast cancer cells inhibits pulmonary metastasis. The zebrafish-metastasis model confirms that the ARF1 gene depletion suppresses breast cancer cells to metastatic disseminate throughout fish body, indicating that ARF1 is a very compelling target to limit metastasis. ARF1 function largely dependents on its activation and LM11, a cell-active inhibitor that specifically inhibits ARF1 activation through targeting the ARF1-GDP/ARNO complex at the Golgi, significantly impairs metastatic capability of breast cancer cell in zebrafish. These findings underline the importance of ARF1 in promoting metastasis and suggest that LM11 that inhibits ARF1 activation may represent a potential therapeutic approach to prevent or treat breast cancer metastasis.

  18. Methotrexate induces poly(ADP-ribose) polymerase-dependent, caspase 3-independent apoptosis in subsets of proliferating CD4+ T cells

    DEFF Research Database (Denmark)

    Nielsen, C H; Albertsen, L; Bendtzen, K

    2007-01-01

    The mechanism of action of methotrexate (MTX) in autoimmune diseases (AID) is unclear. A pro-apoptotic effect has been demonstrated in mitogen-stimulated peripheral blood mononuclear cells (PBMC), but studies employing conventional antigens have disputed a pro-apoptotic effect. CD4+ T helper (Th....... Exposure of CA-stimulated PBMC to MTX significantly increased their level of cleaved poly(ADP-ribose) polymerase (PARP), and a similar tendency was observed in TT-stimulated cells. Unlike CA and TT, the mitogen phytohaemagglutinin (PHA) induced proliferation of both CD4- and CD4+ T cells, and induced......) cells play a significant role in most AID. We therefore examined directly, by flow cytometry, the uptake of MTX by the T helper (Th) cells stimulated for 6 days with Candida albicans (CA) or tetanus toxoid (TT), and its consequences with respect to induction of apoptosis. While none of the resting Th...

  19. Diversity and distribution of cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae.

    Science.gov (United States)

    Purdy, Alexandra E; Balch, Deborah; Lizárraga-Partida, Marcial Leonardo; Islam, Mohammad Sirajul; Martinez-Urtaza, Jaime; Huq, Anwar; Colwell, Rita R; Bartlett, Douglas H

    2010-02-01

    Non-toxigenic non-O1, non-O139 Vibrio cholerae strains isolated from both environmental and clinical settings carry a suite of virulence factors aside from cholera toxin. Among V. cholerae strains isolated from coastal waters of southern California, this includes cholix toxin, an ADP-ribosylating factor that is capable of halting protein synthesis in eukaryotic cells. The prevalence of the gene encoding cholix toxin, chxA, was assessed among a collection of 155 diverse V. cholerae strains originating from both clinical and environmental settings in Bangladesh and Mexico and other countries around the globe. The chxA gene was present in 47% of 83 non-O1, non-O139 strains and 16% of 72 O1/O139 strains screened as part of this study. A total of 86 chxA gene sequences were obtained, and phylogenetic analysis revealed that they fall into two distinct clades. These two clades were also observed in the phylogenies of several housekeeping genes, suggesting that the divergence observed in chxA extends to other regions of the V. cholerae genome, and most likely has arisen from vertical descent rather than horizontal transfer. Our results clearly indicate that ChxA is a major toxin of V. cholerae with a worldwide distribution that is preferentially associated with non-pandemic strains. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  20. Mitochondrial ADP/ATP exchange inhibition: a novel off-target mechanism underlying ibipinabant-induced myotoxicity.

    Science.gov (United States)

    Schirris, Tom J J; Ritschel, Tina; Herma Renkema, G; Willems, Peter H G M; Smeitink, Jan A M; Russel, Frans G M

    2015-09-29

    Cannabinoid receptor 1 (CB1R) antagonists appear to be promising drugs for the treatment of obesity, however, serious side effects have hampered their clinical application. Rimonabant, the first in class CB1R antagonist, was withdrawn from the market because of psychiatric side effects. This has led to the search for more peripherally restricted CB1R antagonists, one of which is ibipinabant. However, this 3,4-diarylpyrazoline derivative showed muscle toxicity in a pre-clinical dog study with mitochondrial dysfunction. Here, we studied the molecular mechanism by which ibipinabant induces mitochondrial toxicity. We observed a strong cytotoxic potency of ibipinabant in C2C12 myoblasts. Functional characterization of mitochondria revealed increased cellular reactive oxygen species generation and a decreased ATP production capacity, without effects on the catalytic activities of mitochondrial enzyme complexes I-V or the complex specific-driven oxygen consumption. Using in silico off-target prediction modelling, combined with in vitro validation in isolated mitochondria and mitoplasts, we identified adenine nucleotide translocase (ANT)-dependent mitochondrial ADP/ATP exchange as a novel molecular mechanism underlying ibipinabant-induced toxicity. Minor structural modification of ibipinabant could abolish ANT inhibition leading to a decreased cytotoxic potency, as observed with the ibipinabant derivative CB23. Our results will be instrumental in the development of new types of safer CB1R antagonists.

  1. Zinc finger nuclease mediated knockout of ADP-dependent glucokinase in cancer cell lines: effects on cell survival and mitochondrial oxidative metabolism.

    Directory of Open Access Journals (Sweden)

    Susan Richter

    Full Text Available Zinc finger nucleases (ZFN are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK, in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116. All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002 and 4.3±0.8% (p = 0.001 for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.

  2. Zinc finger nuclease mediated knockout of ADP-dependent glucokinase in cancer cell lines: effects on cell survival and mitochondrial oxidative metabolism.

    Science.gov (United States)

    Richter, Susan; Morrison, Shona; Connor, Tim; Su, Jiechuang; Print, Cristin G; Ronimus, Ron S; McGee, Sean L; Wilson, William R

    2013-01-01

    Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.

  3. Poly(ADP-ribosyl)ation links the chromatin remodeler SMARCA5/SNF2H to RNF168-dependent DNA damage signaling

    DEFF Research Database (Denmark)

    Smeenk, G.; Wiegant, W.W.; Luijsterburg, M.S.

    2013-01-01

    Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely...... unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of νH2AX into damaged chromatin......, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA...

  4. Cyclic ADP-ribose and IP3 mediate abscisic acid-induced isoflavone accumulation in soybean sprouts

    International Nuclear Information System (INIS)

    Jiao, Caifeng; Yang, Runqiang; Gu, Zhenxin

    2016-01-01

    In this study, the roles of ABA-cADPR-Ca 2+ and ABA-IP3-Ca 2+ signaling pathways in UV-B-induced isoflavone accumulation in soybean sprouts were investigated. Results showed that abscisic acid (ABA) up regulated cyclic ADP-ribose (cADPR) and inositol 1,4,5-trisphosphate (IP3) levels in soybean sprouts under UV-B radiation. Furthermore, cADPR and IP3, as second messengers of UV-B-triggered ABA, induced isoflavone accumulation by up-regulating proteins and genes expression and activity of isoflavone biosynthetic-enzymes (chalcone synthase, CHS; isoflavone synthase, IFS). After Ca 2+ was chelated by EGTA, isoflavone content decreased. Overall, ABA-induced cADPR and IP3 up regulated isoflavone accumulation which was mediated by Ca 2+ signaling via enhancing the expression of proteins and genes participating in isoflavone biosynthesis in soybean sprouts under UV-B radiation. - Highlights: • UV-B-induced cADPR and IP3 synthesis was mediated by ABA. • cADPR and IP3 were involved in UV-B-ABA-induced isoflavone accumulation. • cADPR and IP3-induced isoflavone accumulation may be mediated by Ca 2+ . • ABA, cADPR, IP3 and Ca 2+ could activate proteins expression of CHS and IFS.

  5. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Karen L.; Dashner, Erica J. [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Tsosie, Ranalda [Department of Chemistry and Biochemistry, University of Montana, Missoula, MT 59812 (United States); Cho, Young Mi [Department of Food and Nutrition, College of Human Ecology, Hanyang University, Seoul 133-791 (Korea, Republic of); Lewis, Johnnye [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States); Community Environmental Health Program, University of New Mexico Health Sciences Center College of Pharmacy, Albuquerque, NM 87131 (United States); Hudson, Laurie G., E-mail: lhudson@salud.unm.edu [Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131 (United States)

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; < 10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. - Highlights: • Low micromolar concentration of uranium inhibits polymerase-1 (PARP-1) activity. • Uranium causes zinc loss from multiple DNA repair proteins. • Uranium enhances retention of DNA damage caused by ultraviolet radiation. • Zinc reverses the effects of uranium on PARP activity and DNA damage repair.

  6. Habitat assessment of non-wadeable rivers in Michigan.

    Science.gov (United States)

    Wilhelm, Jennifer G O; Allan, J David; Wessell, Kelly J; Merritt, Richard W; Cummins, Kenneth W

    2005-10-01

    Habitat evaluation of wadeable streams based on accepted protocols provides a rapid and widely used adjunct to biological assessment. However, little effort has been devoted to habitat evaluation in non-wadeable rivers, where it is likely that protocols will differ and field logistics will be more challenging. We developed and tested a non-wadeable habitat index (NWHI) for rivers of Michigan, where non-wadeable rivers were defined as those of order >or=5, drainage area >or=1600 km2, mainstem lengths >or=100 km, and mean annual discharge >or=15 m3/s. This identified 22 candidate rivers that ranged in length from 103 to 825 km and in drainage area from 1620 to 16,860 km2. We measured 171 individual habitat variables over 2-km reaches at 35 locations on 14 rivers during 2000-2002, where mean wetted width was found to range from 32 to 185 m and mean thalweg depth from 0.8 to 8.3 m. We used correlation and principal components analysis to reduce the number of variables, and examined the spatial pattern of retained variables to exclude any that appeared to reflect spatial location rather than reach condition, resulting in 12 variables to be considered in the habitat index. The proposed NWHI included seven variables: riparian width, large woody debris, aquatic vegetation, bottom deposition, bank stability, thalweg substrate, and off-channel habitat. These variables were included because of their statistical association with independently derived measures of human disturbance in the riparian zone and the catchment, and because they are considered important in other habitat protocols or to the ecology of large rivers. Five variables were excluded because they were primarily related to river size rather than anthropogenic disturbance. This index correlated strongly with indices of disturbance based on the riparian (adjusted R2 = 0.62) and the catchment (adjusted R2 = 0.50), and distinguished the 35 river reaches into the categories of poor (2), fair (19), good (13), and

  7. A novel and selective poly (ADP-ribose polymerase inhibitor ameliorates chemotherapy-induced painful neuropathy.

    Directory of Open Access Journals (Sweden)

    Lauren E Ta

    Full Text Available Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose polymerase (PARP inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888 would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice.An established chemotherapy-induced painful neuropathy model of two weekly cycles of 10 intraperitoneal (i.p. injections separated by 5 days rest was used to examine the therapeutic potential of the PARP inhibitor compound 4a. Behavioral testing using von Frey, paw radiant heat, cold plate, and exploratory behaviors were taken at baseline, and followed by testing at 3, 6, and 8 weeks from the beginning of drug treatment.Cisplatin-treated mice developed heat hyperalgesia and mechanical allodynia while oxaliplatin-treated mice exhibited cold hyperalgesia and mechanical allodynia. Co-administration of 50 mg/kg or 25 mg/kg compound 4a with platinum regimen, attenuated cisplatin-induced heat hyperalgesia and mechanical allodynia in a dose dependent manner. Similarly, co-administration of 50 mg/kg compound 4a attenuated oxaliplatin-induced cold hyperalgesia and mechanical allodynia. These data indicate that administration of a novel PARP inhibitor may have important applications as a therapeutic agent for human chemotherapy-induced painful neuropathy.

  8. A novel and selective poly (ADP-ribose) polymerase inhibitor ameliorates chemotherapy-induced painful neuropathy.

    Science.gov (United States)

    Ta, Lauren E; Schmelzer, James D; Bieber, Allan J; Loprinzi, Charles L; Sieck, Gary C; Brederson, Jill D; Low, Philip A; Windebank, Anthony J

    2013-01-01

    Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose) polymerase (PARP) inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888) would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice. An established chemotherapy-induced painful neuropathy model of two weekly cycles of 10 intraperitoneal (i.p.) injections separated by 5 days rest was used to examine the therapeutic potential of the PARP inhibitor compound 4a. Behavioral testing using von Frey, paw radiant heat, cold plate, and exploratory behaviors were taken at baseline, and followed by testing at 3, 6, and 8 weeks from the beginning of drug treatment. Cisplatin-treated mice developed heat hyperalgesia and mechanical allodynia while oxaliplatin-treated mice exhibited cold hyperalgesia and mechanical allodynia. Co-administration of 50 mg/kg or 25 mg/kg compound 4a with platinum regimen, attenuated cisplatin-induced heat hyperalgesia and mechanical allodynia in a dose dependent manner. Similarly, co-administration of 50 mg/kg compound 4a attenuated oxaliplatin-induced cold hyperalgesia and mechanical allodynia. These data indicate that administration of a novel PARP inhibitor may have important applications as a therapeutic agent for human chemotherapy-induced painful neuropathy.

  9. Enhanced activity of ADP glucose pyrophosphorylase and formation of starch induced by Azospirillum brasilense in Chlorella vulgaris.

    Science.gov (United States)

    Choix, Francisco J; Bashan, Yoav; Mendoza, Alberto; de-Bashan, Luz E

    2014-05-10

    ADP-glucose pyrophosphorylase (AGPase) regulates starch biosynthesis in higher plants and microalgae. This study measured the effect of the bacterium Azospirillum brasilense on AGPase activity in the freshwater microalga Chlorella vulgaris and formation of starch. This was done by immobilizing both microorganisms in alginate beads, either replete with or deprived of nitrogen or phosphorus and all under heterotrophic conditions, using d-glucose or Na-acetate as the carbon source. AGPase activity during the first 72h of incubation was higher in C. vulgaris when immobilized with A. brasilense. This happened simultaneously with higher starch accumulation and higher carbon uptake by the microalgae. Either carbon source had similar effects on enzyme activity and starch accumulation. Starvation either by N or P had the same pattern on AGPase activity and starch accumulation. Under replete conditions, the population of C. vulgaris immobilized alone was higher than when immobilized together, but under starvation conditions A. brasilense induced a larger population of C. vulgaris. In summary, adding A. brasilense enhanced AGPase activity, starch formation, and mitigation of stress in C. vulgaris. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. CRED Ocean Data Platform (ODP), Acoustic Doppler Profiler (ADP); Guam, SRR; Long: 144.41778, Lat: 12.83819 (WGS84); Sensor Depth: 20.42m; Data Range: 20051007-20070121.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Ocean Data Platform (ODP) is placed on the sea floor to measure water current profiles, waves, temperature and conductivity. The ODP consists of an upward...

  11. CRED Ocean Data Platform (ODP), Acoustic Doppler Profiler (ADP); PRIA, BAK; Long: -176.46025, Lat: 00.19005 (WGS84); Sensor Depth: 18.90m; Data Range: 20020201-20040122.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Ocean Data Platform (ODP) is placed on the sea floor to measure water current profiles, waves, temperature and conductivity. The ODP consists of an upward...

  12. CRED Ocean Data Platform (ODP), Acoustic Doppler Profiler (ADP); AMSM, SWA; Long: -171.09092, Lat: -11.05848 (WGS84); Sensor Depth: 15.00m; Data Range: 20020227-20021207.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Ocean Data Platform (ODP) is placed on the sea floor to measure water current profiles, waves, temperature and conductivity. The ODP consists of an upward...

  13. CRED Ocean Data Platform (ODP), Acoustic Doppler Profiler (ADP); PRIA, JAR; Long: -160.01553, Lat: -00.37917 (WGS84); Sensor Depth: 15.00m; Data Range: 20020311-20040325.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Ocean Data Platform (ODP) is placed on the sea floor to measure water current profiles, waves, temperature and conductivity. The ODP consists of an upward...

  14. CRED Ocean Data Platform (ODP), Acoustic Doppler Profiler (ADP); PRIA, BAK; Long: -176.46012, Lat: 00.18994 (WGS84); Sensor Depth: 19.81m; Data Range: 20080210-20100130.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Ocean Data Platform (ODP) is placed on the sea floor to measure water current profiles, waves, temperature and conductivity. The ODP consists of an upward...

  15. CRED Ocean Data Platform (ODP), Acoustic Doppler Profiler (ADP); PRIA, BAK; Long: -176.46012, Lat: 00.18994 (WGS84); Sensor Depth: 18.80m; Data Range: 20060131-20080209.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Ocean Data Platform (ODP) is placed on the sea floor to measure water current profiles, waves, temperature and conductivity. The ODP consists of an upward...

  16. CRED Ocean Data Platform (ODP), Acoustic Doppler Profiler (ADP); PRIA, BAK; Long: -176.46025, Lat: 00.19005 (WGS84); Sensor Depth: 18.90m; Data Range: 20040123-20060130.

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Ocean Data Platform (ODP) is placed on the sea floor to measure water current profiles, waves, temperature and conductivity. The ODP consists of an upward...

  17. Impact of ticagrelor on P2Y1 and P2Y12 localization and on cholesterol levels in platelet plasma membrane.

    Science.gov (United States)

    Rabani, Vahideh; Montange, Damien; Meneveau, Nicolas; Davani, Siamak

    2017-10-11

    Ticagrelor is an antiplatelet agent that inhibits platelet activation via P2Y12 antagonism. There are several studies showing that P2Y12 needs lipid rafts to be activated, but there are few data about how ticagrelor impacts lipid raft organization. Therefore, we aimed to investigate how ticagrelor could impact the distribution of cholesterol and consequently alter the organization of lipid rafts on platelet plasma membranes. We identified cholesterol-enriched raft fractions in platelet membranes by quantification of their cholesterol levels. Modifications in cholesterol and protein profiles (Flotillin 1, Flotillin 2, CD36, P2Y1, and P2Y12) were studied in platelets stimulated by ADP, treated by ticagrelor, or both. In ADP-stimulated and ticagrelor-treated groups, we found a decreased level of cholesterol in raft fractions of platelet plasma membrane compared to the control group. In addition, the peak of cholesterol in different experimental groups changed its localization on membrane fractions. In the control group, it was situated on fraction 2, while in ADP-stimulated platelets, it was located in fractions 3 to 5, and in fraction 4 in ticagrelor-treated group. The proteins studied also showed changes in their level of expression and localization in fractions of plasma membrane. Cholesterol levels of plasma membranes have a direct role in the organization of platelet membranes and could be modified by stimulation or drug treatment. Since ticagrelor and ADP both changed lipid composition and protein profile, investigating the lipid and protein composition of platelet membranes is of considerable importance as a focus for further research in anti-platelet management.

  18. Differential transactivation by orphan nuclear receptor NOR1 and its fusion gene product EWS/NOR1: possible involvement of poly(ADP-ribose) polymerase I, PARP-1.

    Science.gov (United States)

    Ohkura, Naganari; Nagamura, Yuko; Tsukada, Toshihiko

    2008-10-15

    In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins. (c) 2008 Wiley-Liss, Inc.

  19. Crystallization and preliminary X-ray analysis of the ATPase domain of the σ(54)-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP-BeFx.

    Science.gov (United States)

    Sysoeva, Tatyana A; Yennawar, Neela; Allaire, Marc; Nixon, B Tracy

    2013-12-01

    One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators.

  20. Characterisation of Tidal Flows at the European Marine Energy Centre in the Absence of Ocean Waves

    Directory of Open Access Journals (Sweden)

    Brian G. Sellar

    2018-01-01

    Full Text Available The data analyses and results presented here are based on the field measurement campaign of the Reliable Data Acquisition Platform for Tidal (ReDAPT project (Energy Technologies Institute (ETI, U.K. 2010–2015. During ReDAPT, a 1 MW commercial prototype tidal turbine was deployed and operated at the Fall of Warness tidal test site within the European Marine Energy Centre (EMEC, Orkney, U.K. Mean flow speeds and Turbulence Intensity (TI at multiple positions proximal to the machine are considered. Through the implemented wave identification techniques, the dataset can be filtered into conditions where the effects of waves are present or absent. Due to the volume of results, only flow conditions in the absence of waves are reported here. The analysis shows that TI and mean flows are found to vary considerably between flood and ebb tides whilst exhibiting sensitivity to the tidal phase and to the specification of spatial averaging and velocity binning. The principal measurement technique was acoustic Doppler profiling provided by seabed-mounted Diverging-beam Acoustic Doppler Profilers (D-ADP together with remotely-operable Single-Beam Acoustic Doppler Profilers (SB-ADP installed at mid-depth on the tidal turbine. This novel configuration allows inter-instrument comparisons, which were conducted. Turbulence intensity averaged over the rotor extents of the ReDAPT turbine for flood tides vary between 16.7% at flow speeds above 0.3 m/s and 11.7% when considering only flow speeds in the turbine operating speed range, which reduces to 10.9% (6.8% relative reduction following the implementation of noise correction techniques. Equivalent values for ebb tides are 14.7%, 10.1% and 9.3% (7.9% relative reduction. For flood and ebb tides, TI values resulting from noise correction are reduced in absolute terms by 3% and 2% respectively across a wide velocity range and approximately 1% for turbine operating speeds. Through comparison with SB-ADP-derived mid

  1. ADP-ribosylation factor arf6p may function as a molecular switch of new end take off in fission yeast

    International Nuclear Information System (INIS)

    Fujita, Atsushi

    2008-01-01

    Small GTPases act as molecular switches in a wide variety of cellular processes. In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as 'New End Take Off' (NETO). Here I report the identification of a gene, arf6 + , encoding an ADP-ribosylation factor small GTPase, that may be essential for NETO. arf6Δ cells completely fail to undergo NETO. arf6p localizes at both cell ends and presumptive septa in a cell-cycle dependent manner. And its polarized localization is not dependent on microtubules, actin cytoskeletons and some NETO factors (bud6p, for3p, tea1p, tea3p, and tea4p). Notably, overexpression of a fast GDP/GTP-cycling mutant of arf6p can advance the timing of NETO. These findings suggest that arf6p functions as a molecular switch for the activation of NETO in fission yeast

  2. Targeting poly(ADP-ribose)polymerase1 in neurological diseases: A promising trove for new pharmacological interventions to enter clinical translation.

    Science.gov (United States)

    Sriram, Chandra Shekhar; Jangra, Ashok; Kasala, Eshvendar Reddy; Bodduluru, Lakshmi Narendra; Bezbaruah, Babul Kumar

    2014-10-01

    The highly conserved abundant nuclear protein poly(ADP-ribose)polymerase1 (PARP1) functions at the center of cellular stress response and is mainly implied in DNA damage repair mechanism. Apart from its involvement in DNA damage repair, it does sway multiple vital cellular processes such as cell death pathways, cell aging, insulator function, chromatin modification, transcription and mitotic apparatus function. Since brain is the principal organ vulnerable to oxidative stress and inflammatory responses, upon stress encounters robust DNA damage can occur and intense PARP1 activation may result that will lead to various CNS diseases. In the context of soaring interest towards PARP1 as a therapeutic target for newer pharmacological interventions, here in the present review, we are attempting to give a silhouette of the role of PARP1 in the neurological diseases and the potential of its inhibitors to enter clinical translation, along with its structural and functional aspects. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. The dual role of poly(ADP-ribose) polymerase-1 in modulating parthanatos and autophagy under oxidative stress in rat cochlear marginal cells of the stria vascularis.

    Science.gov (United States)

    Jiang, Hong-Yan; Yang, Yang; Zhang, Yuan-Yuan; Xie, Zhen; Zhao, Xue-Yan; Sun, Yu; Kong, Wei-Jia

    2018-04-01

    Oxidative stress is reported to regulate several apoptotic and necrotic cell death pathways in auditory tissues. Poly(ADP-ribose) polymerase-1 (PARP-1) can be activated under oxidative stress, which is the hallmark of parthanatos. Autophagy, which serves either a pro-survival or pro-death function, can also be stimulated by oxidative stress, but the role of autophagy and its relationship with parthanatos underlying this activation in the inner ear remains unknown. In this study, we established an oxidative stress model in vitro by glucose oxidase/glucose (GO/G), which could continuously generate low concentrations of H 2 O 2 to mimic continuous exposure to H 2 O 2 in physiological conditions, for investigation of oxidative stress-induced cell death mechanisms and the regulatory role of PARP-1 in this process. We observed that GO/G induced stria marginal cells (MCs) death via upregulation of PARP-1 expression, accumulation of polyADP-ribose (PAR) polymers, decline of mitochondrial membrane potential (MMP) and nuclear translocation of apoptosis-inducing factor (AIF), which all are biochemical features of parthanatos. PARP-1 knockdown rescued GO/G-induced MCs death, as well as abrogated downstream molecular events of PARP-1 activation. In addition, we demonstrated that GO/G stimulated autophagy and PARP-1 knockdown suppressed GO/G-induced autophagy in MCs. Interestingly, autophagy suppression by 3-Methyladenine (3-MA) accelerated GO/G-induced parthanatos, indicating a pro-survival function of autophagy in GO/G-induced MCs death. Taken together, these data suggested that PARP-1 played dual roles by modulating parthanatos and autophagy in oxidative stress-induced MCs death, which may be considered as a promising therapeutic target for ameliorating oxidative stress-related hearing disorders. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Poly(ADP-ribosepolymerase-1 modulates microglial responses to amyloid β

    Directory of Open Access Journals (Sweden)

    Kauppinen Tiina M

    2011-11-01

    Full Text Available Abstract Background Amyloid β (Aβ accumulates in Alzheimer's disease (AD brain. Microglial activation also occurs in AD, and this inflammatory response may contribute to disease progression. Microglial activation can be induced by Aβ, but the mechanisms by which this occurs have not been defined. The nuclear enzyme poly(ADP-ribose polymerase-1 (PARP-1 regulates microglial activation in response to several stimuli through its interactions with the transcription factor, NF-κB. The purpose of this study was to evaluate whether PARP-1 activation is involved in Aβ-induced microglial activation, and whether PARP-1 inhibition can modify microglial responses to Aβ. Methods hAPPJ20 mice, which accumulate Aβ with ageing, were crossed with PARP-1-/- mice to assess the effects of PARP-1 depletion on microglial activation, hippocampal synaptic integrity, and cognitive function. Aβ peptide was also injected into brain of wt and PARP-1-/- mice to directly determine the effects of PARP-1 on Aβ-induced microglial activation. The effect of PARP-1 on Aβ-induced microglial cytokine production and neurotoxicity was evaluated in primary microglia cultures and in microglia-neuron co-cultures, utilizing PARP-1-/- cells and a PARP-1 inhibitor. NF-κB activation was evaluated in microglia infected with a lentivirus reporter gene. Results The hAPPJ20 mice developed microglial activation, reduced hippocampal CA1 calbindin expression, and impaired novel object recognition by age 6 months. All of these features were attenuated in hAPPJ20/PARP-1-/- mice. Similarly, Aβ1-42 injected into mouse brain produced a robust microglial response in wild-type mice, and this was blocked in mice lacking PARP-1 expression or activity. Studies using microglial cultures showed that PARP-1 activity was required for Aβ-induced NF-κB activation, morphological transformation, NO release, TNFα release, and neurotoxicity. Conversely, PARP-1 inhibition increased release of the

  5. Poly(ADP-ribose polymerase 1 (PARP1 overexpression in human breast cancer stem cells and resistance to olaparib.

    Directory of Open Access Journals (Sweden)

    Marine Gilabert

    Full Text Available BACKGROUND: Breast cancer stem cells (BCSCs have been recognized as playing a major role in various aspects of breast cancer biology. To identify specific biomarkers of BCSCs, we have performed comparative proteomics of BCSC-enriched and mature cancer cell populations from the human breast cancer cell line (BCL, BrCA-MZ-01. METHODS: ALDEFLUOR assay was used to sort BCSC-enriched (ALDH+ and mature cancer (ALDH- cell populations. Total proteins were extracted from both fractions and subjected to 2-Dimensional Difference In-Gel Electrophoresis (2-D DIGE. Differentially-expressed spots were excised and proteins were gel-extracted, digested and identified using MALDI-TOF MS. RESULTS: 2-D DIGE identified poly(ADP-ribose polymerase 1 (PARP1 as overexpressed in ALDH+ cells from BrCA-MZ-01. This observation was confirmed by western blot and extended to four additional human BCLs. ALDH+ cells from BRCA1-mutated HCC1937, which had the highest level of PARP1 overexpression, displayed resistance to olaparib, a specific PARP1 inhibitor. CONCLUSION: An unbiased proteomic approach identified PARP1 as upregulated in ALDH+, BCSC-enriched cells from various human BCLs, which may contribute to clinical resistance to PARP inhibitors.

  6. Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

    Energy Technology Data Exchange (ETDEWEB)

    Abdelali, Ala [Department of Anatomy, Faculty of Medicine, Kuwait University (Kuwait); Al-Bader, Maie [Department of Physiology, Faculty of Medicine, Kuwait University (Kuwait); Kilarkaje, Narayana, E-mail: knarayana@hsc.edu.kw [Department of Anatomy, Faculty of Medicine, Kuwait University (Kuwait)

    2016-11-15

    Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis. - Highlights: • Resveratrol inhibits diabetes-induced abnormal sperm morphogenesis • Resveratrol recovers

  7. Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

    International Nuclear Information System (INIS)

    Abdelali, Ala; Al-Bader, Maie; Kilarkaje, Narayana

    2016-01-01

    Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis. - Highlights: • Resveratrol inhibits diabetes-induced abnormal sperm morphogenesis • Resveratrol recovers

  8. Rapid release of 42K and 86Rb from an occluded state of the Na,K-pump in the presence of ATP or ADP

    International Nuclear Information System (INIS)

    Forbush, B. III

    1987-01-01

    We have measured the time course of release of 42 K and 86 Rb from an occluded state of the Na,K-pump using a rapid filtration apparatus. We have found that at 20 degrees C and in the presence of ATP, 42 K is released with a rate constant of approximately 45 s-1 and 86 Rb with a rate constant of approximately 20 s-1; both ATP and ADP are effective at a low affinity site (Kd approximately 0.3 and 1 mM, respectively) with the rate of deocclusion being only half as great in ADP as in ATP. Mg2+ stimulates 2-fold at low concentrations probably by forming MgATP, and free Mg2+ is strongly inhibitory at high concentrations (Kd approximately 10 mM). Mg2+ also decreases the affinity for ATP, and the data are consistent with mixed type inhibition; from the analysis the dissociation constant is approximately 1 mM for the inhibitory Mg2+ and the Rb+-occluded form without ATP. The rate of 42 K or 86 Rb release increases monotonically with pH while ATPase activity decreases above pH 8, so that deocclusion is not rate-limiting in the overall cycle at high pH. This is reflected by a convergence of the rate of Na,K-ATPase and Na,Rb-ATPase activities at high pH and by a decrease in the observed steady-state level of the occluded 86 Rb intermediate at high pH. K+, Rb+, Na+, and Cs+, but not Li+, increase the rate of 42 K and 86 Rb release at constant ionic strength, presumably at sites other than the transport sites. The spontaneous rate of deocclusion is only approximately 0.1 s-1 at low ionic strength in the absence of nucleotides, and it is increased markedly by all cations tested except Li+. Overall the data are consistent with deocclusion as a rate-limiting step in the Na,K-pump cycle

  9. Role of glycolysis inhibition and poly(ADP-ribose) polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemic cells.

    Science.gov (United States)

    Verrax, Julien; Vanbever, Stéphanie; Stockis, Julie; Taper, Henryk; Calderon, Pedro Buc

    2007-03-15

    Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells. (c) 2006 Wiley-Liss, Inc.

  10. The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

    Science.gov (United States)

    Rohrbeck, Astrid; Höltje, Markus; Adolf, Andrej; Oms, Elisabeth; Hagemann, Sandra; Ahnert-Hilger, Gudrun; Just, Ingo

    2017-10-27

    The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to β1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. ADP-glucose pyrophosphorylase gene plays a key role in the quality of corm and yield of cormels in gladiolus

    International Nuclear Information System (INIS)

    Seng, Shanshan; Wu, Jian; Sui, Juanjuan; Wu, Chenyu; Zhong, Xionghui; Liu, Chen; Liu, Chao; Gong, Benhe; Zhang, Fengqin; He, Junna; Yi, Mingfang

    2016-01-01

    Starch is the main storage compound in underground organs like corms. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in storage organs and is likely one of the most important determinant of sink strength. Here, we identify an AGPase gene (GhAGPS1) from gladiolus. The highest transcriptional levels of GhAGPS1 were observed in cormels and corms. Transformation of GhAGPS1 into Arabidopsis rescued the phenotype of aps1 mutant. Silencing GhAGPS1 in gladiolus corms by virus-induced gene silencing (VIGS) decreased the transcriptional levels of two genes and starch content. Transmission electron microscopy analyses of leaf and corm sections confirmed that starch biosynthesis was inhibited. Corm weight and cormel number reduced significantly in the silenced plants. Taken together, these results indicate that inhibiting the expression of AGPase gene could impair starch synthesis, which results in the lowered corm quality and cormel yield in gladiolus. -- Highlights: •Cormel quantity was reduced significantly in silenced Gladiolus plants. •Corm quality was declined significantly in silenced Gladiolus plants. •Starch synthesis was inhibited in silenced Gladiolus plants.

  12. ADP-glucose pyrophosphorylase gene plays a key role in the quality of corm and yield of cormels in gladiolus

    Energy Technology Data Exchange (ETDEWEB)

    Seng, Shanshan, E-mail: seshsh108@126.com [Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, Department of Ornamental Horticulture and Landscape Architecture, China Agricultural University, Yuan Mingyuan Western Road 2#, Beijing 100193 (China); Wu, Jian [Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, Department of Ornamental Horticulture and Landscape Architecture, China Agricultural University, Yuan Mingyuan Western Road 2#, Beijing 100193 (China); Sui, Juanjuan [Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, Department of Ornamental Horticulture and Landscape Architecture, China Agricultural University, Yuan Mingyuan Western Road 2#, Beijing 100193 (China); College of Biology, Fuyang Normal College, Qinghe Western Road 100#, Fuyang 236037, Anhui (China); Wu, Chenyu; Zhong, Xionghui; Liu, Chen; Liu, Chao; Gong, Benhe; Zhang, Fengqin; He, Junna [Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, Department of Ornamental Horticulture and Landscape Architecture, China Agricultural University, Yuan Mingyuan Western Road 2#, Beijing 100193 (China); Yi, Mingfang, E-mail: ymfang@cau.edu.cn [Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, Department of Ornamental Horticulture and Landscape Architecture, China Agricultural University, Yuan Mingyuan Western Road 2#, Beijing 100193 (China)

    2016-05-20

    Starch is the main storage compound in underground organs like corms. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in storage organs and is likely one of the most important determinant of sink strength. Here, we identify an AGPase gene (GhAGPS1) from gladiolus. The highest transcriptional levels of GhAGPS1 were observed in cormels and corms. Transformation of GhAGPS1 into Arabidopsis rescued the phenotype of aps1 mutant. Silencing GhAGPS1 in gladiolus corms by virus-induced gene silencing (VIGS) decreased the transcriptional levels of two genes and starch content. Transmission electron microscopy analyses of leaf and corm sections confirmed that starch biosynthesis was inhibited. Corm weight and cormel number reduced significantly in the silenced plants. Taken together, these results indicate that inhibiting the expression of AGPase gene could impair starch synthesis, which results in the lowered corm quality and cormel yield in gladiolus. -- Highlights: •Cormel quantity was reduced significantly in silenced Gladiolus plants. •Corm quality was declined significantly in silenced Gladiolus plants. •Starch synthesis was inhibited in silenced Gladiolus plants.

  13. Mobilization of Ca2+ by Cyclic ADP-Ribose from the Endoplasmic Reticulum of Cauliflower Florets1

    Science.gov (United States)

    Navazio, Lorella; Mariani, Paola; Sanders, Dale

    2001-01-01

    The NAD+ metabolite cADP-Rib (cADPR) elevates cytosolic free Ca2+ in plants and thereby plays a central role in signal transduction pathways evoked by the drought and stress hormone abscisic acid. cADPR is known to mobilize Ca2+ from the large vacuole of mature cells. To determine whether additional sites for cADPR-gated Ca2+ release reside in plant cells, microsomes from cauliflower (Brassica oleracea) inflorescences were subfractionated on sucrose density gradients, and the distribution of cADPR-elicited Ca2+ release was monitored. cADPR-gated Ca2+ release was detected in the heavy-density fractions associated with rough endoplasmic reticulum (ER). cADPR-dependent Ca2+ release co-migrated with two ER markers, calnexin and antimycin A-insensitive NADH-cytochrome c reductase activity. To investigate the possibility that contaminating plasma membrane in the ER-rich fractions was responsible for the observed release, plasma membrane vesicles were purified by aqueous two-phase partitioning, everted with Brij-58, and loaded with Ca2+: These vesicles failed to respond to cADPR. Ca2+ release evoked by cADPR at the ER was fully inhibited by ruthenium red and 8-NH2-cADPR, a specific antagonist of cADPR-gated Ca2+ release in animal cells. The presence of a Ca2+ release pathway activated by cADPR at higher plant ER reinforces the notion that, alongside the vacuole, the ER participates in Ca2+ signaling. PMID:11299392

  14. Identification of a GTP-binding protein α subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

    International Nuclear Information System (INIS)

    Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.; Simon, M.I.

    1988-01-01

    Recent molecular cloning of cDNA for the α subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein α subunit, which they refer to as G/sub z/α, by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ α-subunit cDNA. The deduced amino acid sequence of G/sub z/α is 41-67% identical with those of other known G-protein α subunits. However, the 355-residue G/sub z/α lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein α subunits. They suggest that G/sub z/α, which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems

  15. Global mass spectrometry based metabolomics profiling of erythrocytes infected with Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Theodore R Sana

    Full Text Available Malaria is a global infectious disease that threatens the lives of millions of people. Transcriptomics, proteomics and functional genomics studies, as well as sequencing of the Plasmodium falciparum and Homo sapiens genomes, have shed new light on this host-parasite relationship. Recent advances in accurate mass measurement mass spectrometry, sophisticated data analysis software, and availability of biological pathway databases, have converged to facilitate our global, untargeted biochemical profiling study of in vitro P. falciparum-infected (IRBC and uninfected (NRBC erythrocytes. In order to expand the number of detectable metabolites, several key analytical steps in our workflows were optimized. Untargeted and targeted data mining resulted in detection of over one thousand features or chemical entities. Untargeted features were annotated via matching to the METLIN metabolite database. For targeted data mining, we queried the data using a compound database derived from a metabolic reconstruction of the P. falciparum genome. In total, over one hundred and fifty differential annotated metabolites were observed. To corroborate the representation of known biochemical pathways from our data, an inferential pathway analysis strategy was used to map annotated metabolites onto the BioCyc pathway collection. This hypothesis-generating approach resulted in over-representation of many metabolites onto several IRBC pathways, most prominently glycolysis. In addition, components of the "branched" TCA cycle, partial urea cycle, and nucleotide, amino acid, chorismate, sphingolipid and fatty acid metabolism were found to be altered in IRBCs. Interestingly, we detected and confirmed elevated levels for cyclic ADP ribose and phosphoribosyl AMP in IRBCs, a novel observation. These metabolites may play a role in regulating the release of intracellular Ca(2+ during P. falciparum infection. Our results support a strategy of global metabolite profiling by untargeted

  16. Increased DNA damage in progression of COPD: a response by poly(ADP-ribose polymerase-1.

    Directory of Open Access Journals (Sweden)

    Ingrid Oit-Wiscombe

    Full Text Available Chronic oxidative stress (OS, a major mechanism of chronic obstructive pulmonary disease (COPD, may cause significant damage to DNA. Poly(ADP-ribose polymerase (PARP-1 is rapidly activated by OS-induced DNA lesions. However, the degree of DNA damage along with the evolution of COPD is unclear. In peripheral blood mononuclear cells of non-smoking individuals, non-obstructive smokers, patients with COPD of all stages and those with COPD exacerbation, we evaluated DNA damage, PARP activity and PARP-1 mRNA expression using Comet Assay IV, biotinylated-NAD incorporation assay and qRT-PCR, respectively and subjected results to ordinal logistic regression modelling. Adjusted for demographics, smoking-related parameters and lung function, novel comet parameters, tail length/cell length ratio and tail migration/cell length ratio, showed the greatest increase along the study groups corresponding to the evolution of COPD [odds ratio (OR 7.88, 95% CI 4.26-14.57; p<0.001 and OR 3.91, 95% CI 2.69-5.66; p<0.001, respectively]. Analogously, PARP activity increased significantly over the groups (OR = 1.01; 95%; p<0.001. An antioxidant tetrapeptide UPF17 significantly reduced the PARP-1 mRNA expression in COPD, compared to that in non-obstructive individuals (p = 0.040. Tail length/cell length and tail migration/cell length ratios provide novel progression-sensitive tools for assessment of DNA damage. However, it remains to be elucidated whether inhibition of an elevated PARP-1 activity has a safe enough potential to break the vicious cycle of the development and progression of COPD.

  17. Inhibition of gamma-ray dose-rate effects by D2O and inhibitors of poly(ADP-ribose) synthetase in cultured mammalian L5178Y cells

    International Nuclear Information System (INIS)

    Ueno, A.M.; Tanaka, O.; Matsudaira, H.

    1984-01-01

    Effects of deuterium oxide (D 2 O) and 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, on cell proliferation and survival were studied in cultured mammalian L5178Y cells under growing conditions and after acute and low-dose-rate irradiation at about 0.1 to 0.4 Gy/hr of γ rays. Growth of irradiated and unirradiated cells was inhibited by 45% D 2 O but not by 3-aminobenzamide at 10mM, except for treatments longer than 30 hr. The presence of these agents either alone or in combination during irradiation at low dose rates suppressed almost totally the decrease in cell killing due to the decrease in dose rate. Among other inhibitors tested, theobromine and theophylline were found to be effective in eliminating the dose-rate effects of γ rays. Possible mechanisms underlying the inhibition are discussed

  18. Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism.

    Science.gov (United States)

    Rotin, Lianne E; Gronda, Marcela; MacLean, Neil; Hurren, Rose; Wang, XiaoMing; Lin, Feng-Hsu; Wrana, Jeff; Datti, Alessandro; Barber, Dwayne L; Minden, Mark D; Slassi, Malik; Schimmer, Aaron D

    2016-01-19

    Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor ibrutinib has significantly improved patient outcomes in several B-cell malignancies, with minimal toxicity. Given the reported expression and constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has been recent interest in investigating the anti-AML activity of ibrutinib. We noted that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs. Using a high-throughput combination chemical screen, we identified that the poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of ibrutinib and ethacridine induced a synergistic increase in reactive oxygen species that was functionally important to explain the observed cell death. Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings indicate that ibrutinib may have a BTK-independent role in AML and that PARG inhibitors may have utility as part of a combination therapy for this disease.

  19. Fps/Fes and Fer non-receptor protein-tyrosine kinases regulate collagen- and ADP-induced platelet aggregation.

    Science.gov (United States)

    Senis, Y A; Sangrar, W; Zirngibl, R A; Craig, A W B; Lee, D H; Greer, P A

    2003-05-01

    Fps/Fes and Fer proto-oncoproteins are structurally related non-receptor protein-tyrosine kinases implicated in signaling downstream from cytokines, growth factors and immune receptors. We show that Fps/Fes and Fer are expressed in human and mouse platelets, and are activated following stimulation with collagen and collagen-related peptide (CRP), suggesting a role in GPVI receptor signaling. Fer was also activated following stimulation with thrombin and a protease-activated receptor4 (PAR4)-activating peptide, suggesting a role in signaling downstream from the G protein-coupled PAR4. There were no detectable perturbations in CRP-induced activation of Syk, PLCgamma2, cortactin, Erk, Jnk, Akt or p38 in platelets from mice lacking Fps/Fes, Fer, or both kinases. Platelets lacking Fps/Fes, from a targeted fps/fes null strain of mice, showed increased rates and amplitudes of collagen-induced aggregation, relative to wild-type platelets. P-Selectin expression was also elevated on the surface of Fps/Fes-null platelets in response to CRP. Fer-deficient platelets, from mice targeted with a kinase-inactivating mutation, disaggregated more rapidly than wild-type platelets in response to ADP. This report provides the first evidence that Fps/Fes and Fer are expressed in platelets and become activated downstream from the GPVI collagen receptor, and that Fer is activated downstream from a G-protein coupled receptor. Furthermore, using targeted mouse models we show that deficiency in Fps/Fes or Fer resulted in disregulated platelet aggregation and disaggregation, demonstrating a role for these kinases in regulating platelet functions.

  20. Arsenite-induced ROS/RNS generation causes zinc loss and inhibits the activity of poly(ADP-ribose) polymerase-1.

    Science.gov (United States)

    Wang, Feng; Zhou, Xixi; Liu, Wenlan; Sun, Xi; Chen, Chen; Hudson, Laurie G; Jian Liu, Ke

    2013-08-01

    Arsenic enhances the genotoxicity of other carcinogenic agents such as ultraviolet radiation and benzo[a]pyrene. Recent reports suggest that inhibition of DNA repair is an important aspect of arsenic cocarcinogenesis, and DNA repair proteins such as poly(ADP ribose) polymerase (PARP)-1 are direct molecular targets of arsenic. Although arsenic has been shown to generate reactive oxygen/nitrogen species (ROS/RNS), little is known about the role of arsenic-induced ROS/RNS in the mechanism underlying arsenic inhibition of DNA repair. We report herein that arsenite-generated ROS/RNS inhibits PARP-1 activity in cells. Cellular exposure to arsenite, as well as hydrogen peroxide and NONOate (nitric oxide donor), decreased PARP-1 zinc content, enzymatic activity, and PARP-1 DNA binding. Furthermore, the effects of arsenite on PARP-1 activity, DNA binding, and zinc content were partially reversed by the antioxidant ascorbic acid, catalase, and the NOS inhibitor, aminoguanidine. Most importantly, arsenite incubation with purified PARP-1 protein in vitro did not alter PARP-1 activity or DNA-binding ability, whereas hydrogen peroxide or NONOate retained PARP-1 inhibitory activity. These results strongly suggest that cellular generation of ROS/RNS plays an important role in arsenite inhibition of PARP-1 activity, leading to the loss of PARP-1 DNA-binding ability and enzymatic activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Adipose tissue NAD+-homeostasis, sirtuins and poly(ADP-ribose) polymerases -important players in mitochondrial metabolism and metabolic health.

    Science.gov (United States)

    Jokinen, Riikka; Pirnes-Karhu, Sini; Pietiläinen, Kirsi H; Pirinen, Eija

    2017-08-01

    Obesity, a chronic state of energy overload, is characterized by adipose tissue dysfunction that is considered to be the major driver for obesity associated metabolic complications. The reasons for adipose tissue dysfunction are incompletely understood, but one potential contributing factor is adipose tissue mitochondrial dysfunction. Derangements of adipose tissue mitochondrial biogenesis and pathways associate with obesity and metabolic diseases. Mitochondria are central organelles in energy metabolism through their role in energy derivation through catabolic oxidative reactions. The mitochondrial processes are dependent on the proper NAD + /NADH redox balance and NAD + is essential for reactions catalyzed by the key regulators of mitochondrial metabolism, sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs). Notably, obesity is associated with disturbed adipose tissue NAD + homeostasis and the balance of SIRT and PARP activities. In this review we aim to summarize existing literature on the maintenance of intracellular NAD + pools and the function of SIRTs and PARPs in adipose tissue during normal and obese conditions, with the purpose of comprehending their potential role in mitochondrial derangements and obesity associated metabolic complications. Understanding the molecular mechanisms that are the root cause of the adipose tissue mitochondrial derangements is crucial for developing new effective strategies to reverse obesity associated metabolic complications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Imidazoquinolinone, imidazopyridine, and isoquinolindione derivatives as novel and potent inhibitors of the poly(ADP-ribose) polymerase (PARP): a comparison with standard PARP inhibitors.

    Science.gov (United States)

    Eltze, Tobias; Boer, Rainer; Wagner, Thomas; Weinbrenner, Steffen; McDonald, Michelle C; Thiemermann, Christoph; Bürkle, Alexander; Klein, Thomas

    2008-12-01

    We have identified three novel structures for inhibitors of the poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA and implicated in DNA repair, apoptosis, organ dysfunction or necrosis. 2-[4-(5-Methyl-1H-imidazol-4-yl)-piperidin-1-yl]-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK49187), 2-(4-pyridin-2-yl-phenyl)-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK236864), 6-chloro-8-hydroxy-2,3-dimethyl-imidazo-[1,2-alpha]-pyridine (BYK20370), and 4-(1-methyl-1H-pyrrol-2-ylmethylene)-4H-isoquinolin-1,3-dione (BYK204165) inhibited cell-free recombinant human PARP-1 with pIC(50) values of 8.36, 7.81, 6.40, and 7.35 (pK(i) 7.97, 7.43, 5.90, and 7.05), and murine PARP-2 with pIC(50) values of 7.50, 7.55, 5.71, and 5.38, respectively. BYK49187, BYK236864, and BYK20370 displayed no selectivity for PARP-1/2, whereas BYK204165 displayed 100-fold selectivity for PARP-1. The IC(50) values for inhibition of poly(ADP-ribose) synthesis in human lung epithelial A549 and cervical carcinoma C4I cells as well in rat cardiac myoblast H9c2 cells after PARP activation by H(2)O(2) were highly significantly correlated with those at cell-free PARP-1 (r(2) = 0.89-0.96, P < 0.001) but less with those at PARP-2 (r(2) = 0.78-0.84, P < 0.01). The infarct size caused by coronary artery occlusion and reperfusion in the anesthetized rat was reduced by 22% (P < 0.05) by treatment with BYK49187 (3 mg/kg i.v. bolus and 3 mg/kg/h i.v. during 2-h reperfusion), whereas the weaker PARP inhibitors, BYK236864 and BYK20370, were not cardioprotective. In conclusion, the imidazoquinolinone BYK49187 is a potent inhibitor of human PARP-1 activity in cell-free and cellular assays in vitro and reduces myocardial infarct size in vivo. The isoquinolindione BYK204165 was found to be 100-fold more selective for PARP-1. Thus, both compounds might be novel and valuable tools for investigating PARP-1-mediated effects.

  3. The inhibition of nitric oxide-activated poly(ADP-ribose) synthetase attenuates transsynaptic alteration of spinal cord dorsal horn neurons and neuropathic pain in the rat.

    Science.gov (United States)

    Mao, J; Price, D D; Zhu, J; Lu, J; Mayer, D J

    1997-09-01

    Transsynaptic alteration of spinal cord dorsal horn neurons characterized by hyperchromatosis of cytoplasm and nucleoplasm (so-called 'dark' neurons) occurs in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the common sciatic nerve. The incidence of dark neurons in CCI rats has been proposed to be mediated by glutamate-induced neurotoxicity. In the present study, we examined whether the inhibition of the nitric oxide (NO)-activated poly(ADP-ribose) synthetase (PARS), a nuclear enzyme critical to glutamate-induced neurotoxicity, would both reduce the incidence of dark neurons and attenuate behavioral manifestations of neuropathic pain in CCI rats. Dark neurons were observed bilaterally (with ipsilateral predominance) within the spinal cord dorsal horn, particularly in laminae I-II, of rats 8 days after unilateral sciatic nerve ligation as compared to sham operated rats. The number of dark neurons in the dorsal horn was dose-dependently reduced in CCI rats receiving once daily intrathecal (i.t.) treatment with the PARS inhibitor benzamide (200 or 400 nmol, but not 100 nmol benzamide or saline) for 7 days. Consistent with the histological improvement, thermal hyperalgesia, mechanical hyperalgesia, and low threshold mechano-allodynia also were reliably reduced in CCI rats treated with either 200 or 400 nmol benzamide. Neither dark neurons nor neuropathic pain behaviors were reliably affected by i.t. administration of either 800 nmol novobiocin (a mono(ADP-ribose) synthetase) or 800 nmol benzoic acid (the backbone structure of benzamide), indicating a selective effect of benzamide. Intrathecal treatment with an NO synthase inhibitor NG-nitro-L-arginine methyl ester (40 nmol, but not its inactive D-isomer) utilizing the same benzamide treatment regimen resulted in similar reductions of both dark neurons and neuropathic pain behaviors in CCI rats. These results provide, for the first time, in vivo evidence indicating that benzamide is

  4. Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair, chromosomal alterations, and mutations

    Energy Technology Data Exchange (ETDEWEB)

    Natarajan, A.T.; van Zeeland, A.A.; Zwanenburg, T.S.

    1983-01-01

    The influence of inhibitors of poly(ADP-ribose) polymerase such as 3-aminobenzamide (3AB) and benzamide (B) on the spontaneously occurring as well as mutagen induced chromosomal aberrations, sister chromatid exchanges (SCEs) and point mutations has been studied. In addition, the influence of 3AB on DNA repair was measured following treatment with physical and chemical mutagens. Post treatment of X-irradiated mammalian cells with 3AB increases the frequencies of induced chromosomal aberrations by a factor of 2 to 3. 3AB, when present in the medium containing bromodeoxyuridine(BrdUrd) during two cell cycles, increases the frequencies of SCEs in Chinese hamster ovary cells (CHO) in a concentration dependent manner leading to about a 10-fold increase at 10 mM concentration. The extent of increase in the frequencies of SCEs due to 1 mM 3AB in several human cell lines has been studied, including those derived from patients suffering from genetic diseases such as ataxia telangiectasia (A-T), Fanconi's anemia (FA), and Huntington's chorea. None of these syndromes showed any increased response when compared to normal cells. 3AB, however, increased the frequencies of spontaneously occurring chromosomal aberrations in A-T and FA cells. 3AB does not influence the frequencies of SCEs induced by UV or mitomycin C (MMC) in CHO cells. However, it increases the frequencies of SCEs induced by ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). Under the conditions in which 3AB increases the frequencies of spontaneously occurring as well as induced SCEs, it does not increase the frequencies of point mutations in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus. 3AB does not influence the amount of repair replication following dimethylsulphate (DMS) treatment of human fibroblasts, or UV irradiated human lymphocytes.

  5. Altered chromatographic behaviour of mitochondrial ADP/ATP translocase induced by stabilization of the protein by binding of 6'-O-fluorescein-atractyloside.

    Science.gov (United States)

    Smith, Vernon R; Fearnley, Ian M; Walker, John E

    2003-01-01

    Atractyloside (ATR) is a high-affinity specific inhibitor of the mitochondrial ADP/ATP translocase (AAT). The binding of a fluorescent derivative, 6'- O -fluorescein-ATR (FATR), to mitochondria has been characterized. The binding constants obtained are in agreement with previously published values for ATR, demonstrating that FATR is a suitable probe of the AAT. AAT inhibited by FATR (FATR-AAT) was solubilized in dodecyl maltoside and purified by two separate ion-exchange chromatography steps at different pHs, which allowed FATR-AAT to be purified to homogeneity. The presence of the bound fluorescent probe enabled the inhibited AAT to be distinguished from the unliganded protein during chromatography, as they were markedly different in their chromatographic behaviour. The purified FATR-AAT was dimeric and in a single major conformation containing 1 mole FATR per mole of AAT dimer. In contrast, uninhibited AAT was monomeric and conformationally unstable. Use of the fluorescent ATR derivative in the development of the protocol enabled the stable dimeric AAT to be monitored directly and purified more effectively. The purification protocol was repeated using non-derivatized ATR, and highly pure AAT was obtained that was devoid of other members of the mitochondrial carrier family. PMID:14498831

  6. Data Profiling

    OpenAIRE

    Hladíková, Radka

    2010-01-01

    Title: Data Profiling Author: Radka Hladíková Department: Department of Software Engineering Supervisor: Ing. Vladimír Kyjonka Supervisor's e-mail address: Abstract: This thesis puts mind on problems with data quality and data profiling. This Work analyses and summarizes problems of data quality, data defects, process of data quality, data quality assessment and data profiling. The main topic is data profiling as a process of researching data available in existing...

  7. Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

    Science.gov (United States)

    Robu, Mihaela; Shah, Rashmi G; Purohit, Nupur K; Zhou, Pengbo; Naegeli, Hanspeter; Shah, Girish M

    2017-08-15

    Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

  8. Critical role of poly(ADP-ribose) polymerase-1 in modulating the mode of cell death caused by continuous oxidative stress.

    Science.gov (United States)

    Son, Young-Ok; Kook, Sung-Ho; Jang, Yong-Suk; Shi, Xianglin; Lee, Jeong-Chae

    2009-11-01

    Continuously generated hydrogen peroxide (H(2)O(2)) inhibits typical apoptosis and instead initiates a caspase-independent, apoptosis-inducing factor (AIF)-mediated pyknotic cell death. This may be related to H(2)O(2)-mediated DNA damage and subsequent ATP depletion, although the exact mechanisms by which the mode of cell death is decided after H(2)O(2) exposure are still unclear. Accumulated evidence and our previous data led us to hypothesize that continuously generated H(2)O(2), not an H(2)O(2) bolus, induces severe DNA damage, signaling poly(ADP-ribose) polymerase-1 (PARP-1) activation, ATP depletion, and eventually caspase-independent cell death. Results from the present study support that H(2)O(2) generated continuously by glucose oxidase causes excessive DNA damage and PARP-1 activation. Blockage of PARP-1 by a siRNA transfection or by pharmacological inhibitor resulted in the significant inhibition of ATP depletion, loss of mitochondrial membrane potential, nuclear translocation of AIF and endonuclease G, and eventually conversion to caspase-dependent apoptosis. Overall, the current study demonstrates the different roles of PARP-1 inhibition in modulation of cell death according to the method of H(2)O(2) exposure, that is, continuous generation versus a direct addition. (c) 2009 Wiley-Liss, Inc.

  9. Cyclic ADP-ribose and heat regulate oxytocin release via CD38 and TRPM2 in the hypothalamus during social or psychological stress in mice

    Directory of Open Access Journals (Sweden)

    Jing Zhong

    2016-07-01

    Full Text Available Hypothalamic oxytocin (OT is released into the brain by cyclic ADP-ribose (cADPR with or without depolarizing stimulation. Previously, we showed that the intracellular free calcium concentration ([Ca2+]i that seems to trigger OT release can be elevated by -NAD+, cADPR, and ADP in mouse oxytocinergic neurons. As these -NAD+ metabolites activate warm-sensitive TRPM2 cation channels, when the incubation temperature is increased, the [Ca2+]i in hypothalamic neurons is elevated. However, it has not been determined whether OT release is facilitated by heat in vitro or hyperthermia in vivo in combination with cADPR. Furthermore, it has not been examined whether CD38 and TRPM2 exert their functions on OT release during stress or stress-induced hyperthermia in relation to the anxiolytic roles and social behaviors of OT under stress conditions. Here, we report that OT release from the isolated hypothalami of male mice in culture was enhanced by extracellular application of cADPR or increasing the incubation temperature from 35°C to 38.5°C, and simultaneous stimulation showed a greater effect. This release was inhibited by a cADPR-dependent ryanodine receptor inhibitor and a nonspecific TRPM2 inhibitor. The facilitated release by heat and cADPR was suppressed in the hypothalamus isolated from CD38 knockout mice and CD38- or TRPM2-knockdown mice. In the course of these experiments, we noted that OT release differed markedly between individual mice under stress with group housing. That is, when male mice received cage-switch stress and eliminated due to their social subclass, significantly higher levels of OT release were found in subordinates compared with ordinates. In mice exposed to anxiety stress in an open field, the cerebrospinal fluid (CSF OT level increased transiently at 5 minutes after exposure, and the rectal temperature also increased from 36.6°C to 37.8°C. OT levels in the CSF of mice with lipopolysaccharide-induced fever (+0.8

  10. Different Principles of ADP-Ribose-Mediated Activation and Opposite Roles of the NUDT9 Homology Domain in the TRPM2 Orthologs of Man and Sea Anemone

    Directory of Open Access Journals (Sweden)

    Frank Kühn

    2017-10-01

    Full Text Available A decisive element in the human cation channel TRPM2 is a region in its cytosolic C-terminus named NUDT9H because of its homology to the NUDT9 enzyme, a pyrophosphatase degrading ADP-ribose (ADPR. In hTRPM2, however, the NUDT9H domain has lost its enzymatic activity but serves as a binding domain for ADPR. As consequence of binding, gating of the channel is initiated. Since ADPR is produced after oxidative DNA damage, hTRPM2 mediates Ca2+ influx in response to oxidative stress which may lead to cell death. In the genome of the sea anemone Nematostella vectensis (nv, a preferred model organism for the evolution of key bilaterian features, a TRPM2 ortholog has been identified that contains a NUDT9H domain as well. Heterologous expression of nvTRPM2 in HEK-293 cells reveals a cation channel with many close similarities to the human counterpart. Most notably, nvTRPM2 is activated by ADPR, and Ca2+ is a co-agonist. However, the intramolecular mechanisms of ADPR gating as well as the role of NUDT9H are strikingly different in the two species. Whereas already subtle changes of NUDT9H abolish ADPR gating in hTRPM2, the region can be completely removed from nvTRPM2 without loss of responses to ADPR. An alternative ADPR binding site seems to be present but has not yet been characterized. The ADP-ribose pyrophosphatase (ADPRase function of nvNUDT9H has been preserved but can be abolished by numerous genetic manipulations. All these manipulations create channels that are sensitive to hydrogen peroxide which fails to induce channel activity in wild-type nvTRPM2. Therefore, the function of NUDT9H in nvTRPM2 is the degradation of ADPR, thereby reducing agonist concentration in the presence of oxidative stress. Thus, the two TRPM2 orthologs have evolved divergently but nevertheless gained analogous functional properties, i.e., gating by ADPR with Ca2+ as co-factor. Opposite roles are played by the respective NUDT9H domains, either binding of ADPR and mediating

  11. NMR-based metabolomic studies on the toxicological effects of cadmium and copper on green mussels Perna viridis

    International Nuclear Information System (INIS)

    Wu Huifeng; Wang Wenxiong

    2010-01-01

    Traditional toxicology studies have focused on selected biomarkers to characterize the biological stress induced by metals in marine organisms. In this study, a system biology tool, metabolomics, was applied to the marine mussel Perna viridis to investigate changes in the metabolic profiles of soft tissue as a response to copper (Cu) and cadmium (Cd), both as single metal and as a mixture. The major metabolite changes corresponding to metal exposure are related to amino acids, osmolytes, and energy metabolites. Following metal exposure for 1 week, there was a significant increase in the levels of branched chain amino acids, histidine, glutamate, glutamine, hypotaurine, dimethylglycine, arginine and ATP/ADP. For the Cu + Cd co-exposed mussels, the levels of lactate, branched chain amino acid, succinate, and NAD increased, whereas the levels of glucose, glycogen, and ATP/ADP decreased, indicating a different metabolic profile for the single metal exposure groups. After 2 weeks of exposure, the mussels showed acclimatization to Cd exposure based on the recovery of some metabolites. However, the metabolic profile induced by the metal mixture was very similar to that from Cu exposure, suggesting that Cu dominantly induced the metabolic disturbances. Both Cu and Cd may lead to neurotoxicity, disturbances in energy metabolism, and osmoregulation changes. These results demonstrate the high applicability and reliability of NMR-based metabolomics in interpreting the toxicological mechanisms of metals using global metabolic biomarkers.

  12. Notch signaling protects retina from nuclear factor-kB- and poly-ADP-ribosepolymerase-mediated apoptosis under high-glucose stimulation

    Institute of Scientific and Technical Information of China (English)

    Xiuhong Qin1; Zhenzhen Zhang2; Haitao Xu1; and Yazhen Wu1

    2011-01-01

    Proliferative diabetic retinopathy,the primary cause of vision loss in adults,is one of serious microvascular complications caused by diabetes.Both poly-ADP-ribosepolymerase (PARP) and nuclear factor (NF)-kB signaling are involved in the injury process.Injury activates PARP,which in turn potentiates NF-kB activation and causes cell apoptosis.Like the NF-kB pathway,Notch1 signaling plays a key role in the regulation of cell proliferation,differentiation,and apoptosis.However,the connections between these signaling pathways are not well understood.In this study,we used both streptozotocin (STZ)-induced diabetic mice and human retinal vascular endothelial cells (HRVECs) cultured in high glucose to detect these relationships.We found that apoptosis was increased in both STZinduced diabetic mice and high-glucose-treated HRVECs,which was due to increased activation of PARP,cleaved caspase3,and reduced expression of Notch1 and p-Akt.The results of Notch1 overexpression and knockdown indicated that Notch1 signaling participated in the interaction of PARP and p50,and inhibited PARP- and p50-mediated apoptosis directly.These phenomena could be blocked by pretreatment with the PI3K inhibitor wortmannin via reducing p-Akt levels.Thus,our study demonstrated that Notch1 signaling protects cells from PARP- and NF-kB-induced apoptosis under high glucose through the activation of Akt.

  13. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: Two guanine nucleotide-dependent activators of cholera toxin

    International Nuclear Information System (INIS)

    Bobak, D.A.; Nightingale, M.S.; Murtagh, J.J.; Price, S.R.; Moss, J.; Vaughan, M.

    1989-01-01

    ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A) + RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A) + RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFS also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs

  14. Minocycline attenuates streptomycin-induced cochlear hair cell death by inhibiting protein nitration and poly (ADP-ribose) polymerase activation.

    Science.gov (United States)

    Wang, Ping; Li, Haonan; Yu, Shuyuan; Jin, Peng; Hassan, Abdurahman; Du, Bo

    2017-08-24

    This study aimed to elucidate the protective effect of minocycline against streptomycin-induced damage of cochlear hair cells and its mechanism. Cochlear membranes were isolated from newborn Wistar rats and randomly divided into control, 500μmol/L streptomycin, 100μmol/L minocycline, and streptomycin and minocycline treatment groups. Hair cell survival was analyzed by detecting the expression of 3-nitrotyrosine (3-NT) in cochlear hair cells by immunofluorescence and an enzyme-linked immunosorbent assay. Expression of 3-NT and inducible nitric oxide synthase (iNOS), and poly (ADP-Ribose) polymerase (PARP) and caspase-3 activation were evaluated by western blotting. The results demonstrated hair cell loss at 24h after streptomycin treatment. No change was found in supporting cells of the cochleae. Minocycline pretreatment improved hair cell survival and significantly reduced the expression of iNOS and 3-NT in cochlear tissues compared with the streptomycin treatment group. PARP and caspase-3 activation was increased in the streptomycin treatment group compared with the control group, and pretreatment with minocycline decreased cleaved PARP and activated caspase-3 expression. Minocycline protected cochlear hair cells from injury caused by streptomycin in vitro. The mechanism underlying the protective effect may be associated with the inhibition of excessive formation of nitric oxide, reduction of the nitration stress reaction, and inhibition of PARP and caspase-3 activation in cochlear hair cells. Combined minocycline therapy can be applied to patients requiring streptomycin treatment. Copyright © 2017. Published by Elsevier B.V.

  15. Inhibition of gamma-ray dose-rate effects by D/sup 2/O and inhibitors of poly(ADP-ribose) synthetase in cultured mammalian L5178Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Ueno, A.M.; Tanaka, O.; Matsudaira, H.

    1984-06-01

    Effects of deuterium oxide (D/sub 2/O) and 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, on cell proliferation and survival were studied in cultured mammalian L5178Y cells under growing conditions and after acute and low-dose-rate irradiation at about 0.1 to 0.4 Gy/hr of ..gamma.. rays. Growth of irradiated and unirradiated cells was inhibited by 45% D/sub 2/O but not by 3-aminobenzamide at 10mM, except for treatments longer than 30 hr. The presence of these agents either alone or in combination during irradiation at low dose rates suppressed almost totally the decrease in cell killing due to the decrease in dose rate. Among other inhibitors tested, theobromine and theophylline were found to be effective in eliminating the dose-rate effects of ..gamma.. rays. Possible mechanisms underlying the inhibition are discussed.

  16. Shave-off depth profiling: Depth profiling with an absolute depth scale

    International Nuclear Information System (INIS)

    Nojima, M.; Maekawa, A.; Yamamoto, T.; Tomiyasu, B.; Sakamoto, T.; Owari, M.; Nihei, Y.

    2006-01-01

    Shave-off depth profiling provides profiling with an absolute depth scale. This method uses a focused ion beam (FIB) micro-machining process to provide the depth profile. We show that the shave-off depth profile of a particle reflected the spherical shape of the sample and signal intensities had no relationship to the depth. Through the introduction of FIB micro-sampling, the shave-off depth profiling of a dynamic random access memory (DRAM) tip was carried out. The shave-off profile agreed with a blue print from the manufacturing process. Finally, shave-off depth profiling is discussed with respect to resolutions and future directions

  17. Searching mixed DNA profiles directly against profile databases.

    Science.gov (United States)

    Bright, Jo-Anne; Taylor, Duncan; Curran, James; Buckleton, John

    2014-03-01

    DNA databases have revolutionised forensic science. They are a powerful investigative tool as they have the potential to identify persons of interest in criminal investigations. Routinely, a DNA profile generated from a crime sample could only be searched for in a database of individuals if the stain was from single contributor (single source) or if a contributor could unambiguously be determined from a mixed DNA profile. This meant that a significant number of samples were unsuitable for database searching. The advent of continuous methods for the interpretation of DNA profiles offers an advanced way to draw inferential power from the considerable investment made in DNA databases. Using these methods, each profile on the database may be considered a possible contributor to a mixture and a likelihood ratio (LR) can be formed. Those profiles which produce a sufficiently large LR can serve as an investigative lead. In this paper empirical studies are described to determine what constitutes a large LR. We investigate the effect on a database search of complex mixed DNA profiles with contributors in equal proportions with dropout as a consideration, and also the effect of an incorrect assignment of the number of contributors to a profile. In addition, we give, as a demonstration of the method, the results using two crime samples that were previously unsuitable for database comparison. We show that effective management of the selection of samples for searching and the interpretation of the output can be highly informative. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  18. Comparison of the release of microRNAs and extracellular vesicles from platelets in response to different agonists.

    Science.gov (United States)

    Ambrose, Ashley R; Alsahli, Mohammed A; Kurmani, Sameer A; Goodall, Alison H

    2018-07-01

    On activation platelets release microRNAs and extracellular vesicles (EV) into circulation. The release of EV from platelets has been shown to be dependent on the agonist; in this study, we investigated whether the microRNA profile or EV released from platelets was also agonist specific. Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (Glycoprotein VI (GPVI)), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block cyclooxygenase-1 and apyrase to remove ADP. The released microRNAs were profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterized by size (Nanoparticle Tracking Analysis, NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70. Platelet activation triggered the release of 57-79 different microRNAs, dependent upon agonist, with a core of 46 microRNAs observed with all agonists. There was a high level of correlation between agonists (r 2  > 0.98; p  0.98; p < 0.0001). The 46 microRNAs seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control, and differentiation. MiR-223-3p was the most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have anti-inflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect. These data suggest that all tested agonists trigger the release of a similar microRNA profile while the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNAs and pdEV.

  19. Profiles

    International Nuclear Information System (INIS)

    2004-01-01

    Profiles is a synthetic overview of more than 100 national energy markets in the world, providing insightful facts and key energy statistics. A Profile is structured around 6 main items and completed by key statistics: Ministries, public agencies, energy policy are concerned; main companies in the oil, gas, electricity and coal sectors, status, shareholders; reserve, production, imports and exports, electricity and refining capacities; deregulation of prices, subsidies, taxes; consumption trends by sector, energy market shares; main energy projects, production and consumption prospects. Statistical Profiles are present in about 3 pages the main data and indicators on oil, gas, coal and electricity. (A.L.B.)

  20. PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

    Science.gov (United States)

    Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

    1992-06-01

    Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

  1. Flourimetric and prototropic studies on the inclusion complexation of 2-amino and 4-aminodiphenyl ethers with {beta}-cyclodextrin: Unusual behavior of 4-aminodiphenyl ether

    Energy Technology Data Exchange (ETDEWEB)

    Enoch, Israel V. Muthu Vijayan [Department of Chemistry, Annamalai University, Annamalainagar 608 002, Tamil Nadu (India); Swaminathan, Meenakshisundaram [Department of Chemistry, Annamalai University, Annamalainagar 608 002, Tamil Nadu (India)], E-mail: chemsam@yahoo.com

    2007-12-15

    The fluorescence characteristics of diphenyl ether (DPE), 2-aminodiphenyl ether (2ADPE) and 4-aminodiphenyl ether (4ADPE) and prototropic behavior of 2ADPE and 4ADPE on inclusion complexation with {beta}-cyclodextrin have been investigated. DPE forms 1:1 complex whereas 2ADPE and 4ADPE form 1:2 complex with {beta}-CDx. The fluorimetric and prototropic behaviors of 4ADPE in {beta}-CDx are different from those in aqueous solution. The dual fluorescence of 4ADPE in {beta}-CDx is found to be due to twisted intramolecular charge transfer (TICT) character induced by inclusion complexation. The two equilibria viz. monocation{r_reversible}monocation solvent exciplex{r_reversible}neutral reported for 4ADPE in aqueous solution are not observed in presence of {beta}-CDx. The ground and excited state pK{sub a} values for monocation-neutral equilibrium of 2ADPE and 4ADPE have been reported.

  2. Karolinske psychodynamic profile (KAPP)

    DEFF Research Database (Denmark)

    Mathiesen, Birgit Bork; Søgaard, Ulf

    2006-01-01

    psykologiske testmetoder, assesment, Karolinska psychodynamic profile (KAPP), psykodynamisk profil......psykologiske testmetoder, assesment, Karolinska psychodynamic profile (KAPP), psykodynamisk profil...

  3. Influence of MLH1 on colon cancer sensitivity to poly(ADP-ribose) polymerase inhibitor combined with irinotecan.

    Science.gov (United States)

    Tentori, Lucio; Leonetti, Carlo; Muzi, Alessia; Dorio, Annalisa Susanna; Porru, Manuela; Dolci, Susanna; Campolo, Federica; Vernole, Patrizia; Lacal, Pedro Miguel; Praz, Françoise; Graziani, Grazia

    2013-07-01

    Poly(ADP-ribose) polymerase inhibitors (PARPi) are currently evaluated in clinical trials in combination with topoisomerase I (Top1) inhibitors against a variety of cancers, including colon carcinoma. Since the mismatch repair component MLH1 is defective in 10-15% of colorectal cancers we have investigated whether MLH1 affects response to the Top1 inhibitor irinotecan, alone or in combination with PARPi. To this end, the colon cancer cell lines HCT116, carrying MLH1 mutations on chromosome 3 and HCT116 in which the wild-type MLH1 gene was replaced via chromosomal transfer (HCT116+3) or by transfection of the corresponding MLH1 cDNA (HCT116 1-2) were used. HCT116 cells or HCT116+3 cells stably silenced for PARP-1 expression were also analysed. The results of in vitro and in vivo experiments indicated that MLH1, together with low levels of Top1, contributed to colon cancer resistance to irinotecan. In the MLH1-proficient cells SN-38, the active metabolite of irinotecan, induced lower levels of DNA damage than in MLH1-deficient cells, as shown by the weaker induction of γ-H2AX and p53 phosphorylation. The presence of MLH1 contributed to induce of prompt Chk1 phosphorylation, restoring G2/M cell cycle checkpoint and repair of DNA damage. On the contrary, in the absence of MLH1, HCT116 cells showed minor Chk1 phosphorylation and underwent apoptosis. Remarkably, inhibition of PARP function by PARPi or by PARP-1 gene silencing always increased the antitumor activity of irinotecan, even in the presence of low PARP-1 expression.

  4. Does S-metolachlor affect the performance of Pseudomonas sp. strain ADP as bioaugmentation bacterium for atrazine-contaminated soils?

    Directory of Open Access Journals (Sweden)

    Cristina A Viegas

    Full Text Available Atrazine (ATZ and S-metolachlor (S-MET are two herbicides widely used, often as mixtures. The present work examined whether the presence of S-MET affects the ATZ-biodegradation activity of the bioaugmentation bacterium Pseudomonas sp. strain ADP in a crop soil. S-MET concentrations were selected for their relevance in worst-case scenarios of soil contamination by a commercial formulation containing both herbicides. At concentrations representative of application of high doses of the formulation (up to 50 µg g(-1 of soil, corresponding to a dose approximately 50× higher than the recommended field dose (RD, the presence of pure S-MET significantly affected neither bacteria survival (~10(7 initial viable cells g(-1 of soil nor its ATZ-mineralization activity. Consistently, biodegradation experiments, in larger soil microcosms spiked with 20× or 50 × RD of the double formulation and inoculated with the bacterium, revealed ATZ to be rapidly (in up to 5 days and extensively (>96% removed from the soil. During the 5 days, concentration of S-MET decreased moderately to about 60% of the initial, both in inoculated and non-inoculated microcosms. Concomitantly, an accumulation of the two metabolites S-MET ethanesulfonic acid and S-MET oxanilic acid was found. Despite the dissipation of almost all the ATZ from the treated soils, the respective eluates were still highly toxic to an aquatic microalgae species, being as toxic as those from the untreated soil. We suggest that this high toxicity may be due to the S-MET and/or its metabolites remaining in the soil.

  5. Cloning of an ADP-ribosylation factor gene from banana (Musa acuminata) and its expression patterns in postharvest ripening fruit.

    Science.gov (United States)

    Wang, Yuan; Wu, Jing; Xu, Bi-Yu; Liu, Ju-Hua; Zhang, Jian-Bin; Jia, Cai-Hong; Jin, Zhi-Qiang

    2010-08-15

    A full-length cDNA encoding an ADP-ribosylation factor (ARF) from banana (Musa acuminata) fruit was cloned and named MaArf. It contains an open reading frame encoding a 181-amino-acid polypeptide. Sequence analysis showed that MaArf shared high similarity with ARF of other plant species. The genomic sequence of MaArf was also obtained using polymerase chain reaction (PCR). Sequence analysis showed that MaArf was a split gene containing five exons and four introns in genomic DNA. Reverse-transcriptase PCR was used to analyze the spatial expression of MaArf. The results showed that MaArf was expressed in all the organs examined: root, rhizome, leaf, flower and fruit. Real-time quantitative PCR was used to explore expression patterns of MaArf in postharvest banana. There was differential expression of MaArf associated with ethylene biosynthesis. In naturally ripened banana, expression of MaArf was in accordance with ethylene biosynthesis. However, in 1-methylcyclopropene-treated banana, the expression of MaArf was inhibited and changed little. When treated with ethylene, MaArf expression in banana fruit significantly increased in accordance with ethylene biosynthesis; the peak of MaArf was 3 d after harvest, 11 d earlier than for naturally ripened banana fruits. These results suggest that MaArf is induced by ethylene in regulating postharvest banana ripening. Finally, subcellular localization assays showed the MaArf protein in the cytoplasm. Copyright 2010 Elsevier GmbH. All rights reserved.

  6. The nuclear protein Poly(ADP-ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana.

    Science.gov (United States)

    Rissel, D; Losch, J; Peiter, E

    2014-11-01

    The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP-ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear-localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3-1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col-0 wild-type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

  7. Profile: Asian Americans

    Science.gov (United States)

    ... and Data > Minority Population Profiles > Asian American Profile: Asian Americans Asian American Profile (Map of the US with the top 10 states displaying the largest Asian American population according to the Census Bureau) CA - ...

  8. Trial watch – inhibiting PARP enzymes for anticancer therapy

    Science.gov (United States)

    Sistigu, Antonella; Manic, Gwenola; Obrist, Florine; Vitale, Ilio

    2016-01-01

    ABSTRACT Poly(ADP-ribose) polymerases (PARPs) are a members of family of enzymes that catalyze poly(ADP-ribosyl)ation (PARylation) and/or mono(ADP-ribosyl)ation (MARylation), two post-translational protein modifications involved in crucial cellular processes including (but not limited to) the DNA damage response (DDR). PARP1, the most abundant family member, is a nuclear protein that is activated upon sensing distinct types of DNA damage and contributes to their resolution by PARylating multiple DDR players. Recent evidence suggests that, along with DDR, activated PARP1 mediates a series of prosurvival and proapoptotic processes aimed at preserving genomic stability. Despite this potential oncosuppressive role, upregulation and/or overactivation of PARP1 or other PARP enzymes has been reported in a variety of human neoplasms. Over the last few decades, several pharmacologic inhibitors of PARP1 and PARP2 have been assessed in preclinical and clinical studies showing potent antineoplastic activity, particularly against homologous recombination (HR)-deficient ovarian and breast cancers. In this Trial Watch, we describe the impact of PARP enzymes and PARylation in cancer, discuss the mechanism of cancer cell killing by PARP1 inactivation, and summarize the results of recent clinical studies aimed at evaluating the safety and therapeutic profile of PARP inhibitors in cancer patients. PMID:27308587

  9. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

    Energy Technology Data Exchange (ETDEWEB)

    La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy); Caligo, Maria Adelaide [Section of Genetic Oncology, University Hospital and University of Pisa, via Roma 57, 56125 Pisa (Italy); Galli, Alvaro, E-mail: alvaro.galli@ifc.cnr.it [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy)

    2015-04-15

    Highlights: • The human poly (ADP-ribose) polymerase 1 (PARP-1) gene affects growth and UV-induced homologous recombination in yeast. • PARP-1 chemical inhibition impacts yeast growth and UV-induced recombination. • A genome-wide screen identifies 99 yeast genes that suppress the growth defect inferred by PARP-1. • Bioinformatics analysis identifies 41 human orthologues that may have a role in PARP-1 intracellular localization. • The findings suggest that PARP-1 nuclear localization may affect the response to PARP inhibitors in cancer therapy. - Abstract: The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the

  10. Design and development of a peptide-based adiponectin receptor agonist for cancer treatment

    Directory of Open Access Journals (Sweden)

    Lovas Sandor

    2011-10-01

    displayed excellent stability (at least 30 min in mouse blood or serum and did not induce gross toxic effects at 5-50 mg/kg bolus doses in normal CBA/J mice. Conclusions ADP 355 is a first-in-class adiponectin receptor agonist. Its biological activity, superior stability in biological fluids as well as acceptable toxicity profile indicate that the peptidomimetic represents a true lead compound for pharmaceutical development to replace low adiponectin levels in cancer and other malignancies.

  11. Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

    Directory of Open Access Journals (Sweden)

    Amanda Piccoli Frasson

    2012-03-01

    Full Text Available Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum. The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

  12. Fitting the IRI F2-profile function to measured profiles

    International Nuclear Information System (INIS)

    Reinisch, B.W.; Huang Xueqin

    1997-01-01

    Comparison with profile data from ionosondes shows that the IRI bottomside F2-profiles can be improved by using better B0 and B1 parameters. The best parameters (in a least-squares sense) can be easily calculated in a numerical procedure from measured profiles presented as a sum of Chebyshev polynomials. 7 refs, 5 figs, 1 tab

  13. Inhibition of potentially lethal radiation damage repair in normal and neoplastic human cells by 3-aminobenzamide: an inhibitor of poly(ADP-ribosylation)

    International Nuclear Information System (INIS)

    Thraves, P.J.; Mossman, K.L.; Frazier, D.T.; Dritschilo, A.

    1986-01-01

    The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, on potentially lethal damage repair (PLDR) was investigated in normal human fibroblasts and four human tumor cell lines from tumors with varying degrees of radiocurability. The tumor lines selected were: Ewing's sarcoma, a bone tumor considered radiocurable and, human lung adenocarcinoma, osteosarcoma, and melanoma, three tumors considered nonradiocurable. PLDR was measured by comparing cell survival when cells were irradiated in a density-inhibited state and replated at appropriate cell numbers at specified times following irradiation to cell survival when cells were replated immediately following irradiation. 3AB was added to cultures 2 hr prior to irradiation and removed at the time of replating. Different test radiation doses were used for the various cell lines to obtain equivalent levels of cell survival. In the absence of inhibitor, PLDR was similar in all cell lines tested. In the presence of 8 mM 3AB, differential inhibition of PLDR was observed. PLDR was almost completely inhibited in Ewing's sarcoma cells and partially inhibited in normal fibroblast cells and osteosarcoma cells. No inhibition of PLDR was observed in the lung adenocarcinoma or melanoma cells. Except for the osteosarcoma cells, inhibition of PLDR by 3AB correlated well with radiocurability

  14. Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

    Energy Technology Data Exchange (ETDEWEB)

    Aoyagi-Scharber, Mika, E-mail: maoyagi@bmrn.com [BioMarin Pharmaceutical Inc., 105 Digital Drive, Novato, CA 94949 (United States); Gardberg, Anna S. [Emerald BioStructures, 7869 NE Day Road West, Bainbridge Island, WA 98110 (United States); Yip, Bryan K.; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A. [BioMarin Pharmaceutical Inc., 105 Digital Drive, Novato, CA 94949 (United States)

    2014-08-29

    BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity. Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity.

  15. Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

    International Nuclear Information System (INIS)

    Aoyagi-Scharber, Mika; Gardberg, Anna S.; Yip, Bryan K.; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A.

    2014-01-01

    BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity. Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity

  16. High-resolution humidity profiles retrieved from wind profiler radar measurements

    Science.gov (United States)

    Saïd, Frédérique; Campistron, Bernard; Di Girolamo, Paolo

    2018-03-01

    The retrieval of humidity profiles from wind profiler radars has already been documented in the past 30 years and is known to be neither as straightforward and nor as robust as the retrieval of the wind velocity. The main constraint to retrieve the humidity profile is the necessity to combine measurements from the wind profiler and additional measurements (such as observations from radiosoundings at a coarser time resolution). Furthermore, the method relies on some assumptions and simplifications that restrict the scope of its application. The first objective of this paper is to identify the obstacles and limitations and solve them, or at least define the field of applicability. To improve the method, we propose using the radar capacity to detect transition levels, such as the top level of the boundary layer, marked by a maximum in the radar reflectivity. This forces the humidity profile from the free troposphere and from the boundary layer to coincide at this level, after an optimization of the calibration coefficients, and reduces the error. The resulting mean bias affecting the specific humidity profile never exceeds 0.25 g kg-1. The second objective is to explore the capability of the algorithm to retrieve the humidity vertical profiles for an operational purpose by comparing the results with observations from a Raman lidar.

  17. Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures.

    Science.gov (United States)

    Souza, Débora Guerini; Bellaver, Bruna; Hansel, Gisele; Arús, Bernardo Assein; Bellaver, Gabriela; Longoni, Aline; Kolling, Janaina; Wyse, Angela T S; Souza, Diogo Onofre; Quincozes-Santos, André

    2016-07-01

    Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na(+)/K(+)-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate-glutamine cycle, as well as glutamate and D-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.

  18. YOUNG ATHLETES' MOTIVATIONAL PROFILES

    Directory of Open Access Journals (Sweden)

    Juan Antonio Moreno Murcia

    2007-06-01

    Full Text Available The aim of this study was to examine the relationship between motivational characteristics and dispositional flow. In order to accomplish this goal, motivational profiles emerging from key constructs within Achievement Goal Theory and Self-Determination Theory were related to the dispositional flow measures. A sample of 413 young athletes (Age range 12 to 16 years completed the PMCSQ-2, POSQ, SMS and DFS measures. Cluster analysis results revealed three profiles: a "self-determined profile" characterised by higher scores on the task-involving climate perception and on the task orientation; a "non-self-determined profile", characterised by higher scores on ego-involving climate perception and ego orientation; and a "low self-determined and low non-self-determined profile" which had the lowest dispositional flow. No meaningful differences were found between the "self-determined profile" and the "non-self-determined profile" in dispositional flow. The "self-determined profile" was more commonly associated with females, athletes practising individual sports and those training more than three days a week. The "non-self-determined profile" was more customary of males and athletes practising team sports as well as those training just two or three days a week

  19. Nicotinamide starvation and inhibition of poly(ADP-Ribose) synthesis enhance the induced mutation in Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Okada, Gensaku; Kaneko, Ichiro; Mitsui, Hideki.

    1987-01-01

    The effects of nicotinamide (NA) deficiency and added NA and 3-aminobenzamide (3AB) on the cytotoxicity and the induction of mutations in Chinese hamster V79-14 cells were investigated. In NA deficiency the addition of NA (up to 4 mM) and 3AB (up to 7.5 mM) was not cytotoxic. The presence of NA prior to exposure to mitomycin C (MMC) or γ-rays produced a dose-dependent increase in the relative cloning ability of DNA-damaged cells. The lethality of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was significantly potentiated by pre-treatment with 5 mM 3AB, but no potentiation by 3AB was observed for MMC, ultraviolet (UV)-B light, or γ-rays. Among cells pre-cultured in NA-free medium there were increased frequencies of mutations at both the hypoxanthineguanine phosphoribosyltransferase (HGPRT) and the adenine phosphoribosyltransferase (APRT) loci following DNA damage. The enhancing effect by NA deficiency was time-dependent. Incubation with NA prior to DNA damage produced a significant reduction in the frequency of mutations. The addition of 3AB to the nicotinamide adenine dinucleotide (NAD + )-depleted cell cultures before or after the DNA damage also strongly increased the frequency of induced mutations, with increasing concentrations of 3AB up to 5 mM, but the frequency was reduced at higher concentrations. The interaction between NA deficiency and the addition of 3AB appears to act synergistically on mutation induction. A correlation was observed between the potential of inhibiting poly (ADP-ribose) polymerase and the enhancement of mutation frequency. (author)

  20. Poly (ADP-ribose polymerase plays an important role in intermittent hypoxia-induced cell death in rat cerebellar granule cells

    Directory of Open Access Journals (Sweden)

    Chiu Sheng-Chun

    2012-03-01

    Full Text Available Abstract Background Episodic cessation of airflow during sleep in patients with sleep apnea syndrome results in intermittent hypoxia (IH. Our aim was to investigate the effects of IH on cerebellar granule cells and to identify the mechanism of IH-induced cell death. Methods Cerebellar granule cells were freshly prepared from neonatal Sprague-Dawley rats. IH was created by culturing the cerebellar granule cells in the incubators with oscillating O2 concentration at 20% and 5% every 30 min for 1-4 days. The results of this study are based on image analysis using a confocal microscope and associated software. Cellular oxidative stress increased with increase in IH. In addition, the occurrence of cell death (apoptosis and necrosis increased as the duration of IH increased, but decreased in the presence of an iron chelator (phenanthroline or poly (ADP-ribose polymerase (PARP inhibitors [3-aminobenzamide (3-AB and DPQ]. The fluorescence of caspase-3 remained the same regardless of the duration of IH, and Western blots did not detect activation of caspase-3. However, IH increased the ratio of apoptosis-inducing factor (AIF translocation to the nucleus, while PARP inhibitors (3-AB reduced this ratio. Results According to our findings, IH increased oxidative stress and subsequently leading to cell death. This effect was at least partially mediated by PARP activation, resulting in ATP depletion, calpain activation leading to AIF translocation to the nucleus. Conclusions We suggest that IH induces cell death in rat primary cerebellar granule cells by stimulating oxidative stress PARP-mediated calpain and AIF activation.

  1. SURFACE BRIGHTNESS PROFILES OF DWARF GALAXIES. I. PROFILES AND STATISTICS

    International Nuclear Information System (INIS)

    Herrmann, Kimberly A.; Hunter, Deidre A.; Elmegreen, Bruce G.

    2013-01-01

    Radial surface brightness profiles of spiral galaxies are classified into three types: (I) single exponential, or the light falls off with one exponential to a break before falling off (II) more steeply, or (III) less steeply. Profile breaks are also found in dwarf disks, but some dwarf Type IIs are flat or increasing out to a break before falling off. Here we re-examine the stellar disk profiles of 141 dwarfs: 96 dwarf irregulars (dIms), 26 Blue Compact Dwarfs (BCDs), and 19 Magellanic-type spirals (Sms). We fit single, double, or even triple exponential profiles in up to 11 passbands: GALEX FUV and NUV, ground-based UBVJHK and Hα, and Spitzer 3.6 and 4.5 μm. We find that more luminous galaxies have brighter centers, larger inner and outer scale lengths, and breaks at larger radii; dwarf trends with M B extend to spirals. However, the V-band break surface brightness is independent of break type, M B , and Hubble type. Dwarf Type II and III profiles fall off similarly beyond the breaks but have different interiors and IIs break ∼twice as far as IIIs. Outer Type II and III scale lengths may have weak trends with wavelength, but pure Type II inner scale lengths clearly decrease from the FUV to visible bands whereas Type III inner scale lengths increase with redder bands. This suggests the influence of different star formation histories on profile type, but nonetheless the break location is approximately the same in all passbands. Dwarfs continue trends between profile and Hubble types such that later-type galaxies have more Type II but fewer Type I and III profiles than early-type spirals. BCDs and Sms are over-represented as Types III and II, respectively, compared to dIms

  2. Minocycline Blocks Asthma-associated Inflammation in Part by Interfering with the T Cell Receptor-Nuclear Factor κB-GATA-3-IL-4 Axis without a Prominent Effect on Poly(ADP-ribose) Polymerase*

    Science.gov (United States)

    Naura, Amarjit S.; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C.; Jordan, Joaquin; Catling, Andrew D.; Rezk, Bashir M.; Elmageed, Zakaria Y. Abd; Pyakurel, Kusma; Tarhuni, Abdelmetalab F.; Abughazleh, Mohammad Q.; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C.; Boulares, A. Hamid

    2013-01-01

    Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N′-nitro-N-nitroso-guanidine-treated mice or H2O2-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production. PMID:23184953

  3. Minocycline blocks asthma-associated inflammation in part by interfering with the T cell receptor-nuclear factor κB-GATA-3-IL-4 axis without a prominent effect on poly(ADP-ribose) polymerase.

    Science.gov (United States)

    Naura, Amarjit S; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C; Jordan, Joaquin; Catling, Andrew D; Rezk, Bashir M; Abd Elmageed, Zakaria Y; Pyakurel, Kusma; Tarhuni, Abdelmetalab F; Abughazleh, Mohammad Q; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C; Boulares, A Hamid

    2013-01-18

    Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

  4. BWR AXIAL PROFILE

    International Nuclear Information System (INIS)

    Huffer, J.

    2004-01-01

    The purpose of this calculation is to develop axial profiles for estimating the axial variation in burnup of a boiling water reactor (BWR) assembly spent nuclear fuel (SNF) given the average burnup of an assembly. A discharged fuel assembly typically exhibits higher burnup in the center and lower burnup at the ends of the assembly. Criticality safety analyses taking credit for SNF burnup must account for axially varying burnup relative to calculations based on uniformly distributed assembly average burnup due to the under-burned tips. Thus, accounting for axially varying burnup in criticality analyses is also referred to as accounting for the ''end effect'' reactivity. The magnitude of the reactivity change due to ''end effect'' is dependent on the initial assembly enrichment, the assembly average burnup, and the particular axial profile characterizing the burnup distribution. The set of bounding axial profiles should incorporate multiple BWR core designs and provide statistical confidence (95 percent confidence that 95 percent of the population is bound by the profile) that end nodes are conservatively represented. The profiles should also conserve the overall burnup of the fuel assembly. More background on BWR axial profiles is provided in Attachment I

  5. Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes.

    Science.gov (United States)

    Vasiljev, Andreja; Ahting, Uwe; Nargang, Frank E; Go, Nancy E; Habib, Shukry J; Kozany, Christian; Panneels, Valérie; Sinning, Irmgard; Prokisch, Holger; Neupert, Walter; Nussberger, Stephan; Rapaport, Doron

    2004-03-01

    Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.

  6. Diagnose Test-Taker's Profile in Terms of Core Profile Patterns: Principal Component (PC) vs. Profile Analysis via MDS (PAMS) Approaches.

    Science.gov (United States)

    Kim, Se-Kang; Davison, Mark L.

    A study was conducted to examine how principal components analysis (PCA) and Profile Analysis via Multidimensional Scaling (PAMS) can be used to diagnose individuals observed score profiles in terms of core profile patterns identified by each method. The standardization sample from the Wechsler Intelligence Scale for Children, Third Edition…

  7. Spectral backward radiation profile

    International Nuclear Information System (INIS)

    Kwon, Sung Duck; Lee, Keun Hyun; Kim, Bo Ra; Yoon, Suk Soo

    2004-01-01

    Ultrasonic backward radiation profile is frequency-dependent when incident region has deptional gradient of acoustical properties or multi-layers. Until now, we have measured the profiles of principal frequencies of used transducers so that it was not easy to understand the change of the frequency component and spectrum of backward radiation from the profile. We tried to measure the spectral backward radiation profiles using DFP(digital filer package) Lecroy DSO. The very big changes in the shape and pattern of spectral backward radiation profiles leads to the conclusion that this new try could be very effective tool to evaluate frequency dependent surface area.

  8. Novel Bioengineered Cassava Expressing an Archaeal Starch Degradation System and a Bacterial ADP-Glucose Pyrophosphorylase for Starch Self-Digestibility and Yield Increase

    Directory of Open Access Journals (Sweden)

    Ayalew Ligaba-Osena

    2018-02-01

    Full Text Available To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava (Manihot esculenta, which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from Pyrococcus furiosus and glucoamylase from Sulfolobus solfataricus, together with the gene encoding a modified ADP-glucose pyrophosphorylase (glgC from Escherichia coli, were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due to glgC expression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activity in vitro than the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes produced in planta maintain their activity and thermostability.

  9. HOPWA Performance Profiles

    Data.gov (United States)

    Department of Housing and Urban Development — HOPWA Performance Profiles are generated quarterly for all agencies receiving HOPWA formula or competitive grants. Performance Profiles are available at the national...

  10. Comparative evaluation of Plateletworks, Multiplate analyzer and Platelet function analyzer-200 in cardiology patients.

    Science.gov (United States)

    Kim, Jeeyong; Cho, Chi Hyun; Jung, Bo Kyeung; Nam, Jeonghun; Seo, Hong Seog; Shin, Sehyun; Lim, Chae Seung

    2018-04-14

    The objective of this study was to comparatively evaluate three commercial whole-blood platelet function analyzer systems: Platelet Function Analyzer-200 (PFA; Siemens Canada, Mississauga, Ontario, Canada), Multiplate analyzer (MP; Roche Diagnostics International Ltd., Rotkreuz, Switzerland), and Plateletworks Combo-25 kit (PLW; Helena Laboratories, Beaumont, TX, USA). Venipuncture was performed on 160 patients who visited a department of cardiology. Pairwise agreement among the three platelet function assays was assessed using Cohen's kappa coefficient and percent agreement within the reference limit. Kappa values with the same agonists were poor between PFA-collagen (COL; agonist)/adenosine diphosphate (ADP) and MP-ADP (-0.147), PFA-COL/ADP and PLW-ADP (0.089), MP-ADP and PLW-ADP (0.039), PFA-COL/ADP and MP-COL (-0.039), and between PFA-COL/ADP and PLW-COL (-0.067). Nonetheless, kappa values for the same assay principle with a different agonist were slightly higher between PFA-COL/ADP and PFA-COL/EPI (0.352), MP-ADP and MP-COL (0.235), and between PLW-ADP and PLW-COL (0.247). The range of percent agreement values was 38.7% to 73.8%. Therefore, various measurements of platelet function by more than one method were needed to obtain a reliable interpretation of platelet function considering low kappa coefficient and modest percent agreement rates among 3 different platelet function tests.

  11. Electron Density Profile Data Contains Virtual Height/Frequency Pairs from a Profile or Profiles (Composite Months) of Ionograms

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Electron Density Profile, N(h), data set contains both individual profiles and composite months. The data consist of virtual height/frequency pairs from a...

  12. Poly[ADP-ribose] polymerase-1 expression is related to cold ischemia, acute tubular necrosis, and delayed renal function in kidney transplantation.

    Directory of Open Access Journals (Sweden)

    Francisco O'Valle

    Full Text Available UNLABELLED: Cold ischemia time especially impacts on outcomes of expanded-criteria donor (ECD transplantation. Ischemia-reperfusion (IR injury produces excessive poly[ADP-Ribose] Polymerase-1 (PARP-1 activation. The present study explored the hypothesis that increased tubular expression of PARP-1 contributes to delayed renal function in suboptimal ECD kidney allografts and in non-ECD allografts that develop posttransplant acute tubular necrosis (ATN. MATERIALS AND METHODS: Nuclear PARP-1 immunohistochemical expression was studied in 326 paraffin-embedded renal allograft biopsies (193 with different degrees of ATN and 133 controls and in murine Parp-1 knockout model of IR injury. RESULTS: PARP-1 expression showed a significant relationship with cold ischemia time (r coefficient = 0.603, time to effective diuresis (r = 0.770, serum creatinine levels at biopsy (r = 0.649, and degree of ATN (r = 0.810 (p = 0.001, Pearson test. In the murine IR model, western blot showed an increase in PARP-1 that was blocked by Parp-1 inhibitor. Immunohistochemical study of PARP-1 in kidney allograft biopsies would allow early detection of possible delayed renal function, and the administration of PARP-1 inhibitors may offer a therapeutic option to reduce damage from IR in donor kidneys by preventing or minimizing ATN. In summary, these results suggest a pivotal role for PARP-1 in the ATN of renal transplantation. We propose the immunohistochemical assessment of PARP-1 in kidney allograft biopsies for early detection of a possible delayed renal function.

  13. Isolation and characterization of two disintegrins inhibiting ADP-induced human platelet aggregation from the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake)

    International Nuclear Information System (INIS)

    Sanchez, Elda E.; Galan, Jacob A.; Russell, William K.; Soto, Julio G.; Russell, David H.; Perez, John C.

    2006-01-01

    Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are nonenzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction, and may have potential in the treatment of strokes, heart attacks, cancers, and osteoporosis. Prior to 1983, the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake) was known to be only neurotoxic; however, now there is evidence that these snakes can contain venom with: (1) neurotoxins; (2) hemorrhagins; and (3) both neurotoxins and hemorrhagins. In this study, two disintegrins, mojastin 1 and mojastin 2, from the venom of a Mohave rattlesnake collected in central Arizona (Pinal County), were isolated and characterized. The disintegrins in these venoms were identified by mass-analyzed laser desorption ionization/time-of-flight/time-of-flight (MALDI/TOF/TOF) mass spectrometry as having masses of 7.436 and 7.636 kDa. Their amino acid sequences are similar to crotratroxin, a disintegrin isolated from the venom of the western diamondback rattlesnake (C. atrox). The amino acid sequence of mojastin 1 was identical to the amino acid sequence of a disintegrin isolated from the venom of the Timber rattlesnake (C. horridus). The disintegrins from the Mohave rattlesnake venom were able to inhibit ADP-induced platelet aggregation in whole human blood both having IC 5 s of 13.8 nM, but were not effective in inhibiting the binding of human urinary bladder carcinoma cells (T24) to fibronectin

  14. Morulustatin, A Disintegrin that Inhibits ADP-Induced Platelet Aggregation, Isolated from the Mexican Tamaulipan Rock Rattlesnake (Crotalus lepidus morulus)

    Science.gov (United States)

    Borja, Miguel; Galan, Jacob Anthony; Cantu, Esteban; Zugasti-Cruz, Alejandro; Rodríguez-Acosta, Alexis; Lazcano, David; Lucena, Sara; Suntravat, Montamas; Sánchez, y Elda Eliza

    2016-01-01

    The Tamaulipan rock rattlesnake (Crotalus lepidus morulus) is a montane snake that occurs in the humid pine-oak forest and the upper cloud forest of the Sierra Madre Oriental in southwestern Tamaulipas, central Nuevo Leon, and southeastern Coahuila in Mexico. Venom from this rattlesnake was fractionated by High-Performance Liquid Chromatography for the purpose of discovering disintegrin molecules. Disintegrins are non-enzymatic, small molecular weight peptides that interfere with cell-cell and cell-matrix interactions by binding to various cell receptors. Eleven fractions were collected by anion exchange chromatography and pooled into six groups (I, II, III, IV, V, and VI). Proteins of the six groups were analyzed by SDS-PAGE and western blot using antibodies raised against a disintegrin. The antibodies recognized different protein bands in five (II, III, IV, V, and VI) of six groups in a molecular mass range of 7 to 105 kDa. Western blot analysis revealed fewer protein bands in the higher molecular mass range and two bands in the disintegrin weight range in group II compared with the other four groups. Proteins in group II were further separated into nine fractions using reverse phase C18 chromatography. Fraction 4 inhibited platelet aggregation and was named morulustatin, which exhibited a single band with a molecular mass of approximately 7 kDa. Mass spectrometry analysis of fraction 4 revealed the identification of disintegrin peptides LRPGAQCADGLCCDQCR (MH+ 2035.84) and AGEECDCGSPANCCDAATCK (MH+ 2328.82). Morulustatin inhibited ADP-induced platelet aggregation in human whole blood and was concentration-dependent with an IC50 of 89.5 nM ± 12. PMID:28713196

  15. A Ravenscar-Java profile implementation

    DEFF Research Database (Denmark)

    Thomsen, Bent; Ravn, Anders Peter; Søndergaard, Hans

    2006-01-01

    This paper presents an implementation of the Ravenscar-Java profile. While most implementations of the profile are reference-implementations showing that it is possible to implement the profile, our implementation is aimed at industrial applications. It uses a dedicated real-time Java processor......, since we want to investigate if the Ravenscar-Java profile, implemented on a Java processor, is efficient for real applications. During the implementation some ambiguities and weaknesses of the profile were uncovered. However, test examples indicate that the profile is suitable for development...... of realistic real-time programs....

  16. Study on Rail Profile Optimization Based on the Nonlinear Relationship between Profile and Wear Rate

    Directory of Open Access Journals (Sweden)

    Jianxi Wang

    2017-01-01

    Full Text Available This paper proposes a rail profile optimization method that takes account of wear rate within design cycle so as to minimize rail wear at the curve in heavy haul railway and extend the service life of rail. Taking rail wear rate as the object function, the vertical coordinate of rail profile at range optimization as independent variable, and the geometric characteristics and grinding depth of rail profile as constraint conditions, the support vector machine regression theory was used to fit the nonlinear relationship between rail profile and its wear rate. Then, the profile optimization model was built. Based on the optimization principle of genetic algorithm, the profile optimization model was solved to achieve the optimal rail profile. A multibody dynamics model was used to check the dynamic performance of carriage running on optimal rail profile. The result showed that the average relative error of support vector machine regression model remained less than 10% after a number of training processes. The dynamic performance of carriage running on optimized rail profile met the requirements on safety index and stability. The wear rate of optimized profile was lower than that of standard profile by 5.8%; the allowable carrying gross weight increased by 12.7%.

  17. Phosphorylation potential in the dominant leg is lower, and [ADPfree] is higher in calf muscles at rest in endurance athletes than in sprinters and in untrained subjects.

    Science.gov (United States)

    Zoladz, J A; Kulinowski, P; Zapart-Bukowska, J; Grandys, M; Majerczak, J; Korzeniewski, B; Jasiński, A

    2007-12-01

    It has been reported that various types of mammalian muscle fibers differ regarding the content of several metabolites at rest. However, to our knowledge no data have been reported in the literature, concerning the muscle energetic status at rest in high class athletes when considering the dominant and non-dominant leg separately. We have hypothesised that due to higher mechanical loads on the dominant leg in athletes, the metabolic profile in the dominant leg at rest in the calf muscles, characterized by [PCr], [ADP(free)], [AMP(free)] and DeltaG(ATP), will significantly differ among endurance athletes, sprinters and untrained individuals. In this study we determined the DeltaG(ATP) and adenine phosphates concentrations in the dominant and non-dominant legs in untrained subjects (n = 6), sprinters (n = 10) and endurance athletes (n = 7) at rest. The (mean +/- SD) age of the subjects was 23.4 +/- 4.3 years. Muscle metabolites were measured in the calf muscles at rest, by means of (31)P-MRS, using a 4.7 T superconducting magnet (Bruker). When taking into account mean values in the left and right leg, phosphocreatine concentration ([PCr]) and DeltaG(ATP) were significantly lower (p<0.05, Wilcoxon-Mann-Whitney test), and [ADP(free)] was significantly higher (p = 0.04) in endurance athletes than in untrained subjects. When considering the differences between the left and right leg, [PCr] in the dominant leg was significantly lower in endurance athletes than in sprinters (p = 0.01) and untrained subjects (p = 0.02) (25.91 +/- 2.87 mM; 30.02 +/- 3.12 mM and 30.71 +/- 2.88 mM, respectively). The [ADP(free)] was significantly higher (p = 0.02) in endurance athletes than in sprinters and untrained subjects (p = 0.02) (42.19 +/- 13.44 microM; 27.86 +/- 10.19 microM; 25.35 +/- 10.97 microM, respectively). The DeltaG(ATP) in the dominant leg was significantly lower (p = 0.02) in endurance athletes than in sprinters and untrained subjects (p = 0.01) (-60.53 +/- 2.03 kJ.M(-1

  18. 48 CFR Appendix A to Part 1219 - Appendix A to Part 1219

    Science.gov (United States)

    2010-10-01

    ...) R414 (3) Radio/TV Communication Equipment (except airborne) 5820 (4) Maintenance, Repair, and Rebuilding of engines, turbines, components and weapons equipment J028/J010 (5) ADP Central Processing Units: Analog 7020 Digital 7021 Hybrid 7022 (6) ADP Support Equipment 7035 (7) ADP Components 7050 (8) ADP...

  19. Polar measurements on profiles

    Energy Technology Data Exchange (ETDEWEB)

    Althaus, D.

    1985-03-01

    Wind tunnel models with a profile depth of t=0.5 m were measured in a laminar wind tunnel by the usual measuring processes. The profile resistance was determined by integration along the width of span. The smooth profiles were examined at Re=0.7/1.0 and 1.5 million. At Re=1.0 million, the position of the changeover points were determined with a stethoscope. Also at this Reynolds number measurements were taken with a trip wire of d=2 mm diameter, directly on the profile nose. The tables contain the co-ordinates of the profiles, the contours, the theoretical speed distributions for 4 different angles of attack, the csub(a)-csub(w) polar measurements and changeover points, and the torque coefficients around the t/4 point. (BR).

  20. Profiling cancer

    DEFF Research Database (Denmark)

    Ciro, Marco; Bracken, Adrian P; Helin, Kristian

    2003-01-01

    In the past couple of years, several very exciting studies have demonstrated the enormous power of gene-expression profiling for cancer classification and prediction of patient survival. In addition to promising a more accurate classification of cancer and therefore better treatment of patients......, gene-expression profiling can result in the identification of novel potential targets for cancer therapy and a better understanding of the molecular mechanisms leading to cancer....

  1. Glucose ameliorates the metabolic profile and mitochondrial function of platelet concentrates during storage in autologous plasma

    Science.gov (United States)

    Amorini, Angela M.; Tuttobene, Michele; Tomasello, Flora M.; Biazzo, Filomena; Gullotta, Stefano; De Pinto, Vito; Lazzarino, Giuseppe; Tavazzi, Barbara

    2013-01-01

    Background It is essential that the quality of platelet metabolism and function remains high during storage in order to ensure the clinical effectiveness of a platelet transfusion. New storage conditions and additives are constantly evaluated in order to achieve this. Using glucose as a substrate is controversial because of its potential connection with increased lactate production and decreased pH, both parameters triggering the platelet lesion during storage. Materials and methods In this study, we analysed the morphological status and metabolic profile of platelets stored for various periods in autologous plasma enriched with increasing glucose concentrations (13.75, 27.5 and 55 mM). After 0, 2, 4, 6 and 8 days, high energy phosphates (ATP, GTP, ADP, AMP), oxypurines (hypoxanthine, xanthine, uric acid), lactate, pH, mitochondrial function, cell lysis and morphology, were evaluated. Results The data showed a significant dose-dependent improvement of the different parameters in platelets stored with increasing glucose, compared to what detected in controls. Interestingly, this phenomenon was more marked at the highest level of glucose tested and in the period of time generally used for platelet transfusion (0–6 days). Conclusion These results indicate that the addition of glucose during platelet storage ameliorates, in a dose-dependent manner, the biochemical parameters related to energy metabolism and mitochondrial function. Since there was no correspondence between glucose addition, lactate increase and pH decrease in our experiments, it is conceivable that platelet derangement during storage is not directly caused by glucose through an increase of anaerobic glycolysis, but rather to a loss of mitochondrial functions caused by reduced substrate availability. PMID:22682337

  2. Dynamic coupling between the LID and NMP domain motions in the catalytic conversion of ATP and AMP to ADP by adenylate kinase.

    Science.gov (United States)

    Jana, Biman; Adkar, Bharat V; Biswas, Rajib; Bagchi, Biman

    2011-01-21

    The catalytic conversion of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP) by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of ATP binding domain (LID) and AMP binding domain (NMP) domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motion of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis, and multidimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two-dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed root mean square deviation variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder-type theoretical model to explain the observed dynamic coupling between the motion of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations.

  3. Beam profile measurements on RHIC

    International Nuclear Information System (INIS)

    Connolly, R.; Michnoff, R.; Moore, T.; Shea, T.; Tepikian, S.

    2000-01-01

    The Relativistic Heavy Ion Collider (RHIC) at Brookhaven National Lab was commissioned during the summer of 1999. Transverse beam profiles on RHIC are measured with ionization profile monitors (IPMs). An IPM measures beam profiles by collecting the electrons liberated by residual gas ionization by the beam. The detector is placed in the gap of a dipole magnet to force the electrons to travel in straight lines from the beamline center to the collector. One IPM was tested and it measured the profiles of a single gold bunch containing 10 8 ions on consecutive turns. We show an example of one of these profiles giving transverse emittance. Also several profiles are combined into a mountain-range plot which shows betatron oscillations at injection

  4. Implicit User Interest Profile

    CERN Document Server

    Chan, K

    2002-01-01

    User interest profile presents items that the users are interested in. Typically those items can be listed or grouped. Listing is good but it does not possess interests at different abstraction levels - the higher-level interests are more general, while the lower-level ones are more specific. Furthermore, more general interests, in some sense, correspond to longer-term interests, while more specific interests correspond to shorter-term interests. This hierarchical user interest profile has obvious advantages: specifying user's specific interests and general interests and representing their relationships. Current user interest profile structures mostly do not use implicit method, nor use an appropriate clustering algorithm especially for conceptually hierarchical structures. This research studies building a hierarchical user interest profile (HUIP) and the hierarchical divisive algorithm (HDC). Several users visit hundreds of web pages and each page is recorded in each users profile. These web pages are used t...

  5. Convolution based profile fitting

    International Nuclear Information System (INIS)

    Kern, A.; Coelho, A.A.; Cheary, R.W.

    2002-01-01

    Full text: In convolution based profile fitting, profiles are generated by convoluting functions together to form the observed profile shape. For a convolution of 'n' functions this process can be written as, Y(2θ)=F 1 (2θ)x F 2 (2θ)x... x F i (2θ)x....xF n (2θ). In powder diffractometry the functions F i (2θ) can be interpreted as the aberration functions of the diffractometer, but in general any combination of appropriate functions for F i (2θ) may be used in this context. Most direct convolution fitting methods are restricted to combinations of F i (2θ) that can be convoluted analytically (e.g. GSAS) such as Lorentzians, Gaussians, the hat (impulse) function and the exponential function. However, software such as TOPAS is now available that can accurately convolute and refine a wide variety of profile shapes numerically, including user defined profiles, without the need to convolute analytically. Some of the most important advantages of modern convolution based profile fitting are: 1) virtually any peak shape and angle dependence can normally be described using minimal profile parameters in laboratory and synchrotron X-ray data as well as in CW and TOF neutron data. This is possible because numerical convolution and numerical differentiation is used within the refinement procedure so that a wide range of functions can easily be incorporated into the convolution equation; 2) it can use physically based diffractometer models by convoluting the instrument aberration functions. This can be done for most laboratory based X-ray powder diffractometer configurations including conventional divergent beam instruments, parallel beam instruments, and diffractometers used for asymmetric diffraction. It can also accommodate various optical elements (e.g. multilayers and monochromators) and detector systems (e.g. point and position sensitive detectors) and has already been applied to neutron powder diffraction systems (e.g. ANSTO) as well as synchrotron based

  6. Performance profiling for brachytherapy applications

    Science.gov (United States)

    Choi, Wonqook; Cho, Kihyeon; Yeo, Insung

    2018-05-01

    In many physics applications, a significant amount of software (e.g. R, ROOT and Geant4) is developed on novel computing architectures, and much effort is expended to ensure the software is efficient in terms of central processing unit (CPU) time and memory usage. Profiling tools are used during the evaluation process to evaluate the efficiency; however, few such tools are able to accommodate low-energy physics regions. To address this limitation, we developed a low-energy physics profiling system in Geant4 to profile the CPU time and memory of software applications in brachytherapy applications. This paper describes and evaluates specific models that are applied to brachytherapy applications in Geant4, such as QGSP_BIC_LIV, QGSP_BIC_EMZ, and QGSP_BIC_EMY. The physics range in this tool allows it to be used to generate low energy profiles in brachytherapy applications. This was a limitation in previous studies, which caused us to develop a new profiling tool that supports profiling in the MeV range, in contrast to the TeV range that is supported by existing high-energy profiling tools. In order to easily compare the profiling results between low-energy and high-energy modes, we employed the same software architecture as that in the SimpliCarlo tool developed at the Fermilab National Accelerator Laboratory (FNAL) for the Large Hadron Collider (LHC). The results show that the newly developed profiling system for low-energy physics (less than MeV) complements the current profiling system used for high-energy physics (greater than TeV) applications.

  7. Estimating Planetary Boundary Layer Heights from NOAA Profiler Network Wind Profiler Data

    Science.gov (United States)

    Molod, Andrea M.; Salmun, H.; Dempsey, M

    2015-01-01

    An algorithm was developed to estimate planetary boundary layer (PBL) heights from hourly archived wind profiler data from the NOAA Profiler Network (NPN) sites located throughout the central United States. Unlike previous studies, the present algorithm has been applied to a long record of publicly available wind profiler signal backscatter data. Under clear conditions, summertime averaged hourly time series of PBL heights compare well with Richardson-number based estimates at the few NPN stations with hourly temperature measurements. Comparisons with clear sky reanalysis based estimates show that the wind profiler PBL heights are lower by approximately 250-500 m. The geographical distribution of daily maximum PBL heights corresponds well with the expected distribution based on patterns of surface temperature and soil moisture. Wind profiler PBL heights were also estimated under mostly cloudy conditions, and are generally higher than both the Richardson number based and reanalysis PBL heights, resulting in a smaller clear-cloudy condition difference. The algorithm presented here was shown to provide a reliable summertime climatology of daytime hourly PBL heights throughout the central United States.

  8. Country nuclear power profiles

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-03-01

    The preparation of Country Nuclear Power Profiles was initiated within the framework of the IAEA`s programme for nuclear power plant performance assessment and feedback. It responded to a need for a database and a technical document containing a description of the energy and economic situation and the primary organizations involved in nuclear power in IAEA Member States. The task was included in the IAEA`s programmes for 1993/1994 and 1995/1996. In March 1993, the IAEA organized a Technical Committee meeting to discuss the establishment of country data ``profiles``, to define the information to be included in the profiles and to review the information already available in the IAEA. Two expert meetings were convened in November 1994 to provide guidance to the IAEA on the establishment of the country nuclear profiles, on the structure and content of the profiles, and on the preparation of the publication and the electronic database. In June 1995, an Advisory Group meeting provided the IAEA with comprehensive guidance on the establishment and dissemination of an information package on industrial and organizational aspects of nuclear power to be included in the profiles. The group of experts recommended that the profiles focus on the overall economic, energy and electricity situation in the country and on its nuclear power industrial structure and organizational framework. In its first release, the compilation would cover all countries with operating power plants by the end of 1995. It was also recommended to further promote information exchange on the lessons learned from the countries engaged in nuclear programmes. For the preparation of this publication, the IAEA received contributions from the 29 countries operating nuclear power plants and Italy. A database has been implemented and the profiles are supporting programmatic needs within the IAEA; it is expected that the database will be publicly accessible in the future. Refs, figs, tabs.

  9. Country nuclear power profiles

    International Nuclear Information System (INIS)

    1998-03-01

    The preparation of Country Nuclear Power Profiles was initiated within the framework of the IAEA's programme for nuclear power plant performance assessment and feedback. It responded to a need for a database and a technical document containing a description of the energy and economic situation and the primary organizations involved in nuclear power in IAEA Member States. The task was included in the IAEA's programmes for 1993/1994 and 1995/1996. In March 1993, the IAEA organized a Technical Committee meeting to discuss the establishment of country data ''profiles'', to define the information to be included in the profiles and to review the information already available in the IAEA. Two expert meetings were convened in November 1994 to provide guidance to the IAEA on the establishment of the country nuclear profiles, on the structure and content of the profiles, and on the preparation of the publication and the electronic database. In June 1995, an Advisory Group meeting provided the IAEA with comprehensive guidance on the establishment and dissemination of an information package on industrial and organizational aspects of nuclear power to be included in the profiles. The group of experts recommended that the profiles focus on the overall economic, energy and electricity situation in the country and on its nuclear power industrial structure and organizational framework. In its first release, the compilation would cover all countries with operating power plants by the end of 1995. It was also recommended to further promote information exchange on the lessons learned from the countries engaged in nuclear programmes. For the preparation of this publication, the IAEA received contributions from the 29 countries operating nuclear power plants and Italy. A database has been implemented and the profiles are supporting programmatic needs within the IAEA; it is expected that the database will be publicly accessible in the future

  10. Personal summarization from profile networks

    Institute of Scientific and Technical Information of China (English)

    Zhongqing WANG; Shoushan LI; Guodong ZHOU

    2017-01-01

    Personal profile information on social media like Linkedln.com and Facebook.com is at the core of many interesting applications,such as talent recommendation and contextual advertising.However,personal profiles usually lack consistent organization confronted with the large amount of available information.Therefore,it is always a challenge for people to quickly find desired information from them.In this paper,we address the task of personal profile summarization by leveraging both textual information and social connection information in social networks from both unsupervised and supervised learning paradigms.Here,using social connection information is motivated by the intuition that people with similar academic,business or social background (e.g.,comajor,co-university,and co-corporation) tend to have similar experiences and should have similar summaries.For unsupervised learning,we propose a collective ranking approach,called SocialRank,to combine textual information in an individual profile and social context information from relevant profiles in generating a personal profile summary.For supervised learning,we propose a collective factor graph model,called CoFG,to summarize personal profiles with local textual attribute functions and social connection factors.Extensive evaluation on a large dataset from LinkedIn.com demonstrates the usefulness of social connection information in personal profile summarization and the effectiveness of our proposed unsupervised and supervised learning approaches.

  11. Nutraceutical Potential of New Alfalfa (Medicago sativa) Ingredients for Beverage Preparations.

    Science.gov (United States)

    Soto-Zarazúa, Maria Guadalupe; Bah, Moustapha; Costa, Anabela Silvia Gomes; Rodrigues, Francisca; Pimentel, Filipa Botelho; Rojas-Molina, Isela; Rojas, Alejandra; Oliveira, Maria Beatriz Prior Pinto

    2017-10-01

    Alfalfa (Medicago sativa) has been extensively used as animal feed, due to its fiber, protein, minerals, and vitamins, being also a useful source of phenolic compounds with potential therapeutic benefits. Nevertheless, its potential use as human ingredient is scarce. The aim of this work was to assess the nutritional composition, amino acid profile, and antioxidant capacity (AOC) of freeze-dried juice (FDJ) and fibrous residual material (RM), two new alfalfa-derived products (Adps) recently launched as ingredients for beverage preparations. Results demonstrated a high content of proteins (23-30 g/100 g FDJ and 13-17 g/100 g RM), crude fiber (29 g/100 g RM), and minerals (such as sodium, calcium, iron, and zinc). No significant difference was found in caloric content (4 kcal/g). Essential and nonessential amino acids were quantified in both Adps being leucine and lysine the most abundant. Total phenolic and total flavonoid contents (TPC and TFC, respectively) and their changes along the different harvesting periods of the year were also examined. FDJ presented the highest TPC in May (19 mg gallic acid equivalents/g dry weight [dw]), while in October TFC had the maximum value (4 mg catechin equivalents/g dw). Both products exhibited an interesting AOC by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power assays. This study reports the nutraceutical potential of two new types of Adps.

  12. Global iTRAQ-based proteomic profiling of Toxoplasma gondii oocysts during sporulation.

    Science.gov (United States)

    Zhou, Chun-Xue; Zhu, Xing-Quan; Elsheikha, Hany M; He, Shuai; Li, Qian; Zhou, Dong-Hui; Suo, Xun

    2016-10-04

    Toxoplasma gondii is a medically and economically important protozoan parasite. However, the molecular mechanisms of its sporulation remain largely unknown. Here, we applied iTRAQ coupled with 2D LC-MS/MS proteomic analysis to investigate the proteomic expression profile of T. gondii oocysts during sporulation. Of the 2095 non-redundant proteins identified, 587 were identified as differentially expressed proteins (DEPs). Based on Gene Ontology enrichment and KEGG pathway analyses the majority of these DEPs were found related to the metabolism of amino acids, carbon and energy. Protein interaction network analysis generated by STRING identified ATP-citrate lyase (ACL), GMP synthase, IMP dehydrogenase (IMPDH), poly (ADP-ribose) glycohydrolase (PARG), and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) as the top five hubs. We also identified 25 parasite virulence factors that were expressed at relatively high levels in sporulated oocysts compared to non-sporulated oocysts, which might contribute to the infectivity of mature oocysts. Considering the importance of oocysts in the dissemination of toxoplasmosis these findings may help in the search of protein targets with a key role in infectiousness and ecological success of oocysts, creating new opportunities for the development of better means for disease prevention. The development of new preventative interventions against T. gondii infection relies on an improved understanding of the proteome and chemical pathways of this parasite. To identify proteins required for the development of environmentally resistant and infective T. gondii oocysts, we compared the proteome of non-sporulated (immature) oocysts with the proteome of sporulated (mature, infective) oocysts. iTRAQ 2D-LC-MS/MS analysis revealed proteomic changes that distinguish non-sporulated from sporulated oocysts. Many of the differentially expressed proteins were involved in metabolic pathways and 25 virulence factors were identified

  13. Professional assessment of facial profile attractiveness.

    Science.gov (United States)

    Soh, Jen; Chew, Ming Tak; Wong, Hwee Bee

    2005-08-01

    The aim of this study was to compare the assessments of Chinese facial profile attractiveness by orthodontists and oral surgeons. The sample comprised 31 dental professionals (20 orthodontists, 11 oral surgeons) in an Asian community. Facial profile photographs and lateral cephalometric radiographs of 2 Chinese adults (1 man, 1 woman) with normal profiles, Class I incisor relationships, and Class I skeletal patterns were digitized. The digital images were modified by altering cephalometric skeletal and dental hard tissue Chinese normative values in increments of 2 standard deviations in the anteroposterior plane to obtain 7 facial profiles for each sex. The images were bimaxillary protrusion, protrusive mandible, retrusive mandible, normal profile (Class I incisor with Class I skeletal pattern), retrusive maxilla, protrusive maxilla, and bimaxillary retrusion. The Mann-Whitney U test was used to determine professional differences in assessment. Multiple regression analysis was performed with age, professional status, sex, and number of years in practice as independent variables. A strong correlation was found in the profile assessment between orthodontists and oral surgeons. Normal and bimaxillary retrusive Chinese male and female profiles were judged to be highly attractive by orthodontists and oral surgeons. Chinese male and female profiles with protrusive mandibles were judged the least attractive. There was a difference in professional opinion about the most attractive male profile (P profile and oral surgeons preferring a fuller normal Chinese profile. Sex of dental professionals and number of years in clinical practice were found to affect profile rankings.

  14. In situ profiling of eastern Arabian Sea coastal waters using a new autonomous vertical profiler

    Digital Repository Service at National Institute of Oceanography (India)

    Desa, E.S.; Madhan, R.; Dabholkar, N.A.; Prabhudesai, S.P.; Navelkar, G.S.; Mascarenhas, A.A.M.Q.; Afzulpurkar, S.; Phaldesai, M.; Maurya, P.

    The autonomous vertical profiler (AVP) presented here offers a fast, cost-effective, optimized approach to profiling in coastal waters. It consists of a hands-free, slightly buoyant, motor-driven in situ robot profiler that requires no operator...

  15. Approximate Stokes Drift Profiles in Deep Water

    Science.gov (United States)

    Breivik, Øyvind; Janssen, Peter A. E. M.; Bidlot, Jean-Raymond

    2014-09-01

    A deep-water approximation to the Stokes drift velocity profile is explored as an alternative to the monochromatic profile. The alternative profile investigated relies on the same two quantities required for the monochromatic profile, viz the Stokes transport and the surface Stokes drift velocity. Comparisons with parametric spectra and profiles under wave spectra from the ERA-Interim reanalysis and buoy observations reveal much better agreement than the monochromatic profile even for complex sea states. That the profile gives a closer match and a more correct shear has implications for ocean circulation models since the Coriolis-Stokes force depends on the magnitude and direction of the Stokes drift profile and Langmuir turbulence parameterizations depend sensitively on the shear of the profile. The alternative profile comes at no added numerical cost compared to the monochromatic profile.

  16. Parent Prevention Communication Profiles and Adolescent Substance Use: A Latent Profile Analysis and Growth Curve Model

    Science.gov (United States)

    Choi, Hye Jeong; Miller-Day, Michelle; Shin, YoungJu; Hecht, Michael L.; Pettigrew, Jonathan; Krieger, Janice L.; Lee, JeongKyu; Graham, John W.

    2017-01-01

    This current study identifies distinct parent prevention communication profiles and examines whether youth with different parental communication profiles have varying substance use trajectories over time. Eleven schools in two rural school districts in the Midwestern United States were selected, and 784 students were surveyed at three time points from the beginning of 7th grade to the end of 8th grade. A series of latent profile analyses were performed to identify discrete profiles/subgroups of substance-specific prevention communication (SSPC). The results revealed a 4-profile model of SSPC: Active-Open, Passive-Open, Active-Silent, and Passive-Silent. A growth curve model revealed different rates of lifetime substance use depending on the youth’s SSPC profile. These findings have implications for parenting interventions and tailoring messages for parents to fit specific SSPC profiles. PMID:29056872

  17. Ti(r) profiles from the JET neutron profile monitor for ohmic discharges

    Energy Technology Data Exchange (ETDEWEB)

    Esposito, B. (ENEA, Frascati (Italy). Centro Ricerche Energia); Marcus, F.B.; Conroy, S.; Jarvis, O.N.; Loughlin, M.J.; Sadler, G.; Belle, P. van (Commission of the European Communities, Abingdon (United Kingdom). JET Joint Undertaking); Adams, J.M.; Watkins, N. (AEA Industrial Technology, Harwell (United Kingdom))

    1991-01-01

    A study has been made of the neutron emissivity, using the JET neutron profile monitor, obtained for ohmically heated deuterium discharges. Both one-dimensional (1-D) best-fit inversion procedures and 2-D tomography have been used to deduce the radial profile of the neutron emission from the line-integral data. The profiles of ion temperature and ion thermal conductivity are then derived. The scaling of the ion thermal conductivity with plasma current is found to be opposite to that of neoclassical theory. (author) 4 refs., 5 figs.

  18. Synthesis of national risk profile

    Science.gov (United States)

    1979-01-01

    The methodology used and results obtained in computing the national risk profile for carbon fibers (CF) released after an aircraft accident (fire or explosion) are presented. The computation was performed by use of twenty-six individual conditional risk profiles, together with the extrapolation of these profiles to other U.S. airports. The risk profile was obtained using 1993 CF utilization forecasts, but numbers of facilities were taken from 1972 and 1975 census data, while losses were expressed in 1977 dollars.

  19. THE BEHAVIORAL PROFILE OF HARVESTER OPERATORS

    Directory of Open Access Journals (Sweden)

    Millana Burger Pagnussat

    Full Text Available ABSTRACT This study aims to characterize the behavioral profile of harvester operators, with the goal of assisting forest managers in selecting and training new teams of employees. A forest company located in central-western Brazil was examined from a sample of 20 harvester operators that did not have experience carrying out the functions of their industry. A behavioral profile evaluation tool was used, consisting of a management system that creates a profile based on behavioral competencies; it was initially used to develop a profile of a high-performing harvester operator; or rather, a reference profile. Next, the behavioral profile of the operators were grouped into distinct classes and compared with the reference profile to identify traits that could positively or negatively affect an operators' performance. An optimal profile had the following qualities: attentive to details, meets deadlines and follows rules, technically-oriented, patient with repetitive tasks, the ability to avoid conflicts, and being an introvert. An improper profile included aspects such as aggressiveness, being argumentative, being persuasive, explosive, and tense at work. The behavioral profile evaluation tool can support the process of choosing forest machine operators; however, it is important to also consider skills and work experience.

  20. Proteome Profiling Outperforms Transcriptome Profiling for Coexpression Based Gene Function Prediction

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jing; Ma, Zihao; Carr, Steven A.; Mertins, Philipp; Zhang, Hui; Zhang, Zhen; Chan, Daniel W.; Ellis, Matthew J. C.; Townsend, R. Reid; Smith, Richard D.; McDermott, Jason E.; Chen, Xian; Paulovich, Amanda G.; Boja, Emily S.; Mesri, Mehdi; Kinsinger, Christopher R.; Rodriguez, Henry; Rodland, Karin D.; Liebler, Daniel C.; Zhang, Bing

    2016-11-11

    Coexpression of mRNAs under multiple conditions is commonly used to infer cofunctionality of their gene products despite well-known limitations of this “guilt-by-association” (GBA) approach. Recent advancements in mass spectrometry-based proteomic technologies have enabled global expression profiling at the protein level; however, whether proteome profiling data can outperform transcriptome profiling data for coexpression based gene function prediction has not been systematically investigated. Here, we address this question by constructing and analyzing mRNA and protein coexpression networks for three cancer types with matched mRNA and protein profiling data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Our analyses revealed a marked difference in wiring between the mRNA and protein coexpression networks. Whereas protein coexpression was driven primarily by functional similarity between coexpressed genes, mRNA coexpression was driven by both cofunction and chromosomal colocalization of the genes. Functionally coherent mRNA modules were more likely to have their edges preserved in corresponding protein networks than functionally incoherent mRNA modules. Proteomic data strengthened the link between gene expression and function for at least 75% of Gene Ontology (GO) biological processes and 90% of KEGG pathways. A web application Gene2Net (http://cptac.gene2net.org) developed based on the three protein coexpression networks revealed novel gene-function relationships, such as linking ERBB2 (HER2) to lipid biosynthetic process in breast cancer, identifying PLG as a new gene involved in complement activation, and identifying AEBP1 as a new epithelial-mesenchymal transition (EMT) marker. Our results demonstrate that proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction. Proteomics should be integrated if not preferred in gene function and human disease studies

  1. Accelerated Profile HMM Searches.

    Directory of Open Access Journals (Sweden)

    Sean R Eddy

    2011-10-01

    Full Text Available Profile hidden Markov models (profile HMMs and probabilistic inference methods have made important contributions to the theory of sequence database homology search. However, practical use of profile HMM methods has been hindered by the computational expense of existing software implementations. Here I describe an acceleration heuristic for profile HMMs, the "multiple segment Viterbi" (MSV algorithm. The MSV algorithm computes an optimal sum of multiple ungapped local alignment segments using a striped vector-parallel approach previously described for fast Smith/Waterman alignment. MSV scores follow the same statistical distribution as gapped optimal local alignment scores, allowing rapid evaluation of significance of an MSV score and thus facilitating its use as a heuristic filter. I also describe a 20-fold acceleration of the standard profile HMM Forward/Backward algorithms using a method I call "sparse rescaling". These methods are assembled in a pipeline in which high-scoring MSV hits are passed on for reanalysis with the full HMM Forward/Backward algorithm. This accelerated pipeline is implemented in the freely available HMMER3 software package. Performance benchmarks show that the use of the heuristic MSV filter sacrifices negligible sensitivity compared to unaccelerated profile HMM searches. HMMER3 is substantially more sensitive and 100- to 1000-fold faster than HMMER2. HMMER3 is now about as fast as BLAST for protein searches.

  2. Development of second generation peptides modulating cellular adiponectin receptor responses

    Directory of Open Access Journals (Sweden)

    Laszlo eOtvos

    2014-10-01

    Full Text Available The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC. In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399. The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM - low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400 was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400 at similar concentrations will be an important target validation tool to study adiponectin functions.

  3. Development of second generation peptides modulating cellular adiponectin receptor responses

    Science.gov (United States)

    Otvos, Laszlo; Knappe, Daniel; Hoffmann, Ralf; Kovalszky, Ilona; Olah, Julia; Hewitson, Tim; Stawikowska, Roma; Stawikowski, Maciej; Cudic, Predrag; Lin, Feng; Wade, John; Surmacz, Eva; Lovas, Sandor

    2014-10-01

    The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM - low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.

  4. New dynamic NNORSY ozone profile climatology

    Science.gov (United States)

    Kaifel, A. K.; Felder, M.; Declercq, C.; Lambert, J.-C.

    2012-01-01

    Climatological ozone profile data are widely used as a-priori information for total ozone using DOAS type retrievals as well as for ozone profile retrieval using optimal estimation, for data assimilation or evaluation of 3-D chemistry-transport models and a lot of other applications in atmospheric sciences and remote sensing. For most applications it is important that the climatology represents not only long term mean values but also the links between ozone and dynamic input parameters. These dynamic input parameters should be easily accessible from auxiliary datasets or easily measureable, and obviously should have a high correlation with ozone. For ozone profile these parameters are mainly total ozone column and temperature profile data. This was the outcome of a user consultation carried out in the framework of developing a new, dynamic ozone profile climatology. The new ozone profile climatology is based on the Neural Network Ozone Retrieval System (NNORSY) widely used for ozone profile retrieval from UV and IR satellite sounder data. NNORSY allows implicit modelling of any non-linear correspondence between input parameters (predictors) and ozone profile target vector. This paper presents the approach, setup and validation of a new family of ozone profile climatologies with static as well as dynamic input parameters (total ozone and temperature profile). The neural network training relies on ozone profile measurement data of well known quality provided by ground based (ozonesondes) and satellite based (SAGE II, HALOE, and POAM-III) measurements over the years 1995-2007. In total, four different combinations (modes) for input parameters (date, geolocation, total ozone column and temperature profile) are available. The geophysical validation spans from pole to pole using independent ozonesonde, lidar and satellite data (ACE-FTS, AURA-MLS) for individual and time series comparisons as well as for analysing the vertical and meridian structure of different modes of

  5. Profiling the Mobile Customer

    DEFF Research Database (Denmark)

    Jessen, Pernille Wegener; King, Nancy J.

    2010-01-01

    of significant concerns about privacy and data protection. This second article in a two part series on "Profiling the Mobile Customer" explores how to best protect consumers' privacy and personal data through available mechanisms that include industry self-regulation, privacy-enhancing technologies...... discusses the current limitations of using technology to protect consumers from privacy abuses related to profiling. Concluding that industry self-regulation and available privacy-enhancing technologies will not be adequate to close important privacy gaps related to consumer profiling without legislative...

  6. Character profiles and life satisfaction.

    Science.gov (United States)

    Park, Hwanjin; Suh, Byung Seong; Kim, Won Sool; Lee, Hye-Kyung; Park, Seon-Cheol; Lee, Kounseok

    2015-04-01

    There is a surge of interest in subjective well-being (SWB), which concerns how individuals feel about their happiness. Life satisfaction tends to be influenced by individual psychological traits and external social factors. The aim of this study was to examine the relationship between individual character and SWB. Data from 3522 university students were analyzed in this study. Character profiles were evaluated using the Temperament and Character Inventory-Revised Short version (TCI-RS). Life satisfaction was assessed using the Satisfaction with Life Scale (SWLS). All statistical tests regarding the correlations between each character profile and life satisfaction were conducted using ANOVAs, t-tests, multiple linear regression models and correlation analyses. The creative (SCT) profile was associated with the highest levels of life satisfaction, whereas the depressive (sct) profile was associated with the lowest levels of life satisfaction. Additionally, high self-directedness, self-transcendence and cooperation were associated with high life satisfaction. The results of gender-adjusted multiple regression analysis showed that the effects of self-directedness were the strongest in the assessment of one's quality of life, followed by self-transcendence and cooperativeness, in that order. All of the three-character profiles were significantly correlated with one's quality of life, and the character profiles of TCI-RS explained 27.6% of life satisfaction in total. Among the three-character profiles, the self-directedness profile was most associated with life satisfaction. Our study was cross-sectional, and self-reported data from students at a single university were analyzed. The results of this study showed that, among the character profiles, the effects of self-directedness were the strongest for predicting life satisfaction. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Modeling Social Influence via Combined Centralized and Distributed Planning Control

    Science.gov (United States)

    Vaccaro, James; Guest, Clark

    2010-01-01

    Real world events are driven by a mixture of both centralized and distributed control of individual agents based on their situational context and internal make up. For example, some people have partial allegiances to multiple, contradictory authorities, as well as to their own goals and principles. This can create a cognitive dissonance that can be exploited by an appropriately directed psychological influence operation (PSYOP). An Autonomous Dynamic Planning and Execution (ADP&E) approach is proposed for modeling both the unperturbed context as well as its reaction to various PSYOP interventions. As an illustrative example, the unrest surrounding the Iranian elections in the summer of 2009 is described in terms applicable to an ADP&E modeling approach. Aspects of the ADP&E modeling process are discussed to illustrate its application and advantages for this example.

  8. Scaling laws for TEXT plasma profiles

    International Nuclear Information System (INIS)

    McCool, S.C.; Bravenec, R.V.; Chen, J.Y.; Foster, M.S.; Li, W.L.; Ouroura, A.; Phillips, P.E.; Richards, B.; Wenzel, K.W.; Zhang, Z.M.

    1994-01-01

    Regression analysis has been performed on a number of measured profiles including temperature and density vs. nominal macroscopic operating parameters for TEXT tokamak (pre-upgrade) ohmic plasmas. The resulting simple empirical model has enabled the authors to quickly approximate profiles of electron temperature and density, ion temperature, and soft x-ray brightness, as well as the scalar quantities: total radiated power, q=1 radius, sawtooth period and amplitude, and energy confinement time as a power law of toroidal field, plasma current, chord average density, and fueling gas atomic weight. The model profiles are only applicable to the plasma interior, i.e. within the limiter radius. In most cases the predicted model profiles are within the experimental error bars of measured profiles and are more accurate at predicting profile variation for small operating parameter changes than the measured profiles

  9. Patterns of coral disease across the Hawaiian archipelago: relating disease to environment.

    Directory of Open Access Journals (Sweden)

    Greta S Aeby

    Full Text Available In Hawaii, coral reefs occur across a gradient of biological (host abundance, climatic (sea surface temperature anomalies and anthropogenic conditions from the human-impacted reefs of the main Hawaiian Islands (MHI to the pristine reefs of the northwestern Hawaiian Islands (NWHI. Coral disease surveys were conducted at 142 sites from across the Archipelago and disease patterns examined. Twelve diseases were recorded from three coral genera (Porites, Montipora, Acropora with Porites having the highest prevalence. Porites growth anomalies (PorGAs were significantly more prevalent within and indicative of reefs in the MHI and Porites trematodiasis (PorTrm was significantly more prevalent within and indicative of reefs in the NWHI. Porites tissue loss syndrome (PorTLS was also important in driving regional differences but that relationship was less clear. These results highlight the importance of understanding disease ecology when interpreting patterns of disease occurrence. PorTrm is caused by a parasitic flatworm that utilizes multiple hosts during its life cycle (fish, mollusk and coral. All three hosts must be present for the disease to occur and higher host abundance leads to higher disease prevalence. Thus, a high prevalence of PorTrm on Hawaiian reefs would be an indicator of a healthy coral reef ecosystem. In contrast, the high occurrence of PorGAs within the MHI suggests that PorGAs are related, directly or indirectly, to some environmental co-factor associated with increased human population sizes. Focusing on the three indicator diseases (PorGAs, PorTrm, PorTLS we used statistical modeling to examine the underlying associations between disease prevalence and 14 different predictor variables (biotic and abiotic. All three diseases showed positive associations with host abundance and negative associations with thermal stress. The association with human population density differed among disease states with PorGAs showing a positive and Por

  10. Patterns of coral disease across the Hawaiian Archipelago: Relating disease to environment

    Science.gov (United States)

    Aeby, G.S.; Williams, G.J.; Franklin, E.C.; Kenyon, J.; Cox, E.F.; Coles, S.; Work, Thierry M.

    2011-01-01

    In Hawaii, coral reefs occur across a gradient of biological (host abundance), climatic (sea surface temperature anomalies) and anthropogenic conditions from the human-impacted reefs of the main Hawaiian Islands (MHI) to the pristine reefs of the northwestern Hawaiian Islands (NWHI). Coral disease surveys were conducted at 142 sites from across the Archipelago and disease patterns examined. Twelve diseases were recorded from three coral genera (Porites, Montipora, Acropora) with Porites having the highest prevalence. Porites growth anomalies (PorGAs) were significantly more prevalent within and indicative of reefs in the MHI and Porites trematodiasis (PorTrm) was significantly more prevalent within and indicative of reefs in the NWHI. Porites tissue loss syndrome (PorTLS) was also important in driving regional differences but that relationship was less clear. These results highlight the importance of understanding disease ecology when interpreting patterns of disease occurrence. PorTrm is caused by a parasitic flatworm that utilizes multiple hosts during its life cycle (fish, mollusk and coral). All three hosts must be present for the disease to occur and higher host abundance leads to higher disease prevalence. Thus, a high prevalence of PorTrm on Hawaiian reefs would be an indicator of a healthy coral reef ecosystem. In contrast, the high occurrence of PorGAs within the MHI suggests that PorGAs are related, directly or indirectly, to some environmental co-factor associated with increased human population sizes. Focusing on the three indicator diseases (PorGAs, PorTrm, PorTLS) we used statistical modeling to examine the underlying associations between disease prevalence and 14 different predictor variables (biotic and abiotic). All three diseases showed positive associations with host abundance and negative associations with thermal stress. The association with human population density differed among disease states with PorGAs showing a positive and PorTrm showing

  11. Discrete-Time Local Value Iteration Adaptive Dynamic Programming: Admissibility and Termination Analysis.

    Science.gov (United States)

    Wei, Qinglai; Liu, Derong; Lin, Qiao

    In this paper, a novel local value iteration adaptive dynamic programming (ADP) algorithm is developed to solve infinite horizon optimal control problems for discrete-time nonlinear systems. The focuses of this paper are to study admissibility properties and the termination criteria of discrete-time local value iteration ADP algorithms. In the discrete-time local value iteration ADP algorithm, the iterative value functions and the iterative control laws are both updated in a given subset of the state space in each iteration, instead of the whole state space. For the first time, admissibility properties of iterative control laws are analyzed for the local value iteration ADP algorithm. New termination criteria are established, which terminate the iterative local ADP algorithm with an admissible approximate optimal control law. Finally, simulation results are given to illustrate the performance of the developed algorithm.In this paper, a novel local value iteration adaptive dynamic programming (ADP) algorithm is developed to solve infinite horizon optimal control problems for discrete-time nonlinear systems. The focuses of this paper are to study admissibility properties and the termination criteria of discrete-time local value iteration ADP algorithms. In the discrete-time local value iteration ADP algorithm, the iterative value functions and the iterative control laws are both updated in a given subset of the state space in each iteration, instead of the whole state space. For the first time, admissibility properties of iterative control laws are analyzed for the local value iteration ADP algorithm. New termination criteria are established, which terminate the iterative local ADP algorithm with an admissible approximate optimal control law. Finally, simulation results are given to illustrate the performance of the developed algorithm.

  12. Tricyclic antidepressant amitriptyline indirectly increases the proliferation of adult dentate gyrus-derived neural precursors: an involvement of astrocytes.

    Directory of Open Access Journals (Sweden)

    Shuken Boku

    Full Text Available Antidepressants increase the proliferation of neural precursors in adult dentate gyrus (DG, which is considered to be involved in the therapeutic action of antidepressants. However, the mechanism underlying it remains unclear. By using cultured adult rat DG-derived neural precursors (ADP, we have already shown that antidepressants have no direct effects on ADP. Therefore, antidepressants may increase the proliferation of neural precursors in adult DG via unknown indirect mechanism. We have also shown that amitriptyline (AMI, a tricyclic antidepressant, induces the expressions of GDNF, BDNF, FGF2 and VEGF, common neurogenic factors, in primary cultured astrocytes (PCA. These suggest that AMI-induced factors in astrocytes may increase the proliferation of neural precursors in adult DG. To test this hypothesis, we examined the effects of AMI-induced factors and conditioned medium (CM from PCA treated with AMI on ADP proliferation. The effects of CM and factors on ADP proliferation were examined with BrdU immunocytochemistry. AMI had no effect on ADP proliferation, but AMI-treated CM increased it. The receptors of GDNF, BDNF and FGF2, but not VEGF, were expressed in ADP. FGF2 significantly increased ADP proliferation, but not BDNF and GDNF. In addition, both of a specific inhibitor of FGF receptors and anti-FGF2 antibody significantly counteracted the increasing effect of CM on ADP proliferation. In addition, FGF2 in brain is mainly derived from astrocytes that are key components of the neurogenic niches in adult DG. These suggest that AMI may increase ADP proliferation indirectly via PCA and that FGF2 may a potential candidate to mediate such an indirect effect of AMI on ADP proliferation via astrocytes.

  13. Ohmic ion temperature and thermal diffusivity profiles from the JET neutron emission profile monitor

    Energy Technology Data Exchange (ETDEWEB)

    Esposito, B. (ENEA, Frascati (Italy). Centro Ricerche Energia); Marcus, F.B.; Conroy, S.; Jarvis, O.N.; Loughlin, M.J.; Sadler, G.; Belle, P. van (Commission of the European Communities, Abingdon (United Kingdom). JET Joint Undertaking); Adams, J.M.; Watkins, N. (AEA Industrial Technology, Harwell (United Kingdom))

    1993-10-01

    The JET neutron emission profile monitor was used to study ohmically heated deuterium discharges. The radial profile of the neutron emissivity is deduced from the line-integral data. The profiles of ion temperature, T[sub i], and ion thermal diffusivity, [chi][sub i], are derived under steady-state conditions. The ion thermal diffusivity is higher than, and its scaling with plasma current opposite to, that predicted by neoclassical theory. (author).

  14. Ohmic ion temperature and thermal diffusivity profiles from the JET neutron emission profile monitor

    International Nuclear Information System (INIS)

    Esposito, B.

    1993-01-01

    The JET neutron emission profile monitor was used to study ohmically heated deuterium discharges. The radial profile of the neutron emissivity is deduced from the line-integral data. The profiles of ion temperature, T i , and ion thermal diffusivity, χ i , are derived under steady-state conditions. The ion thermal diffusivity is higher than, and its scaling with plasma current opposite to, that predicted by neoclassical theory. (author)

  15. Local flow management/profile descent algorithm. Fuel-efficient, time-controlled profiles for the NASA TSRV airplane

    Science.gov (United States)

    Groce, J. L.; Izumi, K. H.; Markham, C. H.; Schwab, R. W.; Thompson, J. L.

    1986-01-01

    The Local Flow Management/Profile Descent (LFM/PD) algorithm designed for the NASA Transport System Research Vehicle program is described. The algorithm provides fuel-efficient altitude and airspeed profiles consistent with ATC restrictions in a time-based metering environment over a fixed ground track. The model design constraints include accommodation of both published profile descent procedures and unpublished profile descents, incorporation of fuel efficiency as a flight profile criterion, operation within the performance capabilities of the Boeing 737-100 airplane with JT8D-7 engines, and conformity to standard air traffic navigation and control procedures. Holding and path stretching capabilities are included for long delay situations.

  16. ORF Alignment: NC_000913 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available 2|D Chain D, ... E. Coli Ribokinase Complexed With Ribose And Adp, Solved ... In Space Group P...d Adp, Solved In ... Space Group P212121 pdb|1RK2|B Chain B, E. Coli ... Ribokinase Complexed ...With Ribose And Adp, Solved In ... Space Group P212121 pdb|1RK2|A Chain A, E. Coli ... Ribokin...ase Complexed With Ribose And Adp, Solved In ... Space Group P212121 pdb|1

  17. ORF Alignment: NC_002695 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available 2|D Chain D, ... E. Coli Ribokinase Complexed With Ribose And Adp, Solved ... In Space Group P...d Adp, Solved In ... Space Group P212121 pdb|1RK2|B Chain B, E. Coli ... Ribokinase Complexed ...With Ribose And Adp, Solved In ... Space Group P212121 pdb|1RK2|A Chain A, E. Coli ... Ribokin...ase Complexed With Ribose And Adp, Solved In ... Space Group P212121 pdb|1

  18. ORF Alignment: NC_002655 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available 2|D Chain D, ... E. Coli Ribokinase Complexed With Ribose And Adp, Solved ... In Space Group P...d Adp, Solved In ... Space Group P212121 pdb|1RK2|B Chain B, E. Coli ... Ribokinase Complexed ...With Ribose And Adp, Solved In ... Space Group P212121 pdb|1RK2|A Chain A, E. Coli ... Ribokin...ase Complexed With Ribose And Adp, Solved In ... Space Group P212121 pdb|1

  19. A predictable Java profile

    DEFF Research Database (Denmark)

    Bøgholm, Thomas; Hansen, Rene Rydhof; Ravn, Anders Peter

    2009-01-01

    A Java profile suitable for development of high integrity embedded systems is presented. It is based on event handlers which are grouped in missions and equipped with respectively private handler memory and shared mission memory. This is a result of our previous work on developing a Java profile......, and is directly inspired by interactions with the Open Group on their on-going work on a safety critical Java profile (JSR-302). The main contribution is an arrangement of the class hierarchy such that the proposal is a generalization of Real-Time Specification for Java (RTSJ). A further contribution...

  20. Detonation Wave Profile

    Energy Technology Data Exchange (ETDEWEB)

    Menikoff, Ralph [Los Alamos National Laboratory

    2015-12-14

    The Zel’dovich-von Neumann-Doering (ZND) profile of a detonation wave is derived. Two basic assumptions are required: i. An equation of state (EOS) for a partly burned explosive; P(V, e, λ). ii. A burn rate for the reaction progress variable; d/dt λ = R(V, e, λ). For a steady planar detonation wave the reactive flow PDEs can be reduced to ODEs. The detonation wave profile can be determined from an ODE plus algebraic equations for points on the partly burned detonation loci with a specified wave speed. Furthermore, for the CJ detonation speed the end of the reaction zone is sonic. A solution to the reactive flow equations can be constructed with a rarefaction wave following the detonation wave profile. This corresponds to an underdriven detonation wave, and the rarefaction is know as a Taylor wave.

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