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Sample records for adp

  1. ADP's ABCs of Training

    Science.gov (United States)

    Weinstein, Margery

    2010-01-01

    When a company's core competence is processing data, it is sometimes easy to lose sight of the obvious--the information right under its nose. In the case of Automatic Data Processing, Inc. (ADP), a business outsourcing company specializing in human resources, payroll, tax, and benefits administrations solutions, that is not a problem. Through…

  2. Antibody-Directed Phototherapy (ADP

    Directory of Open Access Journals (Sweden)

    M. Adil Butt

    2013-04-01

    Full Text Available Photodynamic therapy (PDT is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs, which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs.

  3. Crystallographic Analysis of Tapering of ADP Crystallites

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    On the basis of crystallographic characteristics of ADP (ammonium dihydrogen phosphate) crystals and the selected growth conditions, the growth habit of ADP crystals was studied. In comparison with pyramidal planes, the growth rate of prismatic faces is slower and more sensitive to the additives and impurities for ADP crystals. When the supersaturation is low, the advance of growth steps on prismatic face can be blocked by ethanol or impurities, the crystal morphology is changed from the tetragonal prism to shuttle (i.e., the tapered shape). The tapering formation of ADP crystallites was structurally studied in a novel view.

  4. 45 CFR 95.621 - ADP reviews.

    Science.gov (United States)

    2010-10-01

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION GENERAL ADMINISTRATION-GRANT PROGRAMS (PUBLIC ASSISTANCE, MEDICAL ASSISTANCE AND STATE CHILDREN'S HEALTH INSURANCE PROGRAMS) Automatic Data Processing...; (G) Emergency preparedness; and, (H) Designation of an Agency ADP Security Manager. (iii)...

  5. Wnt pathway activation by ADP-ribosylation.

    Science.gov (United States)

    Yang, Eungi; Tacchelly-Benites, Ofelia; Wang, Zhenghan; Randall, Michael P; Tian, Ai; Benchabane, Hassina; Freemantle, Sarah; Pikielny, Claudio; Tolwinski, Nicholas S; Lee, Ethan; Ahmed, Yashi

    2016-01-01

    Wnt/β-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)-known to target Axin for proteolysis-regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly. PMID:27138857

  6. Poly(ADP-ribose) synthesis, a marker of granulocyte differentiation.

    OpenAIRE

    Ikai, K; Ueda, K; Fukushima, M.; Nakamura, T.; Hayaishi, O

    1980-01-01

    By using an indirect immunofluorescence technique, the distribution of poly(ADP-ribose) synthesis in human blood cells was investigated. The antibody used was reactive with poly(ADP-ribose) larger than trimers. The specific immunofluorescence of poly(ADP-ribose) synthesized in situ from NAD+ was observed in nuclei of lymphocytes and monocytes in normal peripheral blood. No immunofluorescence, however, was detected in granulocytes and erythrocytes. In agreement with this finding, no incorporat...

  7. 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.

  8. 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)

  9. Issues on stability of ADP feedback controllers for dynamical systems.

    Science.gov (United States)

    Balakrishnan, S N; Ding, Jie; Lewis, Frank L

    2008-08-01

    This paper traces the development of neural-network (NN)-based feedback controllers that are derived from the principle of adaptive/approximate dynamic programming (ADP) and discusses their closed-loop stability. Different versions of NN structures in the literature, which embed mathematical mappings related to solutions of the ADP-formulated problems called "adaptive critics" or "action-critic" networks, are discussed. Distinction between the two classes of ADP applications is pointed out. Furthermore, papers in "model-free" development and model-based neurocontrollers are reviewed in terms of their contributions to stability issues. Recent literature suggests that work in ADP-based feedback controllers with assured stability is growing in diverse forms. PMID:18632377

  10. Role of poly(ADP-ribosepolymerase 2 in DNA repair

    Directory of Open Access Journals (Sweden)

    Lavrik O. I.

    2012-06-01

    Full Text Available Poly(ADP-ribosylation is a posttranslational protein modification significant for the genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosylation is catalyzed by poly(ADP-ribosepolymerases (PARPs, which use NAD+ as a substrate, synthesize polymer of (ADP-ribose (PAR covalently attached to nuclear proteins including PARP themselves. PARPs constitute a large family of proteins, in which PARP1 is the most abundant and best-characterized member. In spite of growing body of PARPs’ role in cellular processes, PARP2, the closest homolog of PARP1, still remains poorly characterized at the level of its contribution to different pathways of DNA repair. An overview summarizes in vivo and in vitro data on PARP2 implication in specialized DNA repair processes, base excision repair and double strand break repair.

  11. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation.

    Science.gov (United States)

    Watanabe, Yukihide; Papoutsoglou, Panagiotis; Maturi, Varun; Tsubakihara, Yutaro; Hottiger, Michael O; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-06-10

    We previously established a mechanism of negative regulation of transforming growth factor β signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation. PMID:27129221

  12. ADP1 affects plant architecture by regulating local auxin biosynthesis.

    Directory of Open Access Journals (Sweden)

    Ruixi Li

    2014-01-01

    Full Text Available 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.

  13. Plant extracts inhibit ADP-induced platelet activation in humans: their potential therapeutic role as ADP antagonists.

    Science.gov (United States)

    Jagroop, Indera Anita

    2014-01-01

    Adenosine diphosphate (ADP) plays a pivotal role in platelet activation. Platelet hyperactivity is associated with vascular disease and also has a key role in haemostasis and thrombosis. ADP activates platelets through three purinoceptor subtypes, the G(q)-coupled P2Y(1) receptor, G(i)-coupled P2Y(12) receptor and P2X(1) ligand-gated cation channel. Platelet ADP purinergic receptors are therefore suitable targets for antiplatelet drugs. Thienopyridines such as clopidogrel and ticlopidine, as well as other ADP receptor antagonists like prasugrel, ticagrelor, cangrelor and elinogrel have demonstrated clinical benefits via the inhibition of the selective purinergic ADP receptor, P2Y(12). However, they still have limitations in their mode of action and efficacy, like increased risk of bleeding. Thus, the ongoing pursuit to develop newer and more effective antiplatelet agents continues. There is a growing interest in the purinergic antiplatelet properties exhibited by plant extracts. This article considers the following: pomolic acid isolated from Licania pittieri, brazilin isolated from the heartwood of Caesalpinia sappan L, phylligenin isolated from the twigs of Muraltia vulpina, bark oil of Gonystylus velutinus, seed and bark extracts from Aesculus hippocastanum L. and red wine phenolics and catechins isolated from green tea. Moreover, the method used to investigate platelet purinergic receptors should be considered, since using a more sensitive, high-resolution platelet sizer can sometimes detect platelet variations when the light transmission method was not able to do so. The exact mechanisms by which these plant extracts work need further investigation. They all however inhibit ADP-induced activation in human platelets. This could explain, at least in part, the protective effect of plant extracts as antiplatelet agents. PMID:24190032

  14. File list: Unc.Adp.20.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.20.AllAg.Brown_preadipocytes mm9 Unclassified Adipocyte Brown preadipocytes... http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Adp.20.AllAg.Brown_preadipocytes.bed ...

  15. File list: Unc.Adp.50.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.50.AllAg.Brown_preadipocytes mm9 Unclassified Adipocyte Brown preadipocytes... http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Adp.50.AllAg.Brown_preadipocytes.bed ...

  16. File list: Unc.Adp.05.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.05.AllAg.Brown_preadipocytes mm9 Unclassified Adipocyte Brown preadipocytes... http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Adp.05.AllAg.Brown_preadipocytes.bed ...

  17. File list: Unc.Adp.10.Unclassified.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.10.Unclassified.AllCell hg19 Unclassified Unclassified Adipocyte SRX813776,...SRX813777 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Adp.10.Unclassified.AllCell.bed ...

  18. File list: Unc.Adp.20.Unclassified.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.20.Unclassified.AllCell hg19 Unclassified Unclassified Adipocyte SRX813777,...SRX813776 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Adp.20.Unclassified.AllCell.bed ...

  19. File list: Unc.Adp.05.Unclassified.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.05.Unclassified.AllCell hg19 Unclassified Unclassified Adipocyte SRX813776,...SRX813777 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Adp.05.Unclassified.AllCell.bed ...

  20. An adpA homologue in Streptomyces avermitilis is involved in regulation of morphogenesis and melanogenesis

    Institute of Scientific and Technical Information of China (English)

    ZHAO JinLei; WEN Ying; CHEN Zhi; SONG Yuan; LI JiLun

    2007-01-01

    In Streptomyces griseus, AdpA, the key transcriptional activator in the A-factor regulatory cascade, switches on the transcription of multiple genes required for secondary metabolism and morphological differentiation. Streptomyces avermitilis also contains an ortholog of adpA, which is named adpA-a. To clarify the in vivo function of adpA-a, an adpA-a-disrupted strain was constructed by double crossover recombination. No difference in avermectin production was found between the adpA-a-disruptant and the wild-type strain. However, this disruptant neither formed spores nor produced melanin and its phenotype was restored to the original wild-type by a single copy of the adpA-a gene integrated into the chromosome. This report shows that adpA-a is involved in regulation of morphological differentiation and melanin production in S. avermitilis.

  1. Abiogenic photophosphorylation of ADP to ATP sensitized by flavoproteinoid microspheres.

    Science.gov (United States)

    Kolesnikov, Michael P; Telegina, Taisiya A; Lyudnikova, Tamara A; Kritsky, Mikhail S

    2008-06-01

    A model for abiogenic photophosphorylation of ADP by orthophosphate to yield ATP was studied. The model is based on the photochemical activity of flavoproteinoid microspheres that are formed by aggregation in an aqueous medium of products of thermal condensation of a glutamic acid, glycine and lysine mixture (8:3:1) and contain, along with amino acid polymers (proteinoids), abiogenic isoalloxazine (flavin) pigments. Irradiation of aqueous suspensions of microspheres with blue visible light or ultraviolet in the presence of ADP and orthophosphate resulted in ATP formation. The yield of ATP in aerated suspensions was 10-20% per one mol of starting ADP. Deaeration reduced the photophosphorylating activity of microspheres five to 10 times. Treatment of aerated microsphere suspensions with superoxide dismutase during irradiation partially suppressed ATP formation. Deaerated microspheres restored completely their photophosphorylating activity after addition of hydrogen peroxide to the suspension. The photophosphorylating activity of deaerated suspensions of flavoproteinoid microspheres was also recovered by introduction of Fe3+-cytochrome c, an electron acceptor alternative to oxygen. On the basis of the results obtained, a chemical mechanism of phosphorylation is proposed in which the free radical form of reduced flavin sensitizer (F1H*) and ADP are involved. PMID:18386156

  2. Poly(ADP-ribose) Regulates Stress Responses and microRNA Activity in the Cytoplasm

    OpenAIRE

    Leung, Anthony K. L.; Vyas, Sejal; Rood, Jennifer E.; Bhutkar, Arjun; Sharp, Phillip A.; Chang, Paul

    2011-01-01

    Poly(ADP-ribose) is a major regulatory macromolecule in the nucleus, where it regulates transcription, chromosome structure and DNA damage repair. Functions in the interphase cytoplasm are less understood. Here we identify a requirement for poly(ADP-ribose) in the assembly of cytoplasmic stress granules, which accumulate RNA-binding proteins that regulate the translation and stability of mRNAs upon stress. We show that poly(ADP-ribose), 6 specific poly(ADP-ribose) polymerases and 2 poly(ADP-r...

  3. mADP-RTs: Versatile virulence factors from bacterial pathogens of plants and mammals

    OpenAIRE

    Lennart eWirthmueller; Banfield, Mark J.

    2012-01-01

    Mono ADP-ribosyltransferases (mADP-RTs) are a family of enzymes that cleave NAD+ and covalently attach the ADP-ribosyl moiety to target proteins. mADP-RTs are well established as important virulence factors of bacteria that infect mammals. Cholera toxin, pertussis toxin and diphteria toxin are three of the best-known examples of mADP-RTs. They modify host target proteins in order to promote infection and/or killing of the host cell. Despite low sequence similarity at the primary amino acid le...

  4. mADP-RTs: Versatile virulence factors from bacterial pathogens of plants and mammals

    Directory of Open Access Journals (Sweden)

    Lennart eWirthmueller

    2012-06-01

    Full Text Available Mono ADP-ribosyltransferases (mADP-RTs are a family of enzymes that cleave NAD+ and covalently attach the ADP-ribosyl moiety to target proteins. mADP-RTs are well established as important virulence factors of bacteria that infect mammals. Cholera toxin, pertussis toxin and diphteria toxin are three of the best-known examples of mADP-RTs. They modify host target proteins in order to promote infection and/or killing of the host cell. Despite low sequence similarity at the primary amino acid level, mADP-RTs share a conserved core catalytic fold and structural biology has made important contributions to elucidating how mADP-RTs modify mammalian host targets. Recently, mADP-RTs were shown to be present in plant pathogenic bacteria, suggesting that mADP-RTs are also important virulence factors of plant pathogens. Crystal structures of plant pathogenic bacterial mADP-RTs are also now available. Here we review the structure/function of mADP-RTs from pathogens of mammals and plants, highlighting both commonalities and differences.

  5. Proximal ADP-ribose Hydrolysis in Trypanosomatids is Catalyzed by a Macrodomain.

    Science.gov (United States)

    Haikarainen, Teemu; Lehtiö, Lari

    2016-01-01

    ADP-ribosylation is a ubiquitous protein modification utilized by both prokaryotes and eukaryotes for several cellular functions, such as DNA repair, proliferation, and cell signaling. Higher eukaryotes, such as humans, utilize various enzymes to reverse the modification and to regulate ADP-ribose dependent signaling. In contrast, some lower eukaryotes, including trypanosomatids, lack many of these enzymes and therefore have a much more simplified ADP-ribose metabolism. Here we identified and characterized ADP-ribose hydrolases from Trypanosoma brucei and Trypanosoma cruzi, which are homologous to human O-acetyl-ADP-ribose deacetylases MacroD1 and MacroD2. The enzymes are capable for hydrolysis of protein linked ADP-ribose and a product of sirtuin-mediated lysine deacetylation, O-acetyl-ADP-ribose. Crystal structures of the trypanosomatid macrodomains revealed a conserved catalytic site with distinct differences to human MacroD1 and MacroD2. PMID:27064071

  6. Structure of Plasmodium falciparum ADP-ribosylation factor 1

    Energy Technology Data Exchange (ETDEWEB)

    Cook, William J.; Smith, Craig D.; Senkovich, Olga; Holder, Anthony A.; Chattopadhyay, Debasish (UAB); (NIMR)

    2011-09-26

    Vesicular trafficking may play a crucial role in the pathogenesis and survival of the malaria parasite. ADP-ribosylation factors (ARFs) are among the major components of vesicular trafficking pathways in eukaryotes. The crystal structure of ARF1 GTPase from Plasmodium falciparum has been determined in the GDP-bound conformation at 2.5 {angstrom} resolution and is compared with the structures of mammalian ARF1s.

  7. Yeast mitochondrial ADP/ATP carriers are monomeric in detergents

    OpenAIRE

    Bamber, Lisa; Harding, Marilyn; Butler, P. Jonathan G.; Kunji, Edmund R.S.

    2006-01-01

    Mitochondrial carriers are believed widely to be homodimers both in the inner membrane of the organelle and in detergents. The dimensions and molecular masses of the detergent and protein–detergent micelles were measured for yeast ADP/ATP carriers in a range of different detergents. The radius of the carrier at the midpoint of the membrane, its average radius, its Stokes' radius, its molecular mass, and its excluded volume were determined. These parameters are consistent with the known struct...

  8. 26 CFR 1.401(k)-2 - ADP test.

    Science.gov (United States)

    2010-04-01

    ...)(2)(iv) (as it appeared in the April 1, 2007, edition of 26 CFR part 1). (v) Distribution. Within 12... determined under § 1.401(k)-2(b)(2)(vi) (as it appeared in the April 1, 2007, edition of 26 CFR Part 1). (C... 26 Internal Revenue 5 2010-04-01 2010-04-01 false ADP test. 1.401(k)-2 Section 1.401(k)-2...

  9. A mitochondrial import receptor for the ADP/ATP carrier

    OpenAIRE

    Söllner, Thomas; Griffiths, Gareth; Pfanner, Nikolaus; Neupert, Walter

    1990-01-01

    We have identified a mitochondrial outer membrane protein of 72 kd (MOM72) that exhibits the properties of an import receptor for the ADP/ATP carrier (AAC), the most abundant mitochondrial protein. Monospecific antibodies and Fab fragments against MOM72 selectively inhibit import of AAC at the level of specific binding to the mitochondria. AAC bound to the mitochondrial surface is coprecipitated with antibodies against MOM72 after lysis of mitochondria with detergent. MOM72 thus has a complem...

  10. [Suggested mitochondrial ancestry of non-mitochondrial ATP/ADP].

    Science.gov (United States)

    Emel'ianov, V V

    2007-01-01

    One of the major evolutionary events that transformed endosymbiotic bacterium into mitochondrion was an acquisition of ATP/ADP carrier in order to supply the host with respiration-derived ATP. Along with mitochondrial carrier, unrelated carrier is known which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic alpha-Proteobacteria. This non-mitochondrial ATP/ADP carrier was recently described in rickettsia-like endosymbionts - a group of obligate intracellular bacteria, classified with the order Rickettsiales, which have diverged after free-living alpha-Proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on the non-mitochondrial carrier were reanalysed in the present work using both DNA and protein sequences, and various methods including Bayesian analysis. The data presented are consistent with classic endosymbiont theory for the origin of mitochondria and also suggest that even last but one common ancestor of rickettsiae and organelles may have been an endosymbiotic bacterium in which ATP/ADP carrier has first originated. PMID:17380892

  11. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

    OpenAIRE

    Sharifi, Reza; Morra, Rosa; Denise Appel, C; Tallis, Michael; Chioza, Barry; Jankevicius, Gytis; Simpson, Michael A.; Matic, Ivan; Ozkan, Ege; Golia, Barbara; Schellenberg, Matthew J.; Weston, Ria; Williams, Jason G.; Rossi, Marianna N.; Galehdari, Hamid

    2013-01-01

    Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase...

  12. PARP-1 mechanism for coupling DNA damage detection to poly(ADP-ribose) synthesis

    OpenAIRE

    Langelier, Marie-France; Pascal, John M.

    2013-01-01

    Poly(ADP-ribose) polymerase 1 (PARP-1) regulates gene transcription, cell death signaling, and DNA repair through production of the posttranslational modification poly(ADP-ribose). During the cellular response to genotoxic stress PARP-1 rapidly associates with DNA damage, which robustly stimulates poly(ADP-ribose) production over a low basal level of PARP-1 activity. DNA damage-dependent PARP-1 activity is central to understanding PARP-1 biological function, but structural insights into the m...

  13. Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

    OpenAIRE

    Hu, Biao; Wu, Zhe; Hergert, Polla; Henke, Craig A.; Bitterman, Peter B.; Phan, Sem H.

    2013-01-01

    Poly(ADP-ribosyl)ation (PARylation) is a post-translational protein modification effected by enzymes belonging to the poly(ADP-ribose) polymerase (PARP) superfamily, mainly by PARP-1. The key acceptors of poly(ADP-ribose) include PARP-1 itself, histones, DNA repair proteins, and transcription factors. Because many of these factors are involved in the regulation of myofibroblast differentiation, we examined the role of PARylation on myofibroblast differentiation. Overexpression of PARP-1 with ...

  14. ADP-Glucose Pyrophosphorylase, a Regulatory Enzyme for Bacterial Glycogen Synthesis

    OpenAIRE

    Ballicora, Miguel A; Iglesias, Alberto A.; Preiss, Jack

    2003-01-01

    The accumulation of α-1,4-polyglucans is an important strategy to cope with transient starvation conditions in the environment. In bacteria and plants, the synthesis of glycogen and starch occurs by utilizing ADP-glucose as the glucosyl donor for elongation of the α-1,4-glucosidic chain. The main regulatory step takes place at the level of ADP-glucose synthesis, a reaction catalyzed by ADP-Glc pyrophosphorylase (PPase). Most of the ADP-Glc PPases are allosterically regulated by intermediates ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Okita, Naoyuki, E-mail: nokita7@rs.noda.tus.ac.jp [Genome and Drug Research Center, Tokyo University of Science (Japan); Ashizawa, Daisuke; Ohta, Ryo [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022 (Japan); Abe, Hideaki [Genome and Drug Research Center, Tokyo University of Science (Japan); Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022 (Japan); Tanuma, Sei-ichi, E-mail: tanuma@rs.noda.tus.ac.jp [Genome and Drug Research Center, Tokyo University of Science (Japan); Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022 (Japan)

    2010-02-19

    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{sub max}) and the michaelis constant (k{sub m}) of PARG reaction were 4.46 {mu}M and 128.33 {mu}mol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 {mu}M. 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. Chemical Bond Calculations of Crystal Growth of KDP and ADP

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A novel method was proposed to calculate the crystal morphology (or growth habit) on the basis of chemical bond analysis. All constituent chemical bonds were distinguished as relevant and independent bonds according to their variations during the crystallization process. By employing the current method, the influence of specific growth conditions on the crystal morphology can be considered in the structure analysis process. The ideal morphologies of both KDP (KH2PO4) and ADP (NH4H2PO4) crystals were calculated and compared with our obtained crystallites at room temperature, which validates the present calculation method very well.

  17. Import of ADP/ATP carrier into mitochondria

    OpenAIRE

    Steger, Heinrich F.; Söllner, Thomas; Kiebler, Michael; Dietmeier, Klaus A.; Pfaller, Rupert; Trülzsch, Konrad S.; Tropschug, Maximilian; Neupert, Walter; Pfanner, Nikolaus

    1990-01-01

    We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into ...

  18. 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)

    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

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

    International Nuclear Information System (INIS)

    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)

  20. Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities

    OpenAIRE

    Khadka, Prabhat; Hsu, Joseph K; Veith, Sebastian; Tadokoro, Takashi; Shamanna, Raghavendra A.; Mangerich, Aswin; Croteau, Deborah L.; Bohr, Vilhelm A.

    2015-01-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs ...

  1. File list: Oth.Adp.05.AllAg.Pre-adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.05.AllAg.Pre-adipocytes hg19 TFs and others Adipocyte Pre-adipocytes SRX760...968,SRX760970,SRX760967,SRX760971,SRX760969 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Adp.05.AllAg.Pre-adipocytes.bed ...

  2. File list: His.Adp.10.AllAg.Pre-adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.10.AllAg.Pre-adipocytes hg19 Histone Adipocyte Pre-adipocytes SRX760965,SRX...760962,SRX760966,SRX760964,SRX760963 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.10.AllAg.Pre-adipocytes.bed ...

  3. File list: Oth.Adp.10.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.10.AllAg.Brown_adipocytes mm9 TFs and others Adipocyte Brown adipocytes SRX...RX800014,SRX978690,SRX978689,SRX978688,SRX800019 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Adp.10.AllAg.Brown_adipocytes.bed ...

  4. File list: InP.Adp.10.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.10.AllAg.Brown_preadipocytes mm9 Input control Adipocyte Brown preadipocyte...058,SRX341056,SRX478161 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.10.AllAg.Brown_preadipocytes.bed ...

  5. File list: Pol.Adp.20.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.20.AllAg.Brown_preadipocytes mm9 RNA polymerase Adipocyte Brown preadipocyt...es SRX341031,SRX341032,SRX341029,SRX341030 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Adp.20.AllAg.Brown_preadipocytes.bed ...

  6. File list: ALL.Adp.05.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.05.AllAg.Brown_adipocytes mm9 All antigens Adipocyte Brown adipocytes SRX80...X185879,SRX978689,SRX978688,SRX478162 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Adp.05.AllAg.Brown_adipocytes.bed ...

  7. File list: Unc.Adp.10.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.10.AllAg.Brown_adipocytes mm9 Unclassified Adipocyte Brown adipocytes SRX97...8685,SRX800022 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Adp.10.AllAg.Brown_adipocytes.bed ...

  8. File list: InP.Adp.20.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.20.AllAg.Brown_preadipocytes mm9 Input control Adipocyte Brown preadipocyte...782,SRX341056,SRX478161 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.20.AllAg.Brown_preadipocytes.bed ...

  9. File list: Oth.Adp.20.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.20.AllAg.Brown_adipocytes mm9 TFs and others Adipocyte Brown adipocytes SRX...RX978689,SRX800015,SRX800014,SRX800018,SRX800019 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Adp.20.AllAg.Brown_adipocytes.bed ...

  10. File list: Oth.Adp.05.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.05.AllAg.Brown_preadipocytes mm9 TFs and others Adipocyte Brown preadipocyt...341766,SRX341418,SRX341023,SRX341419,SRX341767 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Adp.05.AllAg.Brown_preadipocytes.bed ...

  11. File list: Pol.Adp.50.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.50.AllAg.Brown_preadipocytes mm9 RNA polymerase Adipocyte Brown preadipocyt...es SRX341031,SRX341032,SRX341029,SRX341030 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Adp.50.AllAg.Brown_preadipocytes.bed ...

  12. File list: Pol.Adp.05.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.05.AllAg.Brown_preadipocytes mm9 RNA polymerase Adipocyte Brown preadipocyt...es SRX341031,SRX341032,SRX341029,SRX341030 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Adp.05.AllAg.Brown_preadipocytes.bed ...

  13. File list: ALL.Adp.10.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.10.AllAg.Brown_adipocytes mm9 All antigens Adipocyte Brown adipocytes SRX80...X978688,SRX800019,SRX478163,SRX478162 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Adp.10.AllAg.Brown_adipocytes.bed ...

  14. File list: ALL.Adp.05.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.05.AllAg.Brown_preadipocytes mm9 All antigens Adipocyte Brown preadipocytes...RX341419,SRX341767,SRX341421,SRX478161,SRX341039,SRX341040 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Adp.05.AllAg.Brown_preadipocytes.bed ...

  15. File list: Unc.Adp.05.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.05.AllAg.Brown_adipocytes mm9 Unclassified Adipocyte Brown adipocytes SRX97...8685,SRX800022 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Adp.05.AllAg.Brown_adipocytes.bed ...

  16. File list: ALL.Adp.10.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.10.AllAg.Brown_preadipocytes mm9 All antigens Adipocyte Brown preadipocytes...RX341420,SRX478161,SRX478160,SRX341040,SRX341041,SRX341039 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Adp.10.AllAg.Brown_preadipocytes.bed ...

  17. File list: Oth.Adp.05.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.05.AllAg.Brown_adipocytes mm9 TFs and others Adipocyte Brown adipocytes SRX...RX800019,SRX978691,SRX978690,SRX978689,SRX978688 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Adp.05.AllAg.Brown_adipocytes.bed ...

  18. File list: InP.Adp.05.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.05.AllAg.Brown_adipocytes mm9 Input control Adipocyte Brown adipocytes SRX4...78163,SRX143805,SRX185879 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.05.AllAg.Brown_adipocytes.bed ...

  19. File list: DNS.Adp.20.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Adp.20.AllAg.Brown_preadipocytes mm9 DNase-seq Adipocyte Brown preadipocytes ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Adp.20.AllAg.Brown_preadipocytes.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.20.AllAg.Brown_preadipocytes mm9 All antigens Adipocyte Brown preadipocytes...RX341044,SRX341420,SRX341421,SRX341046,SRX478161,SRX341027 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Adp.20.AllAg.Brown_preadipocytes.bed ...

  1. File list: Oth.Adp.50.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.50.AllAg.Brown_preadipocytes mm9 TFs and others Adipocyte Brown preadipocyt...341028,SRX341760,SRX341767,SRX341763,SRX341027 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Adp.50.AllAg.Brown_preadipocytes.bed ...

  2. File list: NoD.Adp.05.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adp.05.AllAg.Brown_preadipocytes mm9 No description Adipocyte Brown preadipocyt...es http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Adp.05.AllAg.Brown_preadipocytes.bed ...

  3. File list: DNS.Adp.05.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Adp.05.AllAg.Brown_preadipocytes mm9 DNase-seq Adipocyte Brown preadipocytes ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Adp.05.AllAg.Brown_preadipocytes.bed ...

  4. File list: Pol.Adp.10.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.10.AllAg.Brown_adipocytes mm9 RNA polymerase Adipocyte Brown adipocytes SRX...800010,SRX800016,SRX800017 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Adp.10.AllAg.Brown_adipocytes.bed ...

  5. File list: Pol.Adp.50.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.50.AllAg.Brown_adipocytes mm9 RNA polymerase Adipocyte Brown adipocytes SRX...800010,SRX800016,SRX800017 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Adp.50.AllAg.Brown_adipocytes.bed ...

  6. File list: NoD.Adp.50.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adp.50.AllAg.Brown_adipocytes mm9 No description Adipocyte Brown adipocytes htt...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Adp.50.AllAg.Brown_adipocytes.bed ...

  7. File list: ALL.Adp.20.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.20.AllAg.Brown_adipocytes mm9 All antigens Adipocyte Brown adipocytes SRX80...X800018,SRX800019,SRX185797,SRX478162 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Adp.20.AllAg.Brown_adipocytes.bed ...

  8. File list: ALL.Adp.50.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.50.AllAg.Brown_preadipocytes mm9 All antigens Adipocyte Brown preadipocytes...RX341420,SRX341421,SRX341046,SRX478161,SRX341027,SRX478160 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Adp.50.AllAg.Brown_preadipocytes.bed ...

  9. File list: His.Adp.20.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.20.AllAg.Brown_preadipocytes mm9 Histone Adipocyte Brown preadipocytes SRX3...RX341420,SRX341421,SRX341046 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Adp.20.AllAg.Brown_preadipocytes.bed ...

  10. File list: Pol.Adp.05.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.05.AllAg.Brown_adipocytes mm9 RNA polymerase Adipocyte Brown adipocytes SRX...800010,SRX800016,SRX800017 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Adp.05.AllAg.Brown_adipocytes.bed ...

  11. File list: NoD.Adp.20.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adp.20.AllAg.Brown_adipocytes mm9 No description Adipocyte Brown adipocytes htt...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Adp.20.AllAg.Brown_adipocytes.bed ...

  12. File list: Unc.Adp.20.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.20.AllAg.Brown_adipocytes mm9 Unclassified Adipocyte Brown adipocytes SRX97...8685,SRX800022 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Adp.20.AllAg.Brown_adipocytes.bed ...

  13. File list: His.Adp.05.AllAg.Brown_preadipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.05.AllAg.Brown_preadipocytes mm9 Histone Adipocyte Brown preadipocytes SRX3...RX341421,SRX341039,SRX341040 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Adp.05.AllAg.Brown_preadipocytes.bed ...

  14. File list: NoD.Adp.10.AllAg.Brown_adipocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adp.10.AllAg.Brown_adipocytes mm9 No description Adipocyte Brown adipocytes htt...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Adp.10.AllAg.Brown_adipocytes.bed ...

  15. File list: ALL.Adp.20.AllAg.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.20.AllAg.AllCell mm9 All antigens Adipocyte SRX821805,SRX821802,SRX800012,S...1420,SRX341421,SRX341046,SRX478161,SRX478162,SRX341027 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Adp.20.AllAg.AllCell.bed ...

  16. File list: ALL.Adp.10.AllAg.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.10.AllAg.AllCell mm9 All antigens Adipocyte SRX821805,SRX821802,SRX800012,S...7383,SRX478160,SRX127381,SRX341040,SRX341041,SRX341039 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Adp.10.AllAg.AllCell.bed ...

  17. File list: Oth.Adp.20.Cebpb.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.20.Cebpb.AllCell mm9 TFs and others Cebpb Adipocyte SRX341415,SRX341026,SRX...341417,SRX341414,SRX341025,SRX341416 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Adp.20.Cebpb.AllCell.bed ...

  18. File list: Oth.Adp.50.Cebpb.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.50.Cebpb.AllCell mm9 TFs and others Cebpb Adipocyte SRX341417,SRX341415,SRX...341026,SRX341414,SRX341416,SRX341025 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Adp.50.Cebpb.AllCell.bed ...

  19. File list: ALL.Adp.50.AllAg.Capan-1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.50.AllAg.Capan-1 hg19 All antigens Adipocyte Capan-1 SRX825378,SRX825364,SR...X825385,SRX825392,SRX825371 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.50.AllAg.Capan-1.bed ...

  20. 7 CFR 277.18 - Establishment of an Automated Data Processing (ADP) and Information Retrieval System.

    Science.gov (United States)

    2010-01-01

    ...) and Information Retrieval System. 277.18 Section 277.18 Agriculture Regulations of the Department of... Data Processing (ADP) and Information Retrieval System. (a) Scope and application. This section... costs of planning, design, development or installation of ADP and information retrieval systems if...

  1. 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 ...

  2. File list: Pol.Adp.20.RNA_polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.20.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Adipoc...yte http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Adp.20.RNA_polymerase_III.AllCell.bed ...

  3. File list: Pol.Adp.10.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.10.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Adipocy...te http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Adp.10.RNA_Polymerase_III.AllCell.bed ...

  4. File list: Unc.Adp.20.Unclassified.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.20.Unclassified.AllCell mm9 Unclassified Unclassified Adipocyte SRX978685,S...RX800022 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Adp.20.Unclassified.AllCell.bed ...

  5. File list: Unc.Adp.05.Unclassified.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: Oth.Adp.10.AllAg.SGBS [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Oth.Adp.20.AllAg.SGBS [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: ALL.Adp.50.AllAg.SGBS [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: NoD.Adp.50.NA.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available His.Adp.05.AllAg.Adipose_Tissue,_White hg19 Histone Adipocyte Adipose Tissue, White... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.05.AllAg.Adipose_Tissue,_White.bed ...

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available DNS.Adp.05.AllAg.Adipose_Tissue,_White hg19 DNase-seq Adipocyte Adipose Tissue, Whi...te http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Adp.05.AllAg.Adipose_Tissue,_White.bed ...

  13. File list: NoD.Adp.20.AllAg.Adipose_Tissue [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adp.20.AllAg.Adipose_Tissue hg19 No description Adipocyte Adipose Tissue SRX134...732 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Adp.20.AllAg.Adipose_Tissue.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.10.AllAg.Adipose_Tissue,_White hg19 RNA polymerase Adipocyte Adipose Tissue..., White http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Adp.10.AllAg.Adipose_Tissue,_White.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.10.AllAg.Adipose_Tissue,_White hg19 All antigens Adipocyte Adipose Tissue, ...X821821,SRX821815,SRX821811,SRX821817,SRX821809,SRX821810 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.10.AllAg.Adipose_Tissue,_White.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available DNS.Adp.20.AllAg.Adipose_Tissue,_White hg19 DNase-seq Adipocyte Adipose Tissue, Whi...te http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Adp.20.AllAg.Adipose_Tissue,_White.bed ...

  17. File list: Unc.Adp.50.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.50.AllAg.Adipose_Tissue,_White hg19 Unclassified Adipocyte Adipose Tissue, ...White http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Adp.50.AllAg.Adipose_Tissue,_White.bed ...

  18. 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 ...

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

    Lifescience Database Archive (English)

    Full Text Available His.Adp.10.AllAg.Adipose_Tissue,_White hg19 Histone Adipocyte Adipose Tissue, White... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.10.AllAg.Adipose_Tissue,_White.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.10.AllAg.Adipose_Tissue,_White hg19 Unclassified Adipocyte Adipose Tissue, ...White http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Adp.10.AllAg.Adipose_Tissue,_White.bed ...

  1. File list: His.Adp.20.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.20.AllAg.Adipose_Tissue,_White hg19 Histone Adipocyte Adipose Tissue, White... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.20.AllAg.Adipose_Tissue,_White.bed ...

  2. File list: Unc.Adp.20.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.20.AllAg.Adipose_Tissue,_White hg19 Unclassified Adipocyte Adipose Tissue, ...White http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Adp.20.AllAg.Adipose_Tissue,_White.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.05.AllAg.Adipose_Tissue,_White hg19 All antigens Adipocyte Adipose Tissue, ...X821815,SRX821821,SRX821816,SRX821809,SRX821817,SRX821810 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.05.AllAg.Adipose_Tissue,_White.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.20.AllAg.Adipose_Tissue,_White hg19 RNA polymerase Adipocyte Adipose Tissue..., White http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Adp.20.AllAg.Adipose_Tissue,_White.bed ...

  5. File list: NoD.Adp.50.AllAg.Adipose_Tissue [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adp.50.AllAg.Adipose_Tissue hg19 No description Adipocyte Adipose Tissue SRX134...732 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Adp.50.AllAg.Adipose_Tissue.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.05.AllAg.Adipose_Tissue,_White hg19 TFs and others Adipocyte Adipose Tissue...SRX821815,SRX821821,SRX821816,SRX821809,SRX821817,SRX821810 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Adp.05.AllAg.Adipose_Tissue,_White.bed ...

  7. 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 ...

  8. File list: Pol.Adp.50.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.50.AllAg.Adipose_Tissue,_White hg19 RNA polymerase Adipocyte Adipose Tissue..., White http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Adp.50.AllAg.Adipose_Tissue,_White.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Pol.Adp.05.AllAg.Adipose_Tissue,_White hg19 RNA polymerase Adipocyte Adipose Tissue..., White http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Adp.05.AllAg.Adipose_Tissue,_White.bed ...

  10. 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 ...

  11. File list: DNS.Adp.10.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Adp.10.AllAg.Adipose_Tissue,_White hg19 DNase-seq Adipocyte Adipose Tissue, Whi...te http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Adp.10.AllAg.Adipose_Tissue,_White.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.20.AllAg.Adipose_Tissue,_White hg19 TFs and others Adipocyte Adipose Tissue...SRX821817,SRX821821,SRX821815,SRX821811,SRX821810,SRX821809 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Adp.20.AllAg.Adipose_Tissue,_White.bed ...

  13. File list: Oth.Adp.10.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adp.10.AllAg.Adipose_Tissue,_White hg19 TFs and others Adipocyte Adipose Tissue...SRX821821,SRX821815,SRX821811,SRX821817,SRX821809,SRX821810 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Adp.10.AllAg.Adipose_Tissue,_White.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.50.AllAg.Adipose_Tissue,_White hg19 All antigens Adipocyte Adipose Tissue, ...X821810,SRX821806,SRX821809,SRX821817,SRX821816,SRX821807 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.50.AllAg.Adipose_Tissue,_White.bed ...

  15. File list: DNS.Adp.50.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Adp.50.AllAg.Adipose_Tissue,_White hg19 DNase-seq Adipocyte Adipose Tissue, Whi...te http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Adp.50.AllAg.Adipose_Tissue,_White.bed ...

  16. File list: His.Adp.50.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.50.AllAg.Adipose_Tissue,_White hg19 Histone Adipocyte Adipose Tissue, White... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.50.AllAg.Adipose_Tissue,_White.bed ...

  17. File list: Unc.Adp.05.AllAg.Adipose_Tissue,_White [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Adp.05.AllAg.Adipose_Tissue,_White hg19 Unclassified Adipocyte Adipose Tissue, ...White http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Adp.05.AllAg.Adipose_Tissue,_White.bed ...

  18. Structural and biochemical characterization of NAR E, an iron containing ADP-ribosyltransferase from neisseria meningitidis

    NARCIS (Netherlands)

    Koehler, C.; Carlier, L.P.A.; Veggi, D.; Balducci, C.; Di Marcello, F.; Ferrer-Navarro, M.; Pizza, M.; Daura, X.; Soriani, M.; Boelens, R.; Bonvin, A.M.J.J.

    2011-01-01

    NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the family of ADP-ribosyltransferases (ADPRT) and catalyzes the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogeni

  19. NMR resonance assignments of NarE, a putative ADP-ribosylating toxin from Neisseria meningitidis

    NARCIS (Netherlands)

    Carlier, L.P.A.; Köhler, Christian; Veggi, D.; Pizza, M.; Soriani, M.; Boelens, R.; Bonvin, A.M.J.J.

    2011-01-01

    NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the ADP-ribosyltransferase family and catalyses the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria u

  20. File list: InP.Adp.10.Input_control.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.10.Input_control.AllCell hg19 Input control Input control Adipocyte SRX0194...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Adp.10.Input_control.AllCell.bed ...

  1. File list: InP.Adp.50.Input_control.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.50.Input_control.AllCell mm9 Input control Input control Adipocyte SRX18587...27367 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.50.Input_control.AllCell.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.05.Input_control.AllCell hg19 Input control Input control Adipocyte SRX0194...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Adp.05.Input_control.AllCell.bed ...

  3. File list: InP.Adp.20.Input_control.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.20.Input_control.AllCell hg19 Input control Input control Adipocyte SRX0194...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Adp.20.Input_control.AllCell.bed ...

  4. File list: InP.Adp.20.Input_control.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.20.Input_control.AllCell mm9 Input control Input control Adipocyte SRX99775...78161 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.20.Input_control.AllCell.bed ...

  5. File list: InP.Adp.10.AllAg.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.10.AllAg.AllCell hg19 Input control Adipocyte SRX019491,SRX660092,SRX127280...1,SRX196110,SRX660091,SRX032892,SRX825392,SRX1272789,SRX469459,SRX469457,SRX027404 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Adp.10.AllAg.AllCell.bed ...

  6. File list: InP.Adp.20.AllAg.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.20.AllAg.AllCell hg19 Input control Adipocyte SRX019491,SRX660092,SRX660091...,SRX1272789,SRX1272801,SRX032892,SRX196110,SRX469459,SRX469457,SRX825392,SRX027404 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Adp.20.AllAg.AllCell.bed ...

  7. File list: InP.Adp.10.Input_control.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.10.Input_control.AllCell mm9 Input control Input control Adipocyte SRX99775...78161 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.10.Input_control.AllCell.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. A SPECTROPHOTOMETRIC ASSAY TO MEASURE RUBISCO ACTIVASE ACTIVATION ACTIVITY UNDER VARYING ATP:ADP RATIOS

    Science.gov (United States)

    The ratio of ATP to ADP in the stroma is an important regulatory mechanism for controlling the activation state of Rubisco via Rubisco activase (activase). Understanding the response of activase to a varying ATP:ADP ratio should reveal insights into the regulation of photosynthesis. However, the cur...

  10. File list: NoD.Adp.05.AllAg.Fetal_Heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adp.05.AllAg.Fetal_Heart hg19 No description Adipocyte Fetal Heart SRX088650,SR...X088647,SRX056801,SRX031428,SRX031386,SRX031444,SRX056800 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Adp.05.AllAg.Fetal_Heart.bed ...

  11. File list: His.Adp.20.AllAg.Fetal_Heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.20.AllAg.Fetal_Heart hg19 Histone Adipocyte Fetal Heart SRX860893,SRX860898...,SRX860890,SRX860889,SRX860894,SRX860892,SRX860896,SRX860895,SRX860891,SRX860897 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.20.AllAg.Fetal_Heart.bed ...

  12. File list: NoD.Adp.20.AllAg.Fetal_Heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Adp.20.AllAg.Fetal_Heart hg19 No description Adipocyte Fetal Heart SRX088650,SR...X031428,SRX031386,SRX056801,SRX031444,SRX056800,SRX088647 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Adp.20.AllAg.Fetal_Heart.bed ...

  13. File list: His.Adp.05.AllAg.Fetal_Heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.05.AllAg.Fetal_Heart hg19 Histone Adipocyte Fetal Heart SRX860893,SRX860894...,SRX860897,SRX860898,SRX860892,SRX860890,SRX860889,SRX860896,SRX860895,SRX860891 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.05.AllAg.Fetal_Heart.bed ...

  14. File list: DNS.Adp.20.AllAg.Fetal_Heart [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Adp.20.AllAg.Fetal_Heart hg19 DNase-seq Adipocyte Fetal Heart SRX040387,SRX0404...0390 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Adp.20.AllAg.Fetal_Heart.bed ...

  15. File list: ALL.Adp.10.AllAg.Capan-1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.10.AllAg.Capan-1 hg19 All antigens Adipocyte Capan-1 SRX825378,SRX825371,SR...X825364,SRX825385,SRX825392 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.10.AllAg.Capan-1.bed ...

  16. File list: ALL.Adp.20.AllAg.Capan-2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.20.AllAg.Capan-2 hg19 All antigens Adipocyte Capan-2 SRX825386,SRX825379,SR...X825365,SRX825372 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.20.AllAg.Capan-2.bed ...

  17. File list: His.Adp.10.AllAg.Capan-2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.10.AllAg.Capan-2 hg19 Histone Adipocyte Capan-2 SRX825386,SRX825379,SRX8253...72,SRX825365 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.10.AllAg.Capan-2.bed ...

  18. File list: ALL.Adp.10.AllAg.Capan-2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.10.AllAg.Capan-2 hg19 All antigens Adipocyte Capan-2 SRX825386,SRX825379,SR...X825372,SRX825365 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.10.AllAg.Capan-2.bed ...

  19. File list: His.Adp.50.AllAg.Capan-1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.50.AllAg.Capan-1 hg19 Histone Adipocyte Capan-1 SRX825378,SRX825364,SRX8253...85,SRX825371 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.50.AllAg.Capan-1.bed ...

  20. File list: ALL.Adp.05.AllAg.Capan-2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.05.AllAg.Capan-2 hg19 All antigens Adipocyte Capan-2 SRX825386,SRX825379,SR...X825372,SRX825365 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.05.AllAg.Capan-2.bed ...

  1. File list: His.Adp.50.AllAg.Capan-2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.50.AllAg.Capan-2 hg19 Histone Adipocyte Capan-2 SRX825386,SRX825379,SRX8253...65,SRX825372 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.50.AllAg.Capan-2.bed ...

  2. File list: His.Adp.05.AllAg.Capan-1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.05.AllAg.Capan-1 hg19 Histone Adipocyte Capan-1 SRX825378,SRX825371,SRX8253...64,SRX825385 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.05.AllAg.Capan-1.bed ...

  3. File list: His.Adp.10.AllAg.Capan-1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.10.AllAg.Capan-1 hg19 Histone Adipocyte Capan-1 SRX825378,SRX825371,SRX8253...64,SRX825385 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.10.AllAg.Capan-1.bed ...

  4. File list: His.Adp.05.AllAg.Capan-2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.05.AllAg.Capan-2 hg19 Histone Adipocyte Capan-2 SRX825386,SRX825379,SRX8253...72,SRX825365 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.05.AllAg.Capan-2.bed ...

  5. File list: ALL.Adp.20.AllAg.Capan-1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: His.Adp.20.AllAg.Capan-1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.20.AllAg.Capan-1 hg19 Histone Adipocyte Capan-1 SRX825378,SRX825364,SRX8253...85,SRX825371 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.20.AllAg.Capan-1.bed ...

  7. File list: His.Adp.20.AllAg.Capan-2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Adp.20.AllAg.Capan-2 hg19 Histone Adipocyte Capan-2 SRX825386,SRX825379,SRX8253...65,SRX825372 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Adp.20.AllAg.Capan-2.bed ...

  8. File list: ALL.Adp.05.AllAg.Capan-1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Adp.05.AllAg.Capan-1 hg19 All antigens Adipocyte Capan-1 SRX825378,SRX825371,SR...X825364,SRX825385,SRX825392 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Adp.05.AllAg.Capan-1.bed ...

  9. The NarE protein of Neisseria gonorrhoeae catalyzes ADP-ribosylation of several ADP-ribose acceptors despite an N-terminal deletion.

    Science.gov (United States)

    Rodas, Paula I; Álamos-Musre, A Said; Álvarez, Francisca P; Escobar, Alejandro; Tapia, Cecilia V; Osorio, Eduardo; Otero, Carolina; Calderón, Iván L; Fuentes, Juan A; Gil, Fernando; Paredes-Sabja, Daniel; Christodoulides, Myron

    2016-09-01

    The ADP-ribosylating enzymes are encoded in many pathogenic bacteria in order to affect essential functions of the host. In this study, we show that Neisseria gonorrhoeae possess a locus that corresponds to the ADP-ribosyltransferase NarE, a previously characterized enzyme in N. meningitidis The 291 bp coding sequence of gonococcal narE shares 100% identity with part of the coding sequence of the meningococcal narE gene due to a frameshift previously described, thus leading to a 49-amino-acid deletion at the N-terminus of gonococcal NarE protein. However, we found a promoter region and a GTG start codon, which allowed expression of the protein as demonstrated by RT-PCR and western blot analyses. Using a gonococcal NarE-6xHis fusion protein, we demonstrated that the gonococcal enzyme underwent auto-ADP-ribosylation but to a lower extent than meningococcal NarE. We also observed that gonoccocal NarE exhibited ADP-ribosyltransferase activity using agmatine and cell-free host proteins as ADP-ribose acceptors, but its activity was inhibited by human β-defensins. Taken together, our results showed that NarE of Neisseria gonorrhoeae is a functional enzyme that possesses key features of bacterial ADP-ribosylating enzymes. PMID:27465490

  10. Molecular and biochemical characterization of the ADP-dependent phosphofructokinase from the hyperthermophilic archaeon Pyrococcus furiosus.

    Science.gov (United States)

    Tuininga, J E; Verhees, C H; van der Oost, J; Kengen, S W; Stams, A J; de Vos, W M

    1999-07-23

    Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively. PMID:10409652

  11. Java-Backend für den ADP-Compiler

    OpenAIRE

    Sauthoff, Georg

    2007-01-01

    Im Mittelpunkt der Arbeit steht das Problem der Übersetzung von Algebraic-Dynamic-Programming-Programmen nach Java. Dazu wird ein Java-Backend für den existierenden ADP-Compiler erstellt. Es wird auf die Motivation für diese Übersetzung, auf Probleme, Optimierungsmöglichkeiten und Designentscheidungen bei der konkreten Implementation des neuen Java-Backends für den existierenden ADP-Compiler eingegangen. Neben Benchmarks von nach Java bzw. nach C übersetzten Bioinformatik-ADP-Algorithmen zur ...

  12. Strategies to Potentiate the Cellular Poly(ADP-ribosyl)ation Response to DNA Damage

    OpenAIRE

    Kunzmann, Andrea

    2009-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification of cellular proteins, which is mainly catalyzed by poly(ADP-ribose) polymerase 1 (PARP1) by using NAD+ as substrate. The catalytic activity of PARP1 is known to be triggered by the binding of PARP1 to broken DNA via its two aminoterminal zinc finger motifs. DNA strand break-induced poly(ADP-ribosyl)ation is linked to DNA repair and maintenance of genomic stability.Up to now, little information exists on the biological consequences of ...

  13. Signaling Mechanism of Poly(ADP-Ribose) Polymerase-1 (PARP-1) in Inflammatory Diseases

    OpenAIRE

    Ba, Xueqing; Garg, Nisha Jain

    2011-01-01

    Poly(ADP-ribosyl)ation, attaching the ADP-ribose polymer chain to the receptor protein, is a unique posttranslational modification. Poly(ADP-ribose) polymerase-1 (PARP-1) is a well-characterized member of the PARP family. In this review, we provide a general update on molecular structure and structure-based activity of this enzyme. However, we mainly focus on the roles of PARP-1 in inflammatory diseases. Specifically, we discuss the signaling pathway context that PARP-1 is involved in to regu...

  14. The energetics of allosteric regulation of ADP release from myosin heads.

    Science.gov (United States)

    Jackson, Del R; Baker, Josh E

    2009-06-28

    Myosin molecules are involved in a wide range of transport and contractile activities in cells. A single myosin head functions through its ATPase reaction as a force generator and as a mechanosensor, and when two or more myosin heads work together in moving along an actin filament, the interplay between these mechanisms contributes to collective myosin behaviors. For example, the interplay between force-generating and force-sensing mechanisms coordinates the two heads of a myosin V molecule in its hand-over-hand processive stepping along an actin filament. In muscle, it contributes to the Fenn effect and smooth muscle latch. In both examples, a key force-sensing mechanism is the regulation of ADP release via interhead forces that are generated upon actin-myosin binding. Here we present a model describing the mechanism of allosteric regulation of ADP release from myosin heads as a change, DeltaDeltaG(-D), in the standard free energy for ADP release that results from the work, Deltamicro(mech), performed by that myosin head upon ADP release, or DeltaDeltaG(-D) = Deltamicro(mech). We show that this model is consistent with previous measurements for strain-dependent kinetics of ADP release in both myosin V and muscle myosin II. The model makes explicit the energetic cost of accelerating ADP release, showing that acceleration of ADP release during myosin V processivity requires approximately 4 kT of energy whereas the energetic cost for accelerating ADP release in a myosin II-based actin motility assay is only approximately 0.4 kT. The model also predicts that the acceleration of ADP release involves a dissipation of interhead forces. To test this prediction, we use an in vitro motility assay to show that the acceleration of ADP release from both smooth and skeletal muscle myosin II correlates with a decrease in interhead force. Our analyses provide clear energetic constraints for models of the allosteric regulation of ADP release and provide novel, testable insights

  15. Guanine nucleotide-binding proteins that enhance choleragen ADP-ribosyltransferase activity: nucleotide and deduced amino acid sequence of an ADP-ribosylation factor cDNA.

    OpenAIRE

    Price, S R; Nightingale, M.; Tsai, S C; Williamson, K. C.; Adamik, R; H. C. Chen; Moss, J; M. Vaughan

    1988-01-01

    Three (two soluble and one membrane) guanine nucleotide-binding proteins (G proteins) that enhance ADP-ribosylation of the Gs alpha stimulatory subunit of the adenylyl cyclase (EC 4.6.1.1) complex by choleragen have recently been purified from bovine brain. To further define the structure and function of these ADP-ribosylation factors (ARFs), we isolated a cDNA clone (lambda ARF2B) from a bovine retinal library by screening with a mixed heptadecanucleotide probe whose sequence was based on th...

  16. 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.

  17. Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs.

    Science.gov (United States)

    Gagné, Jean-Philippe; Ethier, Chantal; Defoy, Daniel; Bourassa, Sylvie; Langelier, Marie-France; Riccio, Amanda A; Pascal, John M; Moon, Kyung-Mee; Foster, Leonard J; Ning, Zhibin; Figeys, Daniel; Droit, Arnaud; Poirier, Guy G

    2015-06-01

    An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADP-ribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process. PMID:25800440

  18. Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex

    OpenAIRE

    Tsurumura, Toshiharu; Tsumori, Yayoi; Qiu, Hao; Oda, Masataka; Sakurai, Jun; Nagahama, Masahiro; Tsuge, Hideaki

    2012-01-01

    Clostridium perfringens iota-toxin (Ia) mono-ADP ribosylates Arg177 of actin, leading to cytoskeletal disorganization and cell death. To fully understand the reaction mechanism of arginine-specific mono-ADP ribosyl transferase, the structure of the toxin-substrate protein complex must be characterized. Recently, we solved the crystal structure of Ia in complex with actin and the nonhydrolyzable NAD+ analog βTAD (thiazole-4-carboxamide adenine dinucleotide); however, the structures of the NAD+...

  19. File list: InP.Adp.05.AllAg.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.05.AllAg.AllCell mm9 Input control Adipocyte SRX997757,SRX478163,SRX821800,...0,SRX341056,SRX341058,SRX821799,SRX341057,SRX341783,SRX341781,SRX478161,SRX127367,SRX127370 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.05.AllAg.AllCell.bed ...

  20. File list: InP.Adp.50.AllAg.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Adp.50.AllAg.AllCell mm9 Input control Adipocyte SRX185879,SRX143805,SRX268023,...7,SRX341056,SRX341058,SRX821800,SRX821801,SRX821799,SRX478163,SRX478161,SRX127370,SRX127367 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Adp.50.AllAg.AllCell.bed ...

  1. ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease.

    Science.gov (United States)

    Sequeira, Vasco; Najafi, Aref; Wijnker, Paul J M; Dos Remedios, Cristobal G; Michels, Michelle; Kuster, Diederik W D; van der Velden, Jolanda

    2015-12-15

    Diastolic dysfunction is general to all idiopathic dilated (IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from increased actin-myosin formation during diastole due to altered tropomyosin position, which blocks myosin binding to actin in the absence of Ca(2+). We investigated whether ADP-stimulated force development (without Ca(2+)) can be used to reveal changes in actin-myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7mut, MYBPC3mut) and thin-filament (TNNT2mut, TNNI3mut) mutations, and IDCM were compared with sarcomere mutation-negative HCM (HCMsmn) and nonfailing donors. Myofilament ADP sensitivity was higher in IDCM and HCM compared with donors, whereas it was lower for MYBPC3. Increased ADP sensitivity in IDCM, HCMsmn, and MYH7mut was caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment. Troponin exchange experiments in a TNNT2mut sample corrected the abnormal actin-myosin blockade. In MYBPC3trunc samples, ADP sensitivity highly correlated with cardiac myosin-binding protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with cMyBP-C antibody against the actin-binding N-terminal region reduced ADP sensitivity, indicative of cMyBP-C's role in actin-myosin regulation. In the presence of Ca(2+), ADP increased myofilament force development and sarcomere stiffness. Enhanced sarcomere stiffness in sarcomere mutation-positive HCM samples was irrespective of the phosphorylation background. In conclusion, ADP-stimulated contraction can be used as a tool to study how protein phosphorylation and mutant proteins alter accessibility of myosin binding on actin. In the presence of Ca(2+), pathologic [ADP] and low PKA-phosphorylation, high actin-myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies. PMID:26621701

  2. Pistacia chinensis Methanolic Extract Attenuated MAPK and Akt Phosphorylations in ADP Stimulated Rat Platelets In Vitro

    OpenAIRE

    Ji Young Park; Mei Hong; Qi Jia; Young-Chul Lee; Taddesse Yayeh; Eujin Hyun; Dong-Mi Kwak; Jae Youl Cho; Man Hee Rhee

    2012-01-01

    Pistacia chinensis (Chinese pistache) is a widely grown plant in southern China where the galls extract is a common practice in folk medicine. However, extracts from this plant have never been attempted for their cardiovascular protective effects in experimental setting. Here therefore we aimed to investigate the antiplatelet activity of Pistacia chinensis methanolic extract (PCME) in ADP stimulated rat platelets in vitro. PCME (2.5–20 μg/mL) inhibited ADP-induced platelet aggregation. While ...

  3. Pistacia chinensis Methanolic Extract Attenuated MAPK and Akt Phosphorylations in ADP Stimulated Rat Platelets In Vitro

    Directory of Open Access Journals (Sweden)

    Ji Young Park

    2012-01-01

    (2.5–20 μg/mL inhibited ADP-induced platelet aggregation. While PCME diminished [Ca2+]i, ATP, and TXA2 release in ADP-activated platelets, it enhanced cAMP production in resting platelets. Likewise, PCME inhibited fibrinogen binding to αIIbβ3 and downregulated JNK, ERK, and Akt phosphorylations. Thus, PCME contains potential antiplatelet compounds that could be deployed for their therapeutic values in cardiovascular pathology.

  4. An affinity matrix for the purification of poly(ADP-ribose) glycohydrolase.

    OpenAIRE

    Thomassin, H; Jacobson, M K; Guay, J; Verreault, A; Aboul-Ela, N; Menard, L.; Poirier, G G

    1990-01-01

    The preparation of quantities of poly(ADP-ribose) glycohydrolase sufficient for detailed structural and enzymatic characterizations has been difficult due to the very low tissue content of the enzyme and its lability in late stages of purification. To date, the only purification of this enzyme to apparent homogeneity has involved a procedure requiring 6 column chromatographic steps. Described here is the preparation of an affinity matrix which consists of ADP-ribose polymers bound to dihydrox...

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

    International Nuclear Information System (INIS)

    Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F1 ATPase (CF1) 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 Mg2+ 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 CF1 that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF1. 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 [32P]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 Mg2+-induced inhibition after addition of CF1 to solutions containing Mg2+ 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 Ca2+ as the activating cation

  6. Poly(ADP-Ribose) Polymerase 1: Cellular Pluripotency, Reprogramming, and Tumorogenesis

    OpenAIRE

    Bo-Hua Jiang; Wei-Lien Tseng; Hsin-Yang Li; Mong-Lien Wang; Yuh-Lih Chang; Yen-Jen Sung; Shih-Hwa Chiou

    2015-01-01

    Poly(ADP-ribos)ylation (PARylation) is the catalytic function of the Poly(ADP-ribose) polymerases (Parps) family for post-translational modification in cellular process. Being a major member of Parps, Parp1 is a crucial nuclear factor with biological significance in modulating DNA repair, DNA replication, transcription, DNA methylation and chromatin remodeling through PARylation of downstream proteins. In addition, high expression level and activity of Parp1 are correlated with pluripotent st...

  7. On PAR with PARP: cellular stress signaling through poly(ADP-ribose) and PARP-1

    OpenAIRE

    Luo, Xin; Kraus, W. Lee

    2012-01-01

    PARP-1 (poly[ADP-ribose] polymerase-1) is a nuclear enzyme that processes varied stress signals and, in response, determines cell fates based on the type and strength of the stress stimulus. PAR (poly[ADP-ribose]) is generated by PARP-1 and regulates many of these stress response pathways. In this review, Kraus and colleagues discuss recent important findings that elucidate the role of PAR and PARP-1 in the regulation of stress response.

  8. Inhibition of Nuclear Receptor Signalling by Poly(ADP-Ribose) Polymerase

    OpenAIRE

    Miyamoto, Takahide; Kakizawa, Tomoko; Hashizume, Kiyoshi

    1999-01-01

    Mammalian poly(ADP-ribose) polymerase (PARP) is a nuclear chromatin-associated protein with a molecular mass of 114 kDa that catalyzes the transfer of ADP-ribose units from NAD+ to nuclear proteins that are located within chromatin. We report here the identification of a novel property of PARP as a modulator of nuclear receptor signalling. PARP bound directly to retinoid X receptors (RXR) and repressed ligand-dependent transcriptional activities mediated by heterodimers of RXR and thyroid hor...

  9. How to kill tumor cells with inhibitors of poly(ADP-ribosyl)ation

    OpenAIRE

    Mangerich, Aswin; Bürkle, Alexander

    2011-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification catalyzed by the enzyme family of poly(ADP-ribose) polymerases (PARPs). PARPs exhibit pleiotropic cellular functions ranging from maintenance of genomic stability and chromatin remodeling to regulation of cell death, thereby rendering PARP homologues promising targets in cancer therapy. Depending on the molecular status of a cancer cell, low-molecular weight PARP inhibitors can (i) either be used as monotherapeutic agents following t...

  10. Multiple forms of ADP-glucose pyrophosphorylase from tomato fruit

    Science.gov (United States)

    Chen, B. Y.; Janes, H. W.

    1997-01-01

    ADP-glucose pyrophosphorylase (AGP) was purified from tomato (Lycopersicon esculentum Mill.) fruit to apparent homogeneity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme migrated as two close bands with molecular weights of 50,000 and 51,000. Two-dimensional polyacrylamide gel electrophoresis analysis of the purified enzyme, however, revealed at least five major protein spots that could be distinguished by their slight differences in net charge and molecular weight. Whereas all of the spots were recognized by the antiserum raised against tomato fruit AGP holoenzyme, only three of them reacted strongly with antiserum raised against the potato tuber AGP large subunit, and the other two spots (with lower molecular weights) reacted specifically with antisera raised against spinach leaf AGP holoenzyme and the potato tuber AGP small subunit. The results suggest the existence of at least three isoforms of the AGP large subunit and two isoforms of the small subunit in tomato fruit in vivo. The native molecular mass of the enzyme determined by gel filtration was 220 +/- 10 kD, indicating a tetrameric structure for AGP from tomato fruit. The purified enzyme is very sensitive to 3-phosphoglycerate/inorganic phosphate regulation.

  11. Modification of ADP extinguishing powder by siliconization in spray drying

    Institute of Scientific and Technical Information of China (English)

    Xiaojing Zhang; Zhigang Shen; Chujiang Cai; Xiaozheng Yu; Jun Du; Yushan Xing; Shulin Ma

    2012-01-01

    Superfine spherical fire-extinguishing powder,ammonium dihydrogen phosphate (ADP,NH4H2PO4),was prepared by spray drying and modified in situ with methyl hydrogen silicone oil (MHSO) emulsion and the fluorinated surfactant FK-510.The influences of the MHSO mass ratio on the hydrophobicity,surface composition,surface morphology,dispersion and particle-size distribution of the NH4H2PO4 were studied,and the influence of the drying air temperature on the decomposition of the NH4H2PO4 was also researched.The results indicate that the MHSO and FK-510 congregate on the particle surfaces and then form a hydrophobic shell.This shell improves the particle hydrophobicity and leads to a fine dispersion of the particles.During the process of preparing the precursor solution,3 wt% (based on the weight of NH4H2PO4) was chosen as the optimum value of the MHSO mass ratio.During the spray drying,a low absolute humidity of the air should be maintained,and it is very important to keep the exit-air temperature below 100℃ to avoid decomposition.

  12. Two Arabidopsis ADP-glucose pyrophosphorylase large subunits (APL1 and APL2) are catalytic.

    Science.gov (United States)

    Ventriglia, Tiziana; Kuhn, Misty L; Ruiz, Ma Teresa; Ribeiro-Pedro, Marina; Valverde, Federico; Ballicora, Miguel A; Preiss, Jack; Romero, José M

    2008-09-01

    ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (alpha(2)beta(2)) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1-APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta. PMID:18614708

  13. Two Arabidopsis ADP-Glucose Pyrophosphorylase Large Subunits (APL1 and APL2) Are Catalytic1

    Science.gov (United States)

    Ventriglia, Tiziana; Kuhn, Misty L.; Ruiz, Ma Teresa; Ribeiro-Pedro, Marina; Valverde, Federico; Ballicora, Miguel A.; Preiss, Jack; Romero, José M.

    2008-01-01

    ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (α2β2) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1–APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta. PMID:18614708

  14. Pierisins and CARP-1: ADP-ribosylation of DNA by ARTCs in butterflies and shellfish.

    Science.gov (United States)

    Nakano, Tsuyoshi; Takahashi-Nakaguchi, Azusa; Yamamoto, Masafumi; Watanabe, Masahiko

    2015-01-01

    The cabbage butterfly, Pieris rapae, and related species possess a previously unknown ADP-ribosylating toxin, guanine specific ADP-ribosyltransferase. This enzyme toxin, known as pierisin, consists of enzymatic N-terminal domain and receptor-binding C-terminal domain, or typical AB-toxin structure. Pierisin efficiently transfers an ADP-ribosyl moiety to the N(2) position of the guanine base of dsDNA. Receptors for pierisin are suggested to be the neutral glycosphingolipids, globotriaosylceramide (Gb3), and globotetraosylceramide (Gb4). This DNA-modifying toxin exhibits strong cytotoxicity and induces apoptosis in various human cell lines, which can be blocked by Bcl-2. Pierisin also produces detrimental effects on the eggs and larvae of the non-habitual parasitoids. In contrast, a natural parasitoid of the cabbage butterfly, Cotesia glomerata, was resistant to this toxin. The physiological role of pierisin in the butterfly is suggested to be a defense factor against parasitization by wasps. Other type of DNA ADP-ribosyltransferase is present in certain kinds of edible clams. For example, the CARP-1 protein found in Meretrix lamarckii consists of an enzymatic domain without a possible receptor-binding domain. Pierisin and CARP-1 are almost fully non-homologous at the amino acid sequence level, but other ADP-ribosyltransferases homologous to pierisin are present in different biological species such as eubacterium Streptomyces. Possible diverse physiological roles of the DNA ADP-ribosyltransferases are discussed. PMID:25033755

  15. Red blood cell ATP/ADP & nitric oxide: The best vasodilators in diabetic patients

    Directory of Open Access Journals (Sweden)

    Bakhtiari Nuredin

    2012-08-01

    Full Text Available Abstract Background Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar (glucose levels that result from defects in insulin secretion, or action, or both. Inspired by previous report the release of ATP from RBCs, which may participate in vessel dilation by stimulating NO production in the endothelium through purinergic receptor signaling and so, the aim of this study is to clearly determined relationship between RBC ATP/ADP ratio with nitric oxide. Methods The ATP/ADP ratio of erythrocytes among four groups of normal individuals (young & middle age, athletes’ subjects and diabetic patients were compared and the relationship between ATP/ADP ratio and NO level of plasma was determined with AVOVA test and bioluminescence method. Results ATP/ADP level in four groups normal (young & middle age, athletes, diabetes] are measured and analyzed with ANOVA test that show a significant difference between groups (P-value Conclusion In this study, a positive relationship between RBC ATP/ADP ratio and NO was found. Based on the obtained result, higher RBC ATP/ADP content may control the ratio of plasma NO in different individuals, also this results show that ATP can activate endothelial cells in NO production and is a main factor in releasing of NO from endothelial cells.

  16. Solution growth kinetics and mechanism: Prismatic face of ADP

    Science.gov (United States)

    Chernov, A. A.; Rashkovich, L. N.; Mkrtchan, A. A.

    1986-01-01

    Laser Michelson interferometry has been applied to in situ study the (001) ADP growth kinetics in aqueous solution in the kinetic regime. The technique allows one to simultaneously measure the slope p of a growth hillock and normal growth rate R provided by this hillock. From these data, the average step growth rate v=R/p has been determined as a function of relative supersaturation σ. The dependencev(σ) is found to be linear, demonstrating the unimportance of surface and bulk diffusion. The direct incorporation at steps is characterized by the step kinetic coefficient βl=(5.1-6.4)X10-3 cm/s. The specific step free energy αl=(1.2-1.9) X10-6 erg/cm was determined from the measured linear dependence of the hillock slope on supersaturation for the hillock around presumably single elementary dislocation. For complex dislocation sources with large total Burgers vectors, the tendency to saturationin the hillock slope-supersaturation curves has been found. The curve perfectly fits the BCF expression which takes into account the perimeter 2L of the region occupied by the points in which the dislocation of the complex step source cross the growing face. For two dislocation sources,L=0.92 μm andL=0.31 μm and total Burgers vectors ⋍12h and 6h (h=7.53Å) have been found. The supersaturation dependence of activities for various complex dislocation sources have been directly demonstrated.

  17. Adenovirus Ad-p53AIP1-mediated gene therapy and its regulation of p53-MDM2 interactions

    OpenAIRE

    Jiang, Yunbo; Chen, Huihua; JIA, HAIQUAN; XU, YUANJI; Liu, Gang; Wang, Yan; Yang, Xiaohe; Yinglin LU

    2010-01-01

    We generated replication-defective adenovirus Ad-p53AIP1 and studied its anti-tumor efficacy both in vitro and in vivo. We demonstrated that Ad-p53AIP1 infection elicited high levels of p53AIP1 expression in cancer cells. We also found that Ad-p53AIP1 expression induced marked apoptosis and cell cycle arrest in HepG2 cells. Moreover, Ad-p53AIP1 infection significantly inhibited the tumorigenesis of 4T1 mouse mammary cancer cells in vivo. In particular, we discovered that p53AIP1 overexpressio...

  18. Deficiency of terminal ADP-ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

    Science.gov (United States)

    Sharifi, Reza; Morra, Rosa; Denise Appel, C; Tallis, Michael; Chioza, Barry; Jankevicius, Gytis; Simpson, Michael A; Matic, Ivan; Ozkan, Ege; Golia, Barbara; Schellenberg, Matthew J; Weston, Ria; Williams, Jason G; Rossi, Marianna N; Galehdari, Hamid; Krahn, Juno; Wan, Alexander; Trembath, Richard C; Crosby, Andrew H; Ahel, Dragana; Hay, Ron; Ladurner, Andreas G; Timinszky, Gyula; Williams, R Scott; Ahel, Ivan

    2013-01-01

    Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADP-ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans. PMID:23481255

  19. 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

  20. Selective down-regulation of nuclear poly(ADP-ribose glycohydrolase.

    Directory of Open Access Journals (Sweden)

    David M Burns

    Full Text Available BACKGROUND: The formation of ADP-ribose polymers on target proteins by poly(ADP-ribose polymerases serves a variety of cell signaling functions. In addition, extensive activation of poly(ADP-ribose polymerase-1 (PARP-1 is a dominant cause of cell death in ischemia-reperfusion, trauma, and other conditions. Poly(ADP-ribose glycohydrolase (PARG degrades the ADP-ribose polymers formed on acceptor proteins by PARP-1 and other PARP family members. PARG exists as multiple isoforms with differing subcellular localizations, but the functional significance of these isoforms is uncertain. METHODS / PRINCIPAL FINDINGS: Primary mouse astrocytes were treated with an antisense phosphorodiamidate morpholino oligonucleotide (PMO targeted to exon 1 of full-length PARG to suppress expression of this nuclear-specific PARG isoform. The antisense-treated cells showed down-regulation of both nuclear PARG immunoreactivity and nuclear PARG enzymatic activity, without significant alteration in cytoplasmic PARG activity. When treated with the genotoxic agent MNNG to induced PARP-1 activation, the antisense-treated cells showed a delayed rate of nuclear PAR degradation, reduced nuclear condensation, and reduced cell death. CONCLUSIONS/SIGNIFICANCE: These results support a preferentially nuclear localization for full-length PARG, and suggest a key role for this isoform in the PARP-1 cell death pathway.

  1. Poly(ADP-ribosyl)ation regulates CTCF-dependent chromatin insulation.

    Science.gov (United States)

    Yu, Wenqiang; Ginjala, Vasudeva; Pant, Vinod; Chernukhin, Igor; Whitehead, Joanne; Docquier, France; Farrar, Dawn; Tavoosidana, Gholamreza; Mukhopadhyay, Rituparna; Kanduri, Chandrasekhar; Oshimura, Mitsuo; Feinberg, Andrew P; Lobanenkov, Victor; Klenova, Elena; Ohlsson, Rolf

    2004-10-01

    Chromatin insulators demarcate expression domains by blocking the cis effects of enhancers or silencers in a position-dependent manner. We show that the chromatin insulator protein CTCF carries a post-translational modification: poly(ADP-ribosyl)ation. Chromatin immunoprecipitation analysis showed that a poly(ADP-ribosyl)ation mark, which exclusively segregates with the maternal allele of the insulator domain in the H19 imprinting control region, requires the bases that are essential for interaction with CTCF. Chromatin immunoprecipitation-on-chip analysis documented that the link between CTCF and poly(ADP-ribosyl)ation extended to more than 140 mouse CTCF target sites. An insulator trap assay showed that the insulator function of most of these CTCF target sites is sensitive to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase activity. We suggest that poly(ADP-ribosyl)ation imparts chromatin insulator properties to CTCF at both imprinted and nonimprinted loci, which has implications for the regulation of expression domains and their demise in pathological lesions. PMID:15361875

  2. Acceptor proteins for poly(ADP-ribose) in irradiated normal human and ataxia telangiectasia (AT) fibroblasts

    International Nuclear Information System (INIS)

    Poly(ADP-ribose) polymerase activity is stimulated by DNA strand breaks and may participate in DNA repair. Since treatment of cells with DNA damaging agents stimulated the poly(ADP-ribosylation) of a specific set of proteins, the authors have analyzed the acceptors in irradiated human fibroblasts from normal individuals and from patients with AT, a disease associated with a hypersensitivity to ionizing radiation. Cells were permeabilized and incubated with /sup 32/P-NAD, proteins were separated by polyacrylamide gel electrophoresis, and the poly (ADP-ribose) acceptors were detected by autoradiography. In all four strains, the major acceptor was the 116 kd auto-modified polymerase, while other prominent radioactive bands were at 2, 45, and 60 kd. Labeling of these bands was increased following irradiation of the cells with 3-30 Gy. Of interest was the detection of a poly (ADP-ribosylated) protein at 19 kd in the two normal strains but not in either AT strain. The results suggest that a defect in the ADP-ribosylation of the 19 kd protein is associated with AT and possible with the hypersensitivity of AT cells to ionizing radiation

  3. The Sound of Silence: RNAi in Poly (ADP-Ribose Research

    Directory of Open Access Journals (Sweden)

    Felix R. Althaus

    2012-12-01

    Full Text Available Poly(ADP-ribosyl-ation is a nonprotein posttranslational modification of proteins and plays an integral part in cell physiology and pathology. The metabolism of poly(ADP-ribose (PAR is regulated by its synthesis by poly(ADP-ribose polymerases (PARPs and on the catabolic side by poly(ADP-ribose glycohydrolase (PARG. PARPs convert NAD+ molecules into PAR chains that interact covalently or noncovalently with target proteins and thereby modify their structure and functions. PAR synthesis is activated when PARP1 and PARP2 bind to DNA breaks and these two enzymes account for almost all PAR formation after genotoxic stress. PARG cleaves PAR molecules into free PAR and finally ADP-ribose (ADPR moieties, both acting as messengers in cellular stress signaling. In this review, we discuss the potential of RNAi to manipulate the levels of PARPs and PARG, and consequently those of PAR and ADPR, and compare the results with those obtained after genetic or chemical disruption.

  4. Effect of Co2+ doping on solubility, crystal growth and properties of ADP crystals

    Science.gov (United States)

    Ganesh, V.; Shkir, Mohd.; AlFaify, S.; Yahia, I. S.

    2016-09-01

    Bulk size crystal growth of ADP with different concentrations doping of cobalt (Co2+) has been done by low cost slow evaporation technique at ambient conditions. The solubility measurement was carried out on pure and doped crystals and found that the solubility is decreasing with doping concentrations. The presence of Co2+ ion in crystalline matrix of ADP has been confirmed by structural, vibrational and elemental analyses. Scanning electron microscopic study reveals that the doping has strong effect on the quality of the crystals. The optical absorbance and transmission confirms the enhancement of quality of ADP crystals due to Co2+ doping and so the optical band gap. Further the dislocation, photoluminescence, dielectric and mechanical studies confirms that the properties of grown crystals with Co2+ doping has been enriched and propose it as a better candidate for optoelectronic applications.

  5. Pistacia chinensis Methanolic Extract Attenuated MAPK and Akt Phosphorylations in ADP Stimulated Rat Platelets In Vitro.

    Science.gov (United States)

    Park, Ji Young; Hong, Mei; Jia, Qi; Lee, Young-Chul; Yayeh, Taddesse; Hyun, Eujin; Kwak, Dong-Mi; Cho, Jae Youl; Rhee, Man Hee

    2012-01-01

    Pistacia chinensis (Chinese pistache) is a widely grown plant in southern China where the galls extract is a common practice in folk medicine. However, extracts from this plant have never been attempted for their cardiovascular protective effects in experimental setting. Here therefore we aimed to investigate the antiplatelet activity of Pistacia chinensis methanolic extract (PCME) in ADP stimulated rat platelets in vitro. PCME (2.5-20 μg/mL) inhibited ADP-induced platelet aggregation. While PCME diminished [Ca(2+)]i, ATP, and TXA2 release in ADP-activated platelets, it enhanced cAMP production in resting platelets. Likewise, PCME inhibited fibrinogen binding to αIIbβ3 and downregulated JNK, ERK, and Akt phosphorylations. Thus, PCME contains potential antiplatelet compounds that could be deployed for their therapeutic values in cardiovascular pathology. PMID:22899962

  6. Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody

    Science.gov (United States)

    Rohrbeck, Astrid; Fühner, Viola; Schröder, Anke; Hagemann, Sandra; Vu, Xuan-Khang; Berndt, Sarah; Hust, Michael; Pich, Andreas; Just, Ingo

    2016-01-01

    Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry. PMID:27043630

  7. Design, Synthesis, and Chemical and Biological Properties of Cyclic ADP-4-Thioribose as a Stable Equivalent of Cyclic ADP-Ribose

    Science.gov (United States)

    Tsuzuki, Takayoshi; Takano, Satoshi; Sakaguchi, Natsumi; Kudoh, Takashi; Murayama, Takashi; Sakurai, Takashi; Hashii, Minako; Higashida, Haruhiro; Weber, Karin; Guse, Andreas H.; Kameda, Tomoshi; Hirokawa, Takatsugu; Kumaki, Yasuhiro; Arisawa, Mitsuhiro; Potter, Barry V. L.; Shuto, Satoshi

    2016-01-01

    Here we describe the successful synthesis of cyclic ADP-4-thioribose (cADPtR, 3), designed as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca2+-mobilizing second messenger, in which the key N1-β-thioribosyladenosine structure was stereoselectively constructed by condensation between the imidazole nucleoside derivative 8 and the 4-thioribosylamine 7 via equilibrium in 7 between the α-anomer (7α) and the β-anomer (7β) during the reaction course. cADPtR is, unlike cADPR, chemically and biologically stable, while it effectively mobilizes intracellular Ca2+ like cADPR in various biological systems, such as sea urchin homogenate, NG108-15 neuronal cells, and Jurkat T-lymphocytes. Thus, cADPtR is a stable equivalent of cADPR, which can be useful as a biological tool for investigating cADPR-mediated Ca2+-mobilizing pathways.

  8. Activation of the Potato Tuber ADP-glucose Pyrophosphorylase by Thioredoxin

    OpenAIRE

    Ballicora, Miguel A.; Frueauf, Jeremiah B.; Fu, Yingbin; Schürmann, Peter; Preiss, Jack

    2007-01-01

    The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase (ADP-GlcPPase) catalyzes the first committed step in starch biosynthesis. The main type of regulation of this enzyme is allosteric, and its activity is controlled by the ratio of activator, 3-phosphoglycerate to inhibitor, Pi. It was reported (Fu, Y., Ballicora, M. A., Leykam, J. F., and Preiss, J. (1998) J. Biol. Chem. 273, 25045-25052) that the enzyme was activated by reduction of the Cys12 disulfide linkage present in t...

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

    OpenAIRE

    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 recogni...

  10. Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure

    OpenAIRE

    Colanzi, Antonino; Silletta, Maria Giuseppina; Fiucci, Giusy; Flati, Silvio; Fusella, Aurora; Polishchuk, Roman; Mironov, Alexander; Tullio, Giuseppe Di; Weigert, Roberto; Malhotra, Vivek; Corda, Daniela; De Matteis, Maria Antonietta; Luini, Alberto

    1997-01-01

    We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss...

  11. Poly (ADP-Ribose) Polymerase 1 Is Required for Protein Localization to Cajal Body

    OpenAIRE

    Kotova, Elena; Jarnik, Michael; Tulin, Alexei V.

    2009-01-01

    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 processe...

  12. File list: InP.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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  19. Correlations of serum levels of leptin and other related factor (NPY, ADP) in female children with simple obesity

    International Nuclear Information System (INIS)

    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)

  20. ADP-bildende Acetyl-CoA Synthetasen aus hyperthermophilen Archaea: Molekularbiologische und biochemische Charakterisierung von neuartigen Enzymen der Acetat-Bildung und ATP-Synthese

    OpenAIRE

    Musfeldt, Meike

    2001-01-01

    Keine deutschsprachige Zusammenfassung vorhanden. Acetyl-CoA synthetase (ADP-forming) (ADP-ACS) represents a novel enzyme of acetate formation and energy conservation (acetyl-CoA + ADP + Pi -> acetate + ATP + CoA) in Archaea and eukaryotic protists. The only characterized ADP-ACS in Archaea, two isoenzymes from the hyperthermophile Pyrococcus furiosus, constitute 145 kDa heterotetramers (a2, b2). By using the N-terminal amino acid sequences of both subunits, which are located at different ...

  1. Poly(ADP-ribosyl)ation of a herpes simplex virus immediate early polypeptide

    International Nuclear Information System (INIS)

    In vitro poly(ADP-ribosyl)ation of the herpes simplex virus type 1 (HSV-1) immediate early polypeptide Vmw175 is reported. The phenomenon was most clearly observed by use of the temperature-sensitive mutant tsK, which overproduces Vmw175 at the nonpermissive temperature (NPT) and has a mutation in the coding sequences for this polypeptide. Nuclei prepared from cells which were infected with tsK at NPT and subsequently downshifted to the permissive temperature incorporated [32P]NAD into Vmw175. This reaction did not occur when nuclei were prepared from cells constantly maintained at NPT, showing that only functional Vmw175 can be radiolabeled with [32P]NAD. The identity of the acceptor protein was confirmed by demonstrating the expected electrophoretic mobility differences between the HSV-1 and HSV-2 counterparts of Vmw175. The use of suitable inhibitors demonstrated that the reaction represented mono- or poly(ADP-ribosyl)ation, and further analysis showed the presence of long poly(ADP-ribose) chains attached to Vmw175. Poly(ADP-ribosyl)ation may be important as a cause or result of the regulation of viral transcription by Vmw175. Radiolabeling of another virus-specified polypeptide (approximate molecular weight 38,000), thought to be a structural component of the input virus, is also reported

  2. Impaired ADP channeling to mitochondria and elevated reactive oxygen species in hypertensive hearts.

    Science.gov (United States)

    Power, Amelia S C; Pham, Toan; Loiselle, Denis S; Crossman, David H; Ward, Marie-Louise; Hickey, Anthony J

    2016-06-01

    Systemic hypertension initially promotes a compensatory cardiac hypertrophy, yet it progresses to heart failure (HF), and energetic deficits appear to be central to this failure. However, the transfer of energy between the mitochondria and the myofibrils is not often considered as part of the energetic equation. We compared hearts from old spontaneously hypertensive rats (SHRs) and normotensive Wistar controls. SHR hearts showed a 35% depression in mitochondrial function, yet produced at least double the amount of reactive oxygen species (ROS) in all respiration states in left ventricular (LV) homogenates. To test the connectivity between mitochondria and myofibrils, respiration was further tested in situ with LV permeabilized fibers by addition of multiple substrates and ATP, which requires hydrolysis to mediate oxidative phosphorylation. By trapping ADP using a pyruvate kinase enzyme system, we tested ADP channeling towards mitochondria, and this suppressed respiration and elevated ROS production more in the SHR fibers. The ADP-trapped state was also less relieved on creatine addition, likely reflecting the 30% depression in total CK activity in the SHR heart fibers. Confocal imaging identified a 34% longer distance between the centers of myofibril to mitochondria in the SHR hearts, which increases transverse metabolite diffusion distances (e.g., for ATP, ADP, and creatine phosphate). We propose that impaired connectivity between mitochondria and myofibrils may contribute to elevated ROS production. Impaired energy exchange could be the result of ultrastructural changes that occur with hypertrophy in this model of hypertension. PMID:27084386

  3. 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.

  4. 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.

  5. 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.

  6. The ancestral activation promiscuity of ADP-glucose pyrophosphorylases from oxygenic photosynthetic organisms

    Directory of Open Access Journals (Sweden)

    Kuhn Misty L

    2013-02-01

    Full Text Available Abstract Background ADP-glucose pyrophosphorylase (ADP-Glc PPase catalyzes the first committed step in the synthesis of glycogen in bacteria and starch in algae and plants. In oxygenic photosynthetic organisms, ADP-Glc PPase is mainly activated by 3-phosphoglycerate (3-PGA and to a lesser extent by other metabolites. In this work, we analyzed the activation promiscuity of ADP-Glc PPase subunits from the cyanobacterium Anabaena PCC 7120, the green alga Ostreococcus tauri, and potato (Solanum tuberosum tuber by comparing a specificity constant for 3-PGA, fructose-1,6-bisphosphate (FBP, fructose-6-phosphate, and glucose-6-phosphate. Results The 3-PGA specificity constant for the enzymes from Anabaena (homotetramer, O. tauri, and potato tuber was considerably higher than for other activators. O. tauri and potato tuber enzymes were heterotetramers comprising homologous small and large subunits. Conversely, the O. tauri small subunit (OtaS homotetramer was more promiscuous because its FBP specificity constant was similar to that for 3-PGA. To explore the role of both OtaS and OtaL (O. tauri large subunit in determining the specificity of the heterotetramer, we knocked out the catalytic activity of each subunit individually by site-directed mutagenesis. Interestingly, the mutants OtaSD148A/OtaL and OtaS/OtaLD171A had higher specificity constants for 3-PGA than for FBP. Conclusions After gene duplication, OtaS seemed to have lost specificity for 3-PGA compared to FBP. This was physiologically and evolutionarily feasible because co-expression of both subunits restored the specificity for 3-PGA of the resulting heterotetrameric wild type enzyme. This widespread promiscuity seems to be ancestral and intrinsic to the enzyme family. Its presence could constitute an efficient evolutionary mechanism to accommodate the ADP-Glc PPase regulation to different metabolic needs.

  7. An Entamoeba histolytica ADP-ribosyl transferase from the diphtheria toxin family modifies the bacterial elongation factor Tu.

    Science.gov (United States)

    Avila, Eva E; Rodriguez, Orlando I; Marquez, Jaqueline A; Berghuis, Albert M

    2016-06-01

    ADP-ribosyl transferases are enzymes involved in the post-translational modification of proteins; they participate in multiple physiological processes, pathogenesis and host-pathogen interactions. Several reports have characterized the functions of these enzymes in viruses, prokaryotes and higher eukaryotes, but few studies have reported ADP-ribosyl transferases in lower eukaryotes, such as parasites. The locus EHI_155600 from Entamoeba histolytica encodes a hypothetical protein that possesses a domain from the ADP-ribosylation superfamily; this protein belongs to the diphtheria toxin family according to a homology model using poly-ADP-ribosyl polymerase 12 (PARP12 or ARTD12) as a template. The recombinant protein expressed in Escherichia coli exhibited in vitro ADP-ribosylation activity that was dependent on the time and temperature. Unlabeled βNAD(+), but not ADP-ribose, competed in the enzymatic reaction using biotin-βNAD(+) as the ADP-ribose donor. The recombinant enzyme, denominated EhToxin-like, auto-ADP-ribosylated and modified an acceptor from E. coli that was identified by MS/MS as the elongation factor Tu (EF-Tu). To the best of our knowledge, this is the first report to identify an ADP-ribosyl transferase from the diphtheria toxin family in a protozoan parasite. The known toxins from this family (i.e., the diphtheria toxin, the Pseudomonas aeruginosa toxin Exo-A, and Cholix from Vibrio cholerae) modify eukaryotic elongation factor two (eEF-2), whereas the amoeba EhToxin-like modified EF-Tu, which is another elongation factor involved in protein synthesis in bacteria and mitochondria. PMID:27234208

  8. 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)

    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

  9. Poly(ADP-Ribose) Polymerase 1 Promotes Oxidative-Stress-Induced Liver Cell Death via Suppressing Farnesoid X Receptor α

    OpenAIRE

    Wang, Cheng; Zhang, Fengxiao; Wang, Lin; Zhang, Yanqing; Li, Xiangrao; Huang, Kun; Du, Meng; Liu, Fangmei; Huang, Shizheng; Guan, Youfei; Huang, Dan; Huang, Kai

    2013-01-01

    Farnesoid X receptor α (FXR) is highly expressed in the liver and regulates the expression of various genes involved in liver repair. In this study, we demonstrated that activated poly(ADP-ribose) polymerase 1 (PARP1) promoted hepatic cell death by inhibiting the expression of FXR-dependent hepatoprotective genes. PARP1 could bind to and poly(ADP-ribosyl)ate FXR. Poly(ADP-ribosyl)ation dissociated FXR from the FXR response element (FXRE), present in the promoters of target genes, and suppress...

  10. Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS

    OpenAIRE

    Colanzi, Antonino; Lalioti, Vasiliky S.; Corda, Daniela

    2013-01-01

    ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. H...

  11. HPF1/C4orf27 Is a PARP-1-Interacting Protein that Regulates PARP-1 ADP-Ribosylation Activity

    OpenAIRE

    Gibbs-Seymour, Ian; Fontana, Pietro; Rack, Johannes Gregor Matthias; Ahel, Ivan

    2016-01-01

    Summary We report the identification of histone PARylation factor 1 (HPF1; also known as C4orf27) as a regulator of ADP-ribosylation signaling in the DNA damage response. HPF1/C4orf27 forms a robust protein complex with PARP-1 in cells and is recruited to DNA lesions in a PARP-1-dependent manner, but independently of PARP-1 catalytic ADP-ribosylation activity. Functionally, HPF1 promotes PARP-1-dependent in trans ADP-ribosylation of histones and limits DNA damage-induced hyper-automodificatio...

  12. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of human ARH3, the first eukaryotic protein-ADP-ribosylhydrolase

    International Nuclear Information System (INIS)

    Human ADP-ribosylhydrolase 3 (ARH3), which has been identified as an ARH by a sequence-similarity search and which cleaves the glycosidic bond of ADP-ribose attached to a protein, has been cloned, expressed, purified and crystallized in two different space groups. ADP-ribosylhydrolases catalyze the release of ADP-ribose from ADP-ribosylated proteins via hydrolysis of the glycosidic bond between ADP-ribose and a specific amino-acid residue in a target protein. Human ADP-ribosylhydrolase 3, consisting of 347 amino-acid residues, has been cloned and heterologously expressed in Escherichia coli, purified and crystallized in two different space groups. Preliminary X-ray diffraction studies yielded excellent diffraction data to a resolution of 1.6 Å

  13. Study of the Five Rickettsia prowazekii Proteins Annotated as ATP/ADP Translocases (Tlc): Only Tlc1 Transports ATP/ADP, While Tlc4 and Tlc5 Transport Other Ribonucleotides

    OpenAIRE

    Audia, Jonathon P.; Winkler, Herbert H.

    2006-01-01

    The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interes...

  14. Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities.

    Science.gov (United States)

    Khadka, Prabhat; Hsu, Joseph K; Veith, Sebastian; Tadokoro, Takashi; Shamanna, Raghavendra A; Mangerich, Aswin; Croteau, Deborah L; Bohr, Vilhelm A

    2015-12-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response. PMID:26391948

  15. Biology of Poly(ADP-Ribose) Polymerases: The Factotums of Cell Maintenance.

    Science.gov (United States)

    Bai, Peter

    2015-06-18

    The protein family of poly(ADP-ribose) polymerases (PARPs) or diphtheria toxin-type ADP-ribose transferases (ARTDs) are multidomain proteins originally identified as DNA repair factors. There are 17 PARP enzymes in humans, and it is now evident that PARPs undertake more tasks than DNA repair. The aim of this review is to give a comprehensive view of the biological roles of the PARP family starting from the simplest biochemical reactions to complex regulatory circuits. Special attention will be laid on discussing linkage of PARP enzymes with tumor biology, oxidative stress, inflammatory, and metabolic diseases. A better understanding of PARP-mediated processes and pathologies may help in identifying new pathways and, by these, new targets to combat diseases that affect large populations and seriously shorten life expectancy and the quality of life, such as cancer, metabolic, or inflammatory diseases. PMID:26091343

  16. Ageing of atrazine in manure amended soils assessed by bioavailability to Pseudomonas sp. strain ADP

    DEFF Research Database (Denmark)

    Glæsner, Nadia; Bælum, Jacob; Strobel, Bjarne W.;

    2014-01-01

    Animal manure is applied to agricultural land in areas of high livestock production. In the present study, we evaluated ageing of atrazine in two topsoils with and without addition of manure and in one subsoil. Ageing was assessed as the bioavailability of atrazine to the atrazine mineralizing...... bacteria Pseudomonas sp. strain ADP. Throughout an ageing period of 90 days bioavailability was investigated at days 1, 10, 32, 60 and 90, where ~108 cells g−1 of the ADP strain was inoculated to the 14C-atrazine exposed soil and 14CO2 was collected over 7 days as a measure of mineralized atrazine. Even...... though the bioavailable residue decreased in all of the three soils as time proceeded, we found that ageing occurred faster in the topsoils rich in organic carbon than in subsoil. For one topsoil rich in organic carbon content, Simmelkær, we observed a higher degree of ageing when treated with manure...

  17.   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; Kristensen, Nethe; Rasmussen, Peter Kristian; Rosenmeier, Jaya Birgitte

    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-triphoshate...... (UTP) are both released during myocardial ischemia, influencing hemodynamics. Both mediate the release of tissue plasminogen activator (t-PA), which can reduce infarct size (IS). The objective of this study was to investigate whether exogenous ADP and UTP administration during reperfusion could reduce...... myocardial IS and whether this correlated to t-PA release or improvements in hemodynamic responses. Hemodynamic variables and t-PA were measured in 22 pigs before, during, and after 45 min of left anterior coronary artery occlusion. During reperfusion, the pigs were randomized to 240 min of intracoronary...

  18. The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity.

    Directory of Open Access Journals (Sweden)

    Maria Valeri

    Full Text Available Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis.

  19. D.C. electrical conductivity measurements on ADP single crystals added with simple organic compounds

    Indian Academy of Sciences (India)

    A Anne Assencia; C Mahadevan

    2005-08-01

    Pure and impurity added (with urea and thiourea) ADP single crystals were grown by the free evaporation method. D.C. electrical conductivity measurements were carried out along both the unique axis and perpendicular directions at various temperatures ranging from 40–150°C by the conventional two-probe method. Activation energies were also determined. The present study indicates that the conductivity increases with the increase in impurity concentration and temperature.

  20. Minocycline inhibits poly(ADP-ribose) polymerase-1 at nanomolar concentrations

    OpenAIRE

    Alano, Conrad C.; Kauppinen, Tiina M; Valls, Andreu Viader; Swanson, Raymond A.

    2006-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1), when activated by DNA damage, promotes both cell death and inflammation. Here we report that PARP-1 enzymatic activity is directly inhibited by minocycline and other tetracycline derivatives that have previously been shown to have neuroprotective and anti-inflammatory actions. These agents were evaluated by using cortical neuron cultures in which PARP-1 activation was induced by the genotoxic agents N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or 3-morph...

  1. The switching mechanism of the mitochondrial ADP/ATP carrier explored by free-energy landscapes.

    Science.gov (United States)

    Pietropaolo, Adriana; Pierri, Ciro Leonardo; Palmieri, Ferdinando; Klingenberg, Martin

    2016-06-01

    The ADP/ATP carrier (AAC) of mitochondria has been an early example for elucidating the transport mechanism alternating between the external (c-) and internal (m-) states (M. Klingenberg, Biochim. Biophys. Acta 1778 (2008) 1978-2021). An atomic resolution crystal structure of AAC is available only for the c-state featuring a three repeat transmembrane domain structure. Modeling of transport mechanism remained hypothetical for want of an atomic structure of the m-state. Previous molecular dynamics studies simulated the binding of ADP or ATP to the AAC remaining in the c-state. Here, a full description of the AAC switching from the c- to the m-state is reported using well-tempered metadynamics simulations. Free-energy landscapes of the entire translocation from the c- to the m-state, based on the gyration radii of the c- and m-gates and of the center of mass, were generated. The simulations revealed three free-energy basins attributed to the c-, intermediate- and m-states separated by activation barriers. These simulations were performed with the empty and with the ADP- and ATP-loaded AAC as well as with the poorly transported AMP and guanine nucleotides, showing in the free energy landscapes that ADP and ATP lowered the activation free-energy barriers more than the other substrates. Upon binding AMP and guanine nucleotides a deeper free-energy level stabilized the intermediate-state of the AAC2 hampering the transition to the m-state. The structures of the substrate binding sites in the different states are described producing a full picture of the translocation events in the AAC. PMID:26874054

  2. Treatment with insulin inhibits poly(ADP-ribose)polymerase activation in a rat model of endotoxemia

    OpenAIRE

    Horváth, Eszter M; Benk, Rita; Ger, Domonkos; Kiss, Levente; Szabó, Csaba

    2007-01-01

    In critically ill patients various conditions may lead to the activation of poly(ADP-ribose) polymerase (PARP). By promoting cellular energetic dysfunction, and by enhancing pro-inflammatory gene expression, PARP activation significantly contributes to the pathogenesis of shock. PARP activation is usually triggered by DNA strand breakage, which is typically the result of the overproduction of various reactive oxidant species. One of the pathophysiological conditions associated with PARP activ...

  3. The Effect of Agricultural Development Project (ADP on the Rural Farmers in Adamawa State, Nigeria

    Directory of Open Access Journals (Sweden)

    Umar Adamu Madu

    2012-09-01

    Full Text Available Majority of communities in Nigeria are rural dwellers and agrarian by occupation. Development strategy for a country whose rural population are mainly farmers cannot be achieved without first sustained growth in rural income and standard of living primarily from agriculture. It was based on this that the state wide Agricultural Development Project (ADP was established to raise productivity, income and standard of living of rural farmers in Nigeria. This study assesses the effect of the ADP activities on the wellbeing of the rural farmers in Adamawa State, Nigeria. Data for this study were collect on annual crop output, annual income, farm size, use of improved technology, access to credit among farmers, farmers’ training and rural infrastructure development. The data were sourced using structured questionnaire and personal interviews. The statistical analysis used to determine the effect to the project on the participating farmers include, descriptive statistics and comparability test for difference (T-test analysis. The results indicates that Adamawa ADP had positive and significant impact on rural farmers productivity, income, access to credit, standard of living as measured by assets ownership. However, the project did not have significant impact on the rural infrastructure, adoption of improved technologies and farm sizes, even though the change from before and after ADP activities was positive. The study recommends that much attention should be paid to the provision of rural infrastructure and the needed improved technologies. The study also recommends that the two tiers of government in Nigeria should adequately fund the project to efficiently cope with its responsibility of developing the rural sector.

  4. Cloning and expression of cDNA for human poly(ADP-ribose)polymerase

    International Nuclear Information System (INIS)

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma λgt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly(ADP-ribose) polymerase. To confirm that the 1.3-kb insert from λgt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents

  5. Essential role of poly(ADP-ribosyl)ation in cocaine action

    OpenAIRE

    Scobie, Kimberly N.; Damez-Werno, Diane; Sun, HaoSheng; Shao, NingYi; Gancarz, Amy; Panganiban, Clarisse H.; Dias, Caroline; Koo, Jawook; Caiafa, Paola; Kaufman, Lewis; Neve, Rachael L.; Dietz, David M.; Shen, Li; Nestler, Eric J.

    2014-01-01

    We demonstrate that chronic cocaine, including cocaine self-administration, induces poly(ADP-ribose) polymerase-1 (PARP-1) in the nucleus accumbens (NAc). Using a combination of viral-mediated gene transfer and pharmacological tools, we show that upregulation of PARP-1 in NAc dramatically enhances behavioral responses to cocaine, whereas downregulation of PARP-1 has the opposite effect. We used chromatin immunoprecipitation sequencing to map genome-wide binding of PARP-1 in NAc. The data demo...

  6. Better detection of platelet aggregation in patients with metabolic syndrome using epinephrine and ADP

    OpenAIRE

    Perez-Campos-Mayoral, Laura; Pérez-Campos,Eduardo; Zenteno, Edgar; Majluf-Cruz, Abraham; Perez-Ortega, Eduardo; Matias-Pérez, Diana; Rodal-Canales, Francisco J; Martínez-Cruz, Ruth; Pina-Canseco, Socorro; Reyes Franco, Miguel Angel; Mayoral Andrade, Gabriel; Hernández, Pedro; Gallegos, Belem

    2014-01-01

    Background Patients with metabolic syndrome (MS) often have increased platelet aggregation. In order to determine which concentration detects a higher level of platelet aggregation in patients with MS, the agonists ADP and epinephrine were compared. Methods The study included 56 subjects with MS and 53 healthy subjects. Blood pressure, weight, body-mass index, and hip-to-waist ratio were collected from all subjects. Insulin, glucose, total serum cholesterol, HDL-C, LDL-C, total triglycerides,...

  7. PolyADP-ribose polymerase is a coactivator for AP-2-mediated transcriptional activation.

    OpenAIRE

    Kannan, P; Yu, Y; Wankhade, S; Tainsky, M A

    1999-01-01

    Overexpression of transcription factor AP-2 has been implicated in the tumorigenicity of the human teratocarcinoma cell lines PA-1 that contain an activated ras oncogene. Here we show evidence that overexpression of AP-2 sequesters transcriptional coactivators which results in self-inhibition. We identified AP-2-interacting proteins and determined whether these proteins were coactivators for AP-2-mediated transcription. One such interacting protein is polyADP-ribose polymerase (PARP). PARP su...

  8. Evidence that phospholipase D mediates ADP ribosylation factor- dependent formation of Golgi coated vesicles

    OpenAIRE

    1996-01-01

    Formation of coatomer-coated vesicles from Golgi-enriched membranes requires the activation of a small GTP-binding protein, ADP ribosylation factor (ARF). ARF is also an efficacious activator of phospholipase D (PLD), an activity that is relatively abundant on Golgi- enriched membranes. It has been proposed that ARF, which is recruited onto membranes from cytosolic pools, acts directly to promote coatomer binding and is in a 3:1 stoichiometry with coatomer on coated vesicles. We present evide...

  9. Poly(ADP-Ribose) Polymerase 1 Promotes Transcriptional Repression of Integrated Retroviruses

    OpenAIRE

    Bueno, Murilo T. D.; Reyes, Daniel; Valdes, Luis; Saheba, Adarsh; Urias, Eduardo; Mendoza, Crystal; Fregoso, Oliver I.; de Llano, Manuel

    2013-01-01

    Poly(ADP-ribose) polymerase 1 (PARP-1) is a cellular enzyme with a fundamental role in DNA repair and the regulation of chromatin structure, processes involved in the cellular response to retroviral DNA integration. However, the function of PARP-1 in retroviral DNA integration is controversial, probably due to the functional redundancy of the PARP family in mammalian cells. We evaluated the function of PARP-1 in retroviral infection using the chicken B lymphoblastoid cell line DT40. These cel...

  10. A novel crosstalk between BRCA1 and poly (ADP-ribose) polymerase 1 in breast cancer

    OpenAIRE

    Li, Da; Bi, Fang-Fang; Chen, Na-Na; Cao, Ji-Min; Sun, Wu-Ping; Zhou, Yi-Ming; Li, Chun-Yan; Yang, Qing

    2014-01-01

    BRCA mutations are the main known hereditary factor for breast cancer. Notably, poly (ADP-ribose) polymerase 1 (PARP1) expression status plays a critical role in breast cancer progression and the clinical development of PARP1 inhibitors to treat BRCA-mutated breast cancer has advanced rapidly. However, dynamic crosstalk between BRCA1 and PARP1 remains largely unknown. Here, we showed that: (i) BRCA1 inactivation events (mutation, promoter methylation, or knockdown) were accompanied by increas...

  11. Effect and mechanism of poly (ADP-ribose) polymerase-1 in aldosterone-induced apoptosis

    OpenAIRE

    Qiao, Weiwei; Zhang, Weili; Shao, Shuhong; GAI, YUSHENG; Zhang, Mingxiang

    2015-01-01

    The present study aimed to investigate the effects of aldosterone on vascular endothelial cells and the viability of poly (ADP-ribose) polymerase 1 (PARP1) in cells, and to examine the molecular mechanisms underlying the effects of aldosterone on vascular endothelial cell injury. Cultured endothelial cells were treated either with different concentrations of aldosterone for the same duration or with the same concentrations of aldosterone for different durations, and the levels of apoptosis an...

  12. Relationships between optical aggregometry (type Born) and flow cytometry in evaluating ADP-induced platelet activation

    OpenAIRE

    Sbrana, Silverio; Della Pina, Francesca; Rizza, Antonio; Buffa, Manuela; De Filippis, Rossella; Gianetti, Jacopo; Clerico, Aldo

    2008-01-01

    Background: Platelet response to activating agents is used to monitor the efficacy of anti-aggregation therapies. The aim of our study has been to demonstrate the existence of relationships between early events of ADP-induced platelet activation, measured by flow cytometry and platelet-rich plasma aggregation,quantified by optical aggregometry. Methods: We evaluated peripheral blood of 12 donors. The following parameters were quantified by cytometry after stimulation with adenosine diphosphat...

  13. Role of cytoplasmic and releasable ADP in platelet aggregation induced by laminar shear stress

    International Nuclear Information System (INIS)

    We examined the extent of lytic and sublytic platelet injury after exposure of platelets to shear stress and the role in shear-induced PAG of ADP liberated from platelets as a result of shear-induced platelet dense body release and/or platelet damage. Platelets in C-PRP or TAS were subjected to well-defined, laminar shear stress in a rotational viscometer, and PAG (loss of single, nonaggregated platelets), 14C-serotonin release, and loss from platelets of LDH and 51Cr were determined. Increased PAG with increasing shear stresses was associated with progressive loss of LDH and 51Cr. Loss of 51Cr was consistently in excess of that of LDH, indicating sublytic platelet injury, which was confirmed by electron microscopy. At the lowest shear stress used (50 dynes/cm2), PAG in C-PRP was observed in the absence of detectable loss of 51Cr or LDH. When platelets in TAS were sheared in the presence of CP/CPK, an enzyme system capable of removing extracellular ADP, PAG was only partially (approximately 40%) inhibited. However, when platelets were preincubated with CP/CPK and ATP (to saturate platelet ADP receptors), shear-induced PAG was almost completely suppressed. Similar results were obtained with PAG induced by collagen in the aggregometer. The findings indicate that (1) shear-induced PAG in this system may occur without measurable lytic or sublytic platelet damage and (2) ADP liberated from platelets as a result of shear-induced release or damage may represent the major if not sole mediator of shear-induced PAG

  14. The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells

    OpenAIRE

    Lianhua Piao; Daechun Kang; Takehiro Suzuki; Akiko Masuda; Naoshi Dohmae; Yusuke Nakamura; Ryuji Hamamoto

    2014-01-01

    Poly(ADP-ribose) polymerase-1 (PARP1) catalyzes the poly(ADP-ribosyl)ation of protein acceptors using NAD+ as the substrate is now considered as an important target for development of anticancer therapy. PARP1 is known to be post-translationally modified in various ways including phosphorylation and ubiquitination, but the physiological role of PARP1 methylation is not well understood. Herein we demonstrated that the histone methyltransferase SMYD2, which plays critical roles in human carcino...

  15. The Histone Methyltransferase SMYD2 Methylates PARP1 and Promotes Poly(ADP-ribosyl)ation Activity in Cancer Cells12

    OpenAIRE

    Piao, Lianhua; Kang, Daechun; Suzuki, Takehiro; Masuda, Akiko; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2014-01-01

    Poly(ADP-ribose) polymerase-1 (PARP1) catalyzes the poly(ADP-ribosyl)ation of protein acceptors using NAD+ as the substrate is now considered as an important target for development of anticancer therapy. PARP1 is known to be post-translationally modified in various ways including phosphorylation and ubiquitination, but the physiological role of PARP1 methylation is not well understood. Herein we demonstrated that the histone methyltransferase SMYD2, which plays critical roles in human carcino...

  16. Effects of dietary linoleic acid on rat platelet ADP-induced aggregation and binding of 125I-fibrinogen

    International Nuclear Information System (INIS)

    Platelets from rats fed a diet high in linoleic acid (6%) bound increased amounts of fibrinogen on stimulation with ADP, compared to those from rats fed diets with low (2%) or no linoleic acid. However, this increased fibrinogen binding was associated with a decrease in platelet aggregation induced by ADP. Changes in the linoleic acid concentration in platelet membranes may cause changes in this relationship

  17. Pre-steady-state studies of the adenosine triphosphatase activity of coupled submitochondrial particles. Regulation by ADP.

    Science.gov (United States)

    Martins, O B; Tuena de Gómez-Puyou, M; Gómez-Puyou, A

    1988-09-20

    ATPase activities were measured in 10 mM MgCl2, 5 mM ATP, 1 mM ADP, and 1 microM FCCP with submitochondrial particles from bovine heart that had been stimulated by delta mu H+-forming substrates and with particles whose natural inhibitor protein was partially removed by heating. The activities were not linear with time. With both particles, the rate of ATP hydrolysis in the 7-fold greater than that in the steady state. Pre-steady-state and steady-state kinetic studies showed that the decrease of ATPase activity was due to the binding of ADP in a high-affinity site of the enzyme (K0.5 of 10 microM). Inhibition of ATP hydrolysis was accompanied by the binding of approximately 1 mol of ADP/mol of particulate F1; 10 microM ADP gave half-maximal binding. ADP could be replaced by IDP, but with an affinity 50-fold lower (K0.5 of 0.5 mM). Maximal inhibition by ADP and IDP was achieved in less than 5 s. Inhibition was enhanced by uncouplers. Even in the presence of pyruvate kinase and phosphoenolpyruvate, the rates of hydrolysis were about 2.5-fold higher in the first seconds of reaction than in the steady state. This decrease of ATPase activity also correlated with the binding of nearly 1 mol of ADP/mol of F1. This inhibitory ADP remained bound to the enzyme after several thousand turnovers. Apparently, it is possible to observe maximal rates of hydrolysis only in the first few catalytic cycles of the enzyme. PMID:2974725

  18. 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.

  19. Structural analysis of poly(ADP-ribose)polymerase in higher and lower eukaryotes.

    Science.gov (United States)

    Scovassi, A I; Izzo, R; Franchi, E; Bertazzoni, U

    1986-08-15

    A phylogenetic survey for the poly(ADP-ribose)polymerase has been conducted by analyzing enzyme activity in various organisms and determining the structure of the catalytic peptides by renaturation of functional activities of the enzyme in situ after electrophoresis in denaturing conditions (activity gel). The enzyme is widely distributed in cells from all different classes of vertebrates, from arthropods, mollusks and plant cells but could not be detected in echinoderms, nematodes, platyhelminths, thallophytes (including yeast) and bacteria. The presence on activity gels of a catalytic peptide with Mr = 115,000-120,000 was demonstrated in vertebrates, arthropods and mollusks but no activity bands were recovered in many lower eukaryotes, in plant cells and bacteria. By using an immunological procedure that used an antiserum against homogeneous calf thymus poly(ADP-ribose) polymerase, common immunoreactive peptides were visualized in mammals, avians, reptiles, amphibians and fishes, while lacking in non-vertebrate organisms. Our results indicate that the structure of poly(ADP-ribose) polymerase is conserved down to the mollusks suggesting its important role for DNA metabolism of multicellular organisms. PMID:3091369

  20. Sodium-sodium exchange through the sodium pump: the roles of ATP and ADP.

    Science.gov (United States)

    Cavieres, J D; Glynn, I M

    1979-12-01

    1. We have developed a procedure for preparing resealed red cell ghosts that contain ADP but very little ATP. 2. The procedure involves (i) lysis of the cells in a very large volume of lysing solution, (ii) resuspension of the ghosts in a small volume, (iii) the incorporation into the ghosts, before they are resealed, of the adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (AP5A) and of hexokinase, and (iv) the removal of traces of ATP, formed by residual adenylate kinase activity, by the addition of glucose. 3. Measurements of sodium efflux from ghosts prepared in this way show that sodium-sodium exchange through the sodium pump does not occur in the absence of ATP even if ADP is present. 4. The beta:gamma imido analogue of ATP (AMP.PNP), which is incapable of phosphorylating sodium, potassium-ATPase, cannot replace ATP in supporting sodium-sodium exchange. 5. These findings support the hypothesis that the outward movement of sodium ions through the sodium pump is associated with the transfer of a phosphoryl group from ATP to the enzyme, and that the inward movement of sodium ions through the pump is associated with the return of a phosphoryl group from the phosphoenzyme to ADP. PMID:536926

  1. Poly(ADP-ribose) in the bone: from oxidative stress signal to structural element.

    Science.gov (United States)

    Hegedűs, Csaba; Robaszkiewicz, Agnieszka; Lakatos, Petra; Szabó, Éva; Virág, László

    2015-05-01

    Contrary to common perception bone is a dynamic organ flexibly adapting to changes in mechanical loading by shifting the delicate balance between bone formation and bone resorption carried out by osteoblasts and osteoclasts, respectively. In the past decades numerous studies demonstrating production of reactive oxygen or nitrogen intermediates, effects of different antioxidants, and involvement of prototypical redox control mechanisms (Nrf2-Keap1, Steap4, FoxO, PAMM, caspase-2) have proven the central role of redox regulation in the bone. Poly(ADP-ribosyl)ation (PARylation), a NAD-dependent protein modification carried out by poly(ADP-ribose) polymerase (PARP) enzymes recently emerged as a new regulatory mechanism fine-tuning osteoblast differentiation and mineralization. Interestingly PARylation does not simply serve as a signaling mechanism during osteoblast differentiation but also couples it to osteoblast death. Even more strikingly, the poly(ADP-ribose) polymer likely released from succumbed cells at the terminal stage of differentiation is incorporated into the bone matrix representing the first structural role of this versatile biopolymer. Moreover, this new paradigm explains why and how osteodifferentiation and death of cells entering this pathway are closely coupled to each other. Here we review the role of reactive oxygen and nitrogen intermediates as well as PARylation in osteoblast and osteoclast differentiation, function, and cell death. PMID:25660995

  2. 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.

  3. PARP1 Is a TRF2-associated Poly(ADP-Ribose)Polymerase and Protects Eroded Telomeres

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yie [ORNL; Wu, Jun [ORNL; Schreiber, Valerie [Universite Louis Pasteur, France; Dunlap, John [University of Tennessee, Knoxville (UTK); Dantzer, Francoise [Universite Louis Pasteur, France; Wang, Yisong [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL)

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.

  4. PARP1 is a TRF2-associated poly(ADP-ribose) polymerase and protects eroded telomeres

    Energy Technology Data Exchange (ETDEWEB)

    Gomez, Marla V [ORNL; Wu, Jun [ORNL; Wang, Yisong [ORNL; Liu, Yie [ORNL

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.

  5. Gemcitabine induces poly (ADP-ribose polymerase-1 (PARP-1 degradation through autophagy in pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Yufeng Wang

    Full Text Available Poly (ADP-ribose polymerase-1 (PARP-1 and autophagy play increasingly important roles in DNA damage repair and cell death. Gemcitabine (GEM remains the first-line chemotherapeutic drug for pancreatic cancer (PC. However, little is known about the relationship between PARP-1 expression and autophagy in response to GEM. Here we demonstrate that GEM induces DNA-damage response and degradation of mono-ADP ribosylated PARP-1 through the autophagy pathway in PC cells, which is rescued by inhibiting autophagy. Hypoxia and serum starvation inhibit autophagic activity due to abrogated GEM-induced mono-ADP-ribosylated PARP-1 degradation. Activation of extracellular regulated protein kinases (ERK induced by serum starvation shows differences in intracellular localization as well as modulation of autophagy and PARP-1 degradation in GEM-sensitive KLM1 and -resistant KLM1-R cells. Our study has revealed a novel role of autophagy in PARP-1 degradation in response to GEM, and the different impacts of MEK/ERK signaling pathway on autophagy between GEM-sensitive and -resistant PC cells.

  6. Brefeldin A-induced ADP-ribosylation in the structure and function of the Golgi complex.

    Science.gov (United States)

    Colanzi, A; Mironov, A; Weigert, R; Limina, C; Flati, S; Cericola, C; Di Tullio, G; Di Girolamo, M; Corda, D; De Matteis, M A; Luini, A

    1997-01-01

    Brefeldin A (BFA) is a fungal metabolite that exerts generally inhibitory actions on membrane transport and induces the disappearance of the Golgi complex. Previously we have shown that BFA stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 KD. The BFA-binding components mediating the BFA-sensitive ADP-ribosylation (BAR) and the effect of BFA on ARF binding to Golgi membranes have similar specificities and affinities for BFA and its analogues, suggesting that BAR may have a role in the cellular effects of BFA. To investigate this we used the approach to impair BAR activity by the use of BAR inhibitors. A series of BAR inhibitors was developed and their effects were studied in RBL cells treated with BFA. In addition to the common ADP-ribosylation inhibitors (nicotinamide and aminobenzamide), compounds belonging to the cumarin (novobiocin, cumermycin, dicumarol) class were active BAR inhibitors. All BAR inhibitors were able to prevent the BFA-induced redistribution of a Golgi marker (Helix pomatia lectin) into the endoplasmic reticulum, as assessed in immunofluorescence experiments. At the ultrastructural level, BAR inhibitors prevented the tubular-vesicular transformation of the Golgi complex caused by BFA. The potencies of these compounds in preventing the BFA effects on the Golgi complex were similar to those at which they inhibited BAR. Altogether these data support the hypothesis that BAR mediates at least some of the effects of BFA on the Golgi structure and function. PMID:9193673

  7. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function.

    Science.gov (United States)

    Breslin, Claire; Hornyak, Peter; Ridley, Andrew; Rulten, Stuart L; Hanzlikova, Hana; Oliver, Antony W; Caldecott, Keith W

    2015-08-18

    Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function. PMID:26130715

  8. The XRCC1 phosphate-binding pocket binds poly (ADP-ribose) and is required for XRCC1 function

    Science.gov (United States)

    Breslin, Claire; Hornyak, Peter; Ridley, Andrew; Rulten, Stuart L.; Hanzlikova, Hana; Oliver, Antony W.; Caldecott, Keith W.

    2015-01-01

    Poly (ADP-ribose) is synthesized at DNA single-strand breaks and can promote the recruitment of the scaffold protein, XRCC1. However, the mechanism and importance of this process has been challenged. To address this issue, we have characterized the mechanism of poly (ADP-ribose) binding by XRCC1 and examined its importance for XRCC1 function. We show that the phosphate-binding pocket in the central BRCT1 domain of XRCC1 is required for selective binding to poly (ADP-ribose) at low levels of ADP-ribosylation, and promotes interaction with cellular PARP1. We also show that the phosphate-binding pocket is required for EGFP-XRCC1 accumulation at DNA damage induced by UVA laser, H2O2, and at sites of sub-nuclear PCNA foci, suggesting that poly (ADP-ribose) promotes XRCC1 recruitment both at single-strand breaks globally across the genome and at sites of DNA replication stress. Finally, we show that the phosphate-binding pocket is required following DNA damage for XRCC1-dependent acceleration of DNA single-strand break repair, DNA base excision repair, and cell survival. These data support the hypothesis that poly (ADP-ribose) synthesis promotes XRCC1 recruitment at DNA damage sites and is important for XRCC1 function. PMID:26130715

  9. The PARP1/ARTD1-Mediated Poly-ADP-Ribosylation and DNA Damage Repair in B Cell Diversification

    Directory of Open Access Journals (Sweden)

    Jackline J.M. Lasola

    2014-01-01

    Full Text Available ADP-ribosylation is an essential post-translational modification, mediated by a family of proteins named poly-ADP-ribose polymerases/Diphtheria toxin-like ADP-ribosyltransferases (PARPs/ARTDs, that functions to assist in cellular homeostasis through an array of mechanisms. Although the function of PARP1/ARTD1-mediated poly-ADP-ribosylation (PARylation in response to environmental genotoxic stressors has been extensively studied, its role in the regulation and maintenance of cellular events under times of programmed DNA damage and repair remains to be elucidated. In the case of B cell maturation and differentiation, processes such as V(DJ recombination, somatic hypermutation, and class switch recombination, require the induction of DNA strand breaks for the generation of a varied immunoglobulin repertoire and, thus, serve as a model system to explore the function of PARylation in immunological processes. In this review, we summarize the current understanding of ADP-ribosylation and the PARPs/ARTDs family proteins, in particular PARP1/ARTD1-conferred PARylation, in B cells. Following an overview of PARylation in cellular responses to environmental and spontaneous DNA damage, we discuss the emerging function of PARP1/ARTD1 and PARylation in DNA damage-induced nuclear factor kappaB (NF-κB signaling and B cell maturation and differentiation. Finally, we conclude by underlining further efforts that are needed to understand how the PARPs/ARTDs family proteins and ADP-ribosylation control the development and function of B cells.

  10. 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)

    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)

  11. Cloning, expression, purification and crystallization as well as X-ray fluorescence and preliminary X-ray diffraction analyses of human ADP-ribosylhydrolase 1

    International Nuclear Information System (INIS)

    Human ADP-ribosylhydrolase 1, which cleaves the glycosidic bond between ADP-ribose and specific Arg residues in proteins, has been cloned, expressed, purified and crystallized. Human ADP-ribosylhydrolase 1 (hARH1, ADPRH) cleaves the glycosidic bond of ADP-ribose attached to an Arg residue of a protein. hARH1 has been cloned, expressed heterologously in Escherichia coli, purified and crystallized in complex with K+ and ADP. The orthorhombic crystals contained one monomer per asymmetric unit, exhibited a solvent content of 43% and diffracted X-rays to a resolution of 1.9 Å. A prerequisite for obtaining well diffracting crystals was the performance of X-ray fluorescence analysis on poorly diffracting apo hARH1 crystals, which revealed the presence of trace amounts of K+ in the crystal. Adding K-ADP to the crystallization cocktail then resulted in a crystal of different morphology and with dramatically improved diffraction properties

  12. Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport.

    Science.gov (United States)

    Song, Eun-Kyung; Rah, So-Young; Lee, Young-Rae; Yoo, Chae-Hwa; Kim, Yu-Ri; Yeom, Ji-Hyun; Park, Kwang-Hyun; Kim, Jong-Suk; Kim, Uh-Hyun; Han, Myung-Kwan

    2011-12-30

    The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization. PMID:22033928

  13. 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.

  14. Inhibition of poly(ADP-ribose) polymerase interferes with Trypanosoma cruzi infection and proliferation of the parasite.

    Science.gov (United States)

    Vilchez Larrea, Salomé C; Haikarainen, Teemu; Narwal, Mohit; Schlesinger, Mariana; Venkannagari, Harikanth; Flawiá, Mirtha M; Villamil, Silvia H Fernández; Lehtiö, Lari

    2012-01-01

    Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection. PMID:23049934

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

    International Nuclear Information System (INIS)

    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. 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.

  17. Maximum ADPE Approach for a High Rate CCSDS Return Link Processing System

    Science.gov (United States)

    Krimchansky, Alexander; Moe, Brian; Erickson, David

    1996-01-01

    The earth observing system data and operations system (EDOS) multi-mission data processing and distribution system for the earth observing system is considered. The EDOS was based on the Consultative Committee for Space Data Systems (CCSDS) protocols. The development included the challenge of developing and demonstrating a 150 Mbps CCSDS return link processing capability for the support of the first EDOS delivery. The approach used general-purpose automated data processing equipment (ADPE) and minimized the use of customized hardware. The way in which the system was developed is described. The principle design decisions and the performance benchmark results are presented.

  18. Proteome-wide Identification of Poly(ADP-Ribosyl)ation Targets in Different Genotoxic Stress Responses

    DEFF Research Database (Denmark)

    Jungmichel, S.; Rosenthal, F.; Altmeyer, M.;

    2013-01-01

    Poly(ADP-ribos)ylation (PARylation) is a reversible posttranslational modification found in higher eukaryotes. However, little is known about PARylation acceptor proteins. Here, we describe a sensitive proteomics approach based on high-accuracy quantitative mass spectrometry for the identification...... the PARylation of RNA-processing factors THRAP3 and TAF15 under oxidative stress. High-content imaging reveals that PARylation affects the nuclear relocalization of THRAP3 and TAF15, demonstrating the potential of our approach to uncover hitherto unappreciated processes being controlled by specific...

  19. Dynamics and Control of Orbiting Space Structures NASA Advanced Design Program (ADP)

    Science.gov (United States)

    Cruse, T. A.

    1996-01-01

    The report summarizes the advanced design program in the mechanical engineering department at Vanderbilt University for the academic years 1994-1995 and 1995-1996. Approximately 100 students participated in the two years of the subject grant funding. The NASA-oriented design projects that were selected included lightweight hydrogen propellant tank for the reusable launch vehicle, a thermal barrier coating test facility, a piezoelectric motor for space antenna control, and a lightweight satellite for automated materials processing. The NASA supported advanced design program (ADP) has been a success and a number of graduates are working in aerospace and are doing design.

  20. Poly (ADP-ribose polymerase inhibitor: an evolving paradigm in the treatment of prostate cancer

    Directory of Open Access Journals (Sweden)

    Jingsong Zhang

    2014-06-01

    Full Text Available Recent phase I studies have reported single-agent activities of poly (ADP-ribose polymerase (PARP inhibitor in sporadic and in BRCA-mutant prostate cancers. Two of the most common genetic alterations in prostate cancer, ETS gene rearrangement and loss of PTEN, have been linked to increased sensitivity to PARP inhibitor in preclinical models. Emerging evidence also suggests that PARP1 plays an important role in mediating the transcriptional activities of androgen receptor (AR and ETS gene rearrangement. In this article, the preclinical work and early-phase clinical trials in developing PARP inhibitor-based therapy as a new treatment paradigm for metastatic prostate cancer are reviewed.

  1. Sumoylation of poly(ADP-ribose) polymerase 1 inhibits its acetylation and restrains transcriptional coactivator function

    OpenAIRE

    Messner, S.; Schuermann, D.; Altmeyer, M; Kassner, I; Schmidt, D; Schär, P; Müller, S.; Hottiger, M O

    2009-01-01

    Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated nuclear protein and functions as a molecular stress sensor. At the cellular level, PARP1 has been implicated in a wide range of processes, such as maintenance of genome stability, cell death, and transcription. PARP1 functions as a transcriptional coactivator of nuclear factor B (NF-B) and hypoxia inducible factor 1 (HIF1). In proteomic studies, PARP1 was found to be modified by small ubiquitin-like mod...

  2. Poly(ADP-ribose) polymerase (PARP-1) has a controlling role in homologous recombination

    OpenAIRE

    Schultz, Niklas; Lopez, Elena; Saleh-Gohari, Nasrollah; Helleday, Thomas

    2003-01-01

    Cells with non-functional poly(ADP-ribose) polymerase (PARP-1) show increased levels of sister chromatid exchange, suggesting a hyper recombination phenotype in these cells. To further investigate the involvement of PARP-1 in homologous recombination (HR) we investigated how PARP-1 affects nuclear HR sites (Rad51 foci) and HR repair of an endonuclease-induced DNA double-strand break (DSB). Several proteins involved in HR localise to Rad51 foci and HR-deficient cells fail to form Rad51 foci in...

  3. Recognition of Platinum-DNA Damage by Poly(ADP-Ribose) Polymerase-1†

    OpenAIRE

    Zhu, Guangyu; Chang, Paul; Lippard, Stephen J.

    2010-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) was recently identified as a platinum DNA damage response protein. To investigate the binding properties of PARP-1 to different platinum-DNA adducts in greater detail, biotinylated DNA probes containing a site-specific cisplatin 1,2-d(GpG) or 1,3-d(GpTpG) intrastrand cross-link, or a cisplatin 5’-d(GC)/5’-d(GC) interstrand cross-link (ICL) were utilized in binding assays with cell free extracts (CFEs) in vitro. The activated state of PARP-1 was generated...

  4. Evolutionary history of the poly(ADP-ribose polymerase gene family in eukaryotes

    Directory of Open Access Journals (Sweden)

    Teotia Sachin

    2010-10-01

    Full Text Available Abstract Background The Poly(ADP-ribosepolymerase (PARP superfamily was originally identified as enzymes that catalyze the attachment of ADP-ribose subunits to target proteins using NAD+ as a substrate. The family is characterized by the catalytic site, termed the PARP signature. While these proteins can be found in a range of eukaryotes, they have been best studied in mammals. In these organisms, PARPs have key functions in DNA repair, genome integrity and epigenetic regulation. More recently it has been found that proteins within the PARP superfamily have altered catalytic sites, and have mono(ADP-ribose transferase (mART activity or are enzymatically inactive. These findings suggest that the PARP signature has a broader range of functions that initially predicted. In this study, we investigate the evolutionary history of PARP genes across the eukaryotes. Results We identified in silico 236 PARP proteins from 77 species across five of the six eukaryotic supergroups. We performed extensive phylogenetic analyses of the identified PARPs. They are found in all eukaryotic supergroups for which sequence is available, but some individual lineages within supergroups have independently lost these genes. The PARP superfamily can be subdivided into six clades. Two of these clades were likely found in the last common eukaryotic ancestor. In addition, we have identified PARPs in organisms in which they have not previously been described. Conclusions Three main conclusions can be drawn from our study. First, the broad distribution and pattern of representation of PARP genes indicates that the ancestor of all extant eukaryotes encoded proteins of this type. Second, the ancestral PARP proteins had different functions and activities. One of these proteins was similar to human PARP1 and likely functioned in DNA damage response. The second of the ancestral PARPs had already evolved differences in its catalytic domain that suggest that these proteins may not have

  5. Inactivation of the Elongation Factor Tu by Mosquitocidal Toxin-Catalyzed Mono-ADP-Ribosylation

    OpenAIRE

    Schirmer, Jörg; Wieden, Hans-Joachim; Rodnina, Marina V.; Aktories, Klaus

    2002-01-01

    The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an ∼97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in Escherichia coli expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in E. coli and subsequently cleaved to an active 27-kDa enzyme frag...

  6. Import of ADP/ATP carrier into mitochondria: two receptors act in parallel

    OpenAIRE

    1990-01-01

    We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into ...

  7. 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.

  8. Coexistence of ferroelectric and antiferroelectric microregions in the paraelectric phase of NH4H2PO4 (ADP)

    International Nuclear Information System (INIS)

    Ammonium dihydrogen phosphate NH4H2PO4 (ADP) is an antiferroelectric (AFE) compound belonging to the KDP-type family of hydrogen-bonded ferroelectrics. Recent ab initio results have shown that the optimization of the N-H-O bridges in ADP leads to the stabilization of the AFE state over a FE one. However, electron spin probe measurements have suggested that microregions of both phases may coexist above the critical antiferroelectric-paraelectric transition temperature. We have performed first principles studies of the energetics and relative stability of different AFE and FE defects embedded in a paraelectric (PE) matrix of ADP. Our analysis indicates that FE and AFE clusters are stable and may coexist in the PE phase, thus confirming the above suggestion.

  9. Isolation and properties of ADP-ribosyltransferase from rabbit skeletal muscle

    International Nuclear Information System (INIS)

    Sarcoplasmic reticulum (SR) bound ADP-ribosyltransferase activity is purified approximately 2000-3000 fold from extracts of rabbit skeletal muscle. The overall procedure involves the isolation of microsomal fraction by high speed centrifugation followed by detergent treatment and centrifugation on sucrose density layers to obtain a light SR preparation. The membrane proteins in the light SR are solubilized in 2% polyoxyethylene-9-lauryl ether. The transferase is further purified by chromatography on reactive red 120 agarose and high performance liquid chromatography on TSK-DEAE. The reactive red and anion exchange chromatography are repeated. SDS polyacrylamide gel electrophoresis of the preparation showed only one prominent protein band of approximately 155 K. The purified enzyme preparation can ADP-ribosylate the low molecular weight guanidino compounds such as guanyl hydrazones, polyarginine and the proteins lysozyme, phosphorylase and phosphorylase kinase. When added to the SR, the purified enzyme greatly stimulates the incorporation of 1abel from 32[P]NAD into a 100-110KD protein band which corresponds to the major protein band in the membrane

  10. P2Y12-ADP receptor antagonists: Days of future and past.

    Science.gov (United States)

    Laine, Marc; Paganelli, Franck; Bonello, Laurent

    2016-05-26

    Antiplatelet therapy is the cornerstone of the therapeutic arsenal in coronary artery disease. Thanks to a better understanding in physiology, pharmacology and pharmacogenomics huge progress were made in the field of platelet reactivity inhibition thus allowing the expansion of percutaneous coronary intervention. Stent implantation requires the combination of two antiplatelet agents acting in a synergistic way. Asprin inhibit the cyclo-oxygenase pathway of platelet activation while clopidogrel is a P2Y12 adenosine diphosphate (ADP)-receptor antagonist. This dual antiplatelet therapy has dramatically improved the prognosis of stented patients. However, due to pharmacological limitations of clopidogrel (interindividual variability in its biological efficacy, slow onset of action, mild platelet reactivity inhibition) ischemic recurrences remained high following stent implantation especially in acute coronary syndrome patients. Thus, more potent P2Y12-ADP receptor inhibitors were developped including prasugrel, ticagrelor and more recently cangrelor to overcome these pitfalls. These new agents reduced the rate of thrombotic events in acute coronary syndrome patients at the cost of an increased bleeding risk. The abundance in antiplatelet agents allow us to tailor our strategy based on the thrombotic/bleeding profile of each patient. Recently, the ACCOAST trial cast a doubt on the benefit of pre treatment in non-ST segment elevation acute coronary syndrome. The aim of the present review is to summarize the results of the main studies dealing with antiplatelet therapy in stented/acute coronary syndromes patients. PMID:27231519

  11.  Poly(ADP-ribose polymerase (PARP inhibitors in BRCA1/2 cancer therapy

    Directory of Open Access Journals (Sweden)

    Katarzyna Kluzek

    2012-06-01

    Full Text Available  A majority of currently used anticancer drugs belong to a group of chemical agents that damage DNA. The efficiency of the treatment is limited by effective DNA repair systems functioning in cancer cells. Many chemotherapeutic compounds cause strong systemic toxicity. Therefore, there is still a need for new anticancer agents which are less toxic for nontransformed cells and selectively kill cancer cells. One of the most promising molecular targets in cancer therapy is poly(ADP-ribose polymerases (PARP. PARP play an essential role in repairing DNA strand breaks. Small molecule inhibitors of these enzymes have been developed and have proved to be extremely toxic for cancer cells that lack the functional BRCA1 and BRCA2 proteins that are involved in homologous recombination, a complex repair mechanism of DNA double strand breaks. Mutations in BRCA1/2 genes are associated with genetically inherited breast and ovarian cancers. Therefore PARP inhibitors may prove to be very effective and selective in the treatment of these cancer types. This review is focused on the function of BRCA1/2 proteins and poly(ADP-ribose polymerases in DNA repair systems, especially in the homologous recombination process. A short history of the studies that led to synthesis of high specificity small molecule PARP inhibitors is also presented, as well as the results of clinical trials concerning the most effective PARP inhibitors in view of their potential application in oncological treatment, particularly breast cancers.

  12. Analysis of current situation and perspective of digital preservation in the Social Science Data Archives (ADP

    Directory of Open Access Journals (Sweden)

    Irena Vipavc Brvar

    2011-01-01

    Full Text Available Social science data archives have begun to evolve in 1960’s, thus being one of the pioneers of digital preservation. The Slovenian Data Archive (ADP follows and adjusts best practices that have emerged in this specific field. Its tasks comprise acquisition,processing and long term preservation of valuable primary research data, as well as providing accessibility to the content for users. As in other areas of digital preservation much attention is paid to meeting the requirements of long term preservation as specified in the OAIS (Opean Archival Information System standard, and the use of technological solutions, proposed metadata standards, strategies and policies, and best practices. The purpose of this paper is to present procedures in ADP, to compare them with selected solutions of organizations with the similar mission, and to suggest improvements.Examples are taken from partner organizations within the CESSDA data archives association. These organizations (especially those from the USA, Great Britain and the Netherlands have developed solutions in the field of digital preservation to the highest degree. The paper also reflects on agreed guidelines of reliable services for the archives of research data, which arose in the frame of the FP7 project with the same title (i.e. CESSDA PPP. The results of analysis are useful for all are seeking practical solutions to similar problems.

  13. P2Y12-ADP receptor antagonists: Days of future and past

    Science.gov (United States)

    Laine, Marc; Paganelli, Franck; Bonello, Laurent

    2016-01-01

    Antiplatelet therapy is the cornerstone of the therapeutic arsenal in coronary artery disease. Thanks to a better understanding in physiology, pharmacology and pharmacogenomics huge progress were made in the field of platelet reactivity inhibition thus allowing the expansion of percutaneous coronary intervention. Stent implantation requires the combination of two antiplatelet agents acting in a synergistic way. Asprin inhibit the cyclo-oxygenase pathway of platelet activation while clopidogrel is a P2Y12 adenosine diphosphate (ADP)-receptor antagonist. This dual antiplatelet therapy has dramatically improved the prognosis of stented patients. However, due to pharmacological limitations of clopidogrel (interindividual variability in its biological efficacy, slow onset of action, mild platelet reactivity inhibition) ischemic recurrences remained high following stent implantation especially in acute coronary syndrome patients. Thus, more potent P2Y12-ADP receptor inhibitors were developped including prasugrel, ticagrelor and more recently cangrelor to overcome these pitfalls. These new agents reduced the rate of thrombotic events in acute coronary syndrome patients at the cost of an increased bleeding risk. The abundance in antiplatelet agents allow us to tailor our strategy based on the thrombotic/bleeding profile of each patient. Recently, the ACCOAST trial cast a doubt on the benefit of pre treatment in non-ST segment elevation acute coronary syndrome. The aim of the present review is to summarize the results of the main studies dealing with antiplatelet therapy in stented/acute coronary syndromes patients. PMID:27231519

  14. Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release.

    Science.gov (United States)

    Hagberg, I A; Sølvik, U Ø; Opdahl, H; Roald, H E; Lyberg, T

    1999-01-01

    To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40 ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s(-1)). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear. PMID:16801117

  15. Role of CD38, a cyclic ADP-ribosylcyclase, in morphine antinociception and tolerance.

    Science.gov (United States)

    Hull, Lynn C; Rabender, Christopher; Gabra, Bichoy H; Zhang, Fan; Li, Pin-Lan; Dewey, William L

    2010-09-01

    Our previous studies have demonstrated that an increase in intracellular levels of Ca(2+) in neurons is an important component of both the antinociception produced by morphine and morphine's tolerance. The present study tested the hypothesis that the Ca(2+) signaling second messenger, cyclic ADP-ribose (cADPR), derived from CD38 activation participates in morphine antinociception and tolerance. We first showed that morphine's antinociceptive potency was increased by the intracerebroventricular injection of CD38 substrate beta-NAD(+) in mice. Furthermore, morphine tolerance was reversed by intracerebroventricular administration of each of three different inhibitors of the CD38-cADPR-ryanodine receptor Ca(2+) signaling pathway. These inhibitors were the ADP-ribosylcyclase inhibitor nicotinamide, cADPR analog 8-bromo-cADPR, and a large dose of ryanodine (>50 muM) that blocks the ryanodine receptor. In CD38 gene knockout [CD38(-/-)] mice, the antinociceptive action of morphine was found to be less potent compared with wild-type (WT) mice, as measured by tail-flick response, hypothermia assay, and observations of straub tail. However, there was no difference in locomotor activation between CD38(-/-) and WT animals. It was also found that less tolerance to morphine developed in CD38(-/-) mice compared with WT animals. These results indicate that cADRP-ryanodine receptor Ca(2+) signaling associated with CD38 plays an important role in morphine tolerance. PMID:20551293

  16. Identification of the Escherichia coli ADP-glucose synthetase inhibitor binding site(s)

    International Nuclear Information System (INIS)

    The photoaffinity labeling agent 8-azido adenylate (AMP) is an inhibitor site specific probe of the E. coli ADPG synthetase. In the absence of light, 8-azido AMP exhibits the typical reversible allosteric kinetics of the physiological inhibitor AMP. In the presence of light (254 nm), [2-3H]8-azido AMP specifically and covalently incorporates into the enzyme. Photoincorporation is linearly related to loss of catalytic activity up to at least 65% inactivation. The substrate ADP-glucose (ADPG) provides nearly 100% protection from 8-azido AMP photoinactivation, while the substrate AMP provides approximately 50% protection and the inhibitor AMP provides approximately 30% protection. These three adenylate allosteric effects of E. coli ADPG synthetase also protect it from photoincorporation of 8-azido AMP. The reaction site(s) of [2-3H]-azido AMP with the enzyme was identified by reverse phase HPLC isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease generated peptides containing the labeled analog. This site is the same as the major binding region of the substrate site specific probe, 8-azido ADP-[14C]glucose. Conformational analysis of this region predicts that it is a part of a Rossmann fold, the super-secondary structure found in many adenine nucleotide binding proteins. Two minor reaction regions of the enzyme with [2-3H]8-azido AMP were also identified. The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure

  17. The Structural and Functional Characterization of Mammalian ADP-dependent Glucokinase.

    Science.gov (United States)

    Richter, Jan P; Goroncy, Alexander K; Ronimus, Ron S; Sutherland-Smith, Andrew J

    2016-02-19

    The enzyme-catalyzed phosphorylation of glucose to glucose-6-phosphate is a reaction central to the metabolism of all life. ADP-dependent glucokinase (ADPGK) catalyzes glucose-6-phosphate production, utilizing ADP as a phosphoryl donor in contrast to the more well characterized ATP-requiring hexokinases. ADPGK is found in Archaea and metazoa; in Archaea, ADPGK participates in a glycolytic role, but a function in most eukaryotic cell types remains unknown. We have determined structures of the eukaryotic ADPGK revealing a ribokinase-like tertiary fold similar to archaeal orthologues but with significant differences in some secondary structural elements. Both the unliganded and the AMP-bound ADPGK structures are in the "open" conformation. The structures reveal the presence of a disulfide bond between conserved cysteines that is positioned at the nucleotide-binding loop of eukaryotic ADPGK. The AMP-bound ADPGK structure defines the nucleotide-binding site with one of the disulfide bond cysteines coordinating the AMP with its main chain atoms, a nucleotide-binding motif that appears unique to eukaryotic ADPGKs. Key amino acids at the active site are structurally conserved between mammalian and archaeal ADPGK, and site-directed mutagenesis has confirmed residues essential for enzymatic activity. ADPGK is substrate inhibited by high glucose concentration and shows high specificity for glucose, with no activity for other sugars, as determined by NMR spectroscopy, including 2-deoxyglucose, the glucose analogue used for tumor detection by positron emission tomography. PMID:26555263

  18. C3larvin toxin, an ADP-ribosyltransferase from Paenibacillus larvae.

    Science.gov (United States)

    Krska, Daniel; Ravulapalli, Ravikiran; Fieldhouse, Robert J; Lugo, Miguel R; Merrill, A Rod

    2015-01-16

    C3larvin toxin was identified by a bioinformatic strategy as a putative mono-ADP-ribosyltransferase and a possible virulence factor from Paenibacillus larvae, which is the causative agent of American Foulbrood in honey bees. C3larvin targets RhoA as a substrate for its transferase reaction, and kinetics for both the NAD(+) (Km = 34 ± 12 μm) and RhoA (Km = 17 ± 3 μm) substrates were characterized for this enzyme from the mono-ADP-ribosyltransferase C3 toxin subgroup. C3larvin is toxic to yeast when expressed in the cytoplasm, and catalytic variants of the enzyme lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. A small molecule inhibitor of C3larvin enzymatic activity was discovered called M3 (Ki = 11 ± 2 μm), and to our knowledge, is the first inhibitor of transferase activity of the C3 toxin family. C3larvin was crystallized, and its crystal structure (apoenzyme) was solved to 2.3 Å resolution. C3larvin was also shown to have a different mechanism of cell entry from other C3 toxins. PMID:25477523

  19. C3larvin Toxin, an ADP-ribosyltransferase from Paenibacillus larvae*

    Science.gov (United States)

    Krska, Daniel; Ravulapalli, Ravikiran; Fieldhouse, Robert J.; Lugo, Miguel R.; Merrill, A. Rod

    2015-01-01

    C3larvin toxin was identified by a bioinformatic strategy as a putative mono-ADP-ribosyltransferase and a possible virulence factor from Paenibacillus larvae, which is the causative agent of American Foulbrood in honey bees. C3larvin targets RhoA as a substrate for its transferase reaction, and kinetics for both the NAD+ (Km = 34 ± 12 μm) and RhoA (Km = 17 ± 3 μm) substrates were characterized for this enzyme from the mono-ADP-ribosyltransferase C3 toxin subgroup. C3larvin is toxic to yeast when expressed in the cytoplasm, and catalytic variants of the enzyme lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. A small molecule inhibitor of C3larvin enzymatic activity was discovered called M3 (Ki = 11 ± 2 μm), and to our knowledge, is the first inhibitor of transferase activity of the C3 toxin family. C3larvin was crystallized, and its crystal structure (apoenzyme) was solved to 2.3 Å resolution. C3larvin was also shown to have a different mechanism of cell entry from other C3 toxins. PMID:25477523

  20. How to kill tumor cells with inhibitors of poly(ADP-ribosyl)ation.

    Science.gov (United States)

    Mangerich, Aswin; Bürkle, Alexander

    2011-01-15

    Poly(ADP-ribosyl)ation is a post-translational modification catalyzed by the enzyme family of poly(ADP-ribose) polymerases (PARPs). PARPs exhibit pleiotropic cellular functions ranging from maintenance of genomic stability and chromatin remodeling to regulation of cell death, thereby rendering PARP homologues promising targets in cancer therapy. Depending on the molecular status of a cancer cell, low-molecular weight PARP inhibitors can (i) either be used as monotherapeutic agents following the concept of synthetic lethality or (ii) to support classical chemotherapy or radiotherapy. The rationales are the following: (i) in cancers with selective defects in homologous recombination repair, inactivation of PARPs directly causes cell death. In cancer treatment, this phenomenon can be employed to specifically target tumor cells while sparing nonmalignant tissue. (ii) PARP inhibitors can also be used to sensitize cells to cytotoxic DNA-damaging treatments, as some PARPs actively participate in genomic maintenance. Apart from that, PARP inhibitors possess antiangiogenic functions, thus opening up a further option to inhibit tumor growth. In view of the above, a number of high-potency PARP inhibitors have been developed during the last decade and are currently evaluated as cancer therapeutics in clinical trials by several leading pharmaceutical companies. PMID:20853319

  1. Phosphorylation of Thr18 and Ser20 of p53 in Ad-p53 – induced apoptosis

    OpenAIRE

    Nakamizo, Akira; Amano, Toshiyuko; Zhang, Wei; Zhang, Xin-qiao; Ramdas, Latha; Liu, Ta-Jen; Bekele, B. Nebiyou; Shono, Tadahisa; Sasaki, Tomio; Benedict, William F.; Sawaya, Raymond; Lang, Frederick F.

    2008-01-01

    The p53 protein plays a critical role in inducing cell cycle arrest or apoptosis. Because p53 is inactivated in human gliomas, restoring p53 function is a major focus of glioma therapy. The most clinically tested strategy for replacing p53 has been adenoviral-mediated p53 gene therapy (Ad-p53). In addition to their therapeutic implications, investigations into Ad-p53 provide model systems for understanding p53’s ability to induce cell cycle arrest versus apoptosis, particularly because wild-t...

  2. Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP

    International Nuclear Information System (INIS)

    The amidase domain of the allophanate hydrolase AtzF from Pseudomonas sp. strain ADP has been crystallized and preliminary X-ray diffraction data have been collected. The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Å resolution and adopted space group P21, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, β = 106.6°

  3. Postnatal Age Influences Hypoglycemia-induced Poly(ADP-ribose) Polymerase-1 Activation in the Brain Regions of Rats

    OpenAIRE

    Rao, Raghavendra; Sperr, Dustin; Ennis, Kathleen; Tran, Phu

    2009-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) overactivation plays a significant role in hypoglycemia-induced brain injury in adult rats. To determine the influence of postnatal age on PARP-1 activation, developing and adult male rats were subjected to acute hypoglycemia of equivalent severity and duration. The expression of PARP-1 and its downstream effectors, apoptosis inducing factor (Aifm1), caspase 3 (Casp3), NF-κB (Nfkb1) and bcl-2 (Bcl2), and cellular poly(ADP-ribose) (PAR) polymer expression...

  4. Prediction of the Occurrence of the ADP-binding βαβ-fold in Proteins, Using an Amino Acid Sequence Fingerprint

    NARCIS (Netherlands)

    Wierenga, Rik K.; Terpstra, Peter; Hol, Wim G.J.

    1986-01-01

    An amino acid sequence "fingerprint” has been derived that can be used to test if a particular sequence will fold into a βαβ-unit with ADP-binding properties. It was deduced from a careful analysis of the known three-dimensional structures of ADP-binding βαβ-folds. This fingerprint is in fact a set

  5. Study of the five Rickettsia prowazekii proteins annotated as ATP/ADP translocases (Tlc): Only Tlc1 transports ATP/ADP, while Tlc4 and Tlc5 transport other ribonucleotides.

    Science.gov (United States)

    Audia, Jonathon P; Winkler, Herbert H

    2006-09-01

    The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/ADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tlc homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tlc genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a K(i) similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins. PMID:16923893

  6. Localization of the C3-Like ADP-Ribosyltransferase from Staphylococcus aureus during Bacterial Invasion of Mammalian Cells

    OpenAIRE

    Molinari, Gabriella; Rohde, Manfred; Wilde, Christian; Just, Ingo; Aktories, Klaus; Chhatwal, Gursharan S.

    2006-01-01

    The C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase which possesses no specific receptor-binding domain or translocation unit required for entry in target cells where its substrate is located. Here we show that C3stau2 can reach its target after invasion of staphylococci in eukaryotic cells without needing translocation.

  7. 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

  8. Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells

    International Nuclear Information System (INIS)

    The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms

  9. Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Burgman, P.; Konings, A.W. (State Univ. Groningen (Netherlands))

    1989-08-01

    The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms.

  10. Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells.

    Science.gov (United States)

    Burgman, P; Konings, A W

    1989-08-01

    The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms. PMID:2502817

  11. 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.

  12. Poly(ADP-Ribose) Polymerase in Cervical Cancer Pathogenesis: Mechanism and Potential Role for PARP Inhibitors.

    Science.gov (United States)

    Kotsopoulos, Ioannis C; Kucukmetin, Ali; Mukhopadhyay, Asima; Lunec, John; Curtin, Nicola J

    2016-05-01

    Treatment options for disease recurrence of women treated for locally advanced and advanced cervical cancer are very limited-largely palliative chemotherapy. The low efficacy of the currently available drugs raises the need for new targeted agents. Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi) have emerged as a promising class of chemotherapeutic agents in cancers associated with defects in DNA repair. Their therapeutic potential in cervical cancer is currently being evaluated in 3 ongoing clinical trials. Here we review the available information regarding all the aspects of PARP in cervical intraepithelial neoplasia and invasive cervical cancer, from expression and the mechanism of action to the role of the polymorphisms in the pathogenesis of the disease, as well as the potential of the inhibitors. We finally propose a new unifying theory regarding the role of PARPs in the development of cervical carcinomas. PMID:26905326

  13. Regulatory properties of potato-Arabidopsis hybrid ADP-glucose pyrophosphorylase.

    Science.gov (United States)

    Ventriglia, Tiziana; Ballicora, Miguel A; Crevillén, Pedro; Preiss, Jack; Romero, José M

    2007-06-01

    In higher plants, ADP-glucose pyrophosphorylase (ADPGlc-PPase) is a heterotetrameric enzyme comprised of two small and two large subunits. Potato-Arabidopsis hybrid ADPGlc-PPases were generated and their regulatory properties analyzed. We show that ADPGlc-PPase subunits from two different species can interact, producing active enzymes with new regulatory properties. Depending on the subunit combinations, hybrid heterotetramers showed responses to allosteric effectors [3-phosphoglycerate (3-PGA) and Pi] in the micromolar or millimolar range. While hybrid potato small subunit (PSS) and the Arabidopsis large subunit APL1 showed an extremely sensitive response to 3-PGA and Pi, hybrid PSS/Arabidopsis APL2 was very insensitive to them. Intermediate responses were determined for other subunit combinations. PMID:17452341

  14. Substrate binding properties of potato tuber ADP-glucose pyrophosphorylase as determined by isothermal titration calorimetry.

    Science.gov (United States)

    Cakir, Bilal; Tuncel, Aytug; Green, Abigail R; Koper, Kaan; Hwang, Seon-Kap; Okita, Thomas W; Kang, ChulHee

    2015-06-01

    Substrate binding properties of the large (LS) and small (SS) subunits of potato tuber ADP-glucose pyrophosphorylase were investigated by using isothermal titration calorimetry. Our results clearly show that the wild type heterotetramer (S(WT)L(WT)) possesses two distinct types of ATP binding sites, whereas the homotetrameric LS and SS variant forms only exhibited properties of one of the two binding sites. The wild type enzyme also exhibited significantly increased affinity to this substrate compared to the homotetrameric enzyme forms. No stable binding was evident for the second substrate, glucose-1-phosphate, in the presence or absence of ATPγS suggesting that interaction of glucose-1-phosphate is dependent on hydrolysis of ATP and supports the Theorell-Chance bi bi reaction mechanism. PMID:25953126

  15. ATP- and ADP-dnaA protein, a molecular switch in gene regulation.

    OpenAIRE

    Speck, C.; Weigel, C; Messer, W

    1999-01-01

    DnaA protein functions by binding to asymmetric 9mer DNA sites, the DnaA boxes. ATP-DnaA and ADP-DnaA bind to 9mer DnaA boxes with equal affinity, but only ATP-DnaA protein binds in addition to an as yet unknown 6mer site, the ATP-DnaA box AGATCT, or a close match to it. ATP-DnaA protein binding to ATP-DnaA boxes is restricted to sites located in close proximity to DnaA boxes, suggesting that protein-protein interaction is required for its stabilization. We show that ATP-DnaA represses dnaA t...

  16. Poly(ADP)-Ribose Polymerase-1 Inhibitors as a Potential Treatment for Cocaine Addiction.

    Science.gov (United States)

    Scobie, Kimberly N

    2015-01-01

    As of 2008, according to the National Survey on Drug Use and Health, nearly 1.4 million Americans met the Diagnostic and Statistical Manual of Mental Disorders criteria for dependence or abuse of cocaine (in any form) in the past 12 months. However, there are no treatments for cocaine use disorders approved by the Federal Drug Administration (FDA). Alterations in gene regulation contribute significantly to the changes that occur in the brain, both structurally and functionally, and the resultant addictive phenotype that occurs with chronic exposure to drugs of abuse. The Emerging Targets of Cocaine Use Disorders meeting sought to explore novel targets for the treatment of stimulant use disorder. The evidence for a role of one novel target, Poly(ADP)-ribose polymerase-1 (PARP-1), was presented at the meeting and will be summarized in this review. PMID:26022260

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

    International Nuclear Information System (INIS)

    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

  18. PCR cloning and characterization of multiple ADP-glucose pyrophosphorylase cDNAs from tomato

    Science.gov (United States)

    Chen, B. Y.; Janes, H. W.; Gianfagna, T.

    1998-01-01

    Four ADP-glucose pyrophosphorylase (AGP) cDNAs were cloned from tomato fruit and leaves by the PCR techniques. Three of them (agp S1, agp S2, and agp S3) encode the large subunit of AGP, the fourth one (agp B) encodes the small subunit. The deduced amino acid sequences of the cDNAs show very high identities (96-98%) to the corresponding potato AGP isoforms, although there are major differences in tissue expression profiles. All four tomato AGP transcripts were detected in fruit and leaves; the predominant ones in fruit are agp B and agp S1, whereas in leaves they are agp B and agp S3. Genomic southern analysis suggests that the four AGP transcripts are encoded by distinct genes.

  19. Transcriptional regulation by Poly(ADP-ribose polymerase-1 during T cell activation

    Directory of Open Access Journals (Sweden)

    Parrilla Pascual

    2008-04-01

    Full Text Available Abstract Background Accumulating evidence suggests an important role for the enzyme poly(ADP-ribose polymerase-1 (PARP-1 as an integral part of the gene expression regulatory machinery during development and in response to specific cellular signals. PARP-1 might modulate gene expression through its catalytic activity leading to poly(ADP-ribosylation of nuclear proteins or by its physical association with relevant proteins. Recently, we have shown that PARP-1 is activated during T cell activation. However, the proposed role of PARP-1 in reprogramming T cell gene expression upon activation remains largely unexplored. Results In the present study we use oligonucleotide microarray analysis to gain more insight into the role played by PARP-1 during the gene expression reprogramming that takes place in T cells upon activation with anti-CD3 stimulation alone, or in combination with anti-CD28 co-stimulation. We have identified several groups of genes with expression modulated by PARP-1. The expression of 129 early-response genes to anti-CD3 seems to be regulated by PARP-1 either in a positive (45 genes or in a negative manner (84 genes. Likewise, in the presence of co-stimulation (anti-CD3 + anti-CD28 stimulation, the expression of 203 genes is also regulated by PARP-1 either up (173 genes or down (30 genes. Interestingly, PARP-1 deficiency significantly alters expression of genes associated with the immune response such as chemokines and genes involved in the Th1/Th2 balance. Conclusion This study provides new insights into changes in gene expression mediated by PARP-1 upon T cell activation. Pathway analysis of PARP-1 as a nuclear signalling molecule in T cells would be of relevance for the future development of new therapeutic approaches targeting PARP-1 in the acquired immune response.

  20. Thermoregulatory uncoupling in heart muscle mitochondria: involvement of the ATP/ADP antiporter and uncoupling protein.

    Science.gov (United States)

    Simonyan, R A; Skulachev, V P

    1998-09-25

    Possible involvement of the ATP/ADP antiporter and uncoupling protein (UCP) in thermoregulatory uncoupling of oxidative phosphorylation in heart muscle has been studied. To this end, effects of carboxyatractylate (cAtr) and GDP, specific inhibitors of the antiporter and UCP, on the membrane potential of the oligomycin-treated mitochondria from cold-exposed (6 degrees C, 48 h) and control rats have been measured. It is found that cAtr increases the membrane potential level in both cold-exposed and non-exposed groups, the effect being strongly enhanced by cooling. As for GDP, it is effective only in mitochondria from the cold-exposed rats. In these mitochondria, the coupling effect of GDP is smaller than that of cAtr. CDP, which does not interact with UCP, is without any influence on membrane potential. The cold exposure is found to increase the uncoupling efficiency of added natural (palmitate) or artificial (SF6847) uncouplers, the increase being cAtr- and GDP-sensitive in the case of palmitate. The fatty acid-free bovine serum albumin enhances delta psi in both cold-exposed and control groups, the effect being much larger in the former case. It is concluded that in heart muscle mitochondria the ATP/ADP antiporter is responsible for the 'mild uncoupling' under normal conditions and for major portion of the thermoregulatory uncoupling in the cold whereas the rest of thermoregulatory uncoupling is served by UCP (presumably by UCP2 since the UCP2 mRNA level is shown to strongly increase in rat heart muscle under the cold exposure conditions used). PMID:9771898

  1. The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2.

    Science.gov (United States)

    Croy, Heather E; Fuller, Caitlyn N; Giannotti, Jemma; Robinson, Paige; Foley, Andrew V A; Yamulla, Robert J; Cosgriff, Sean; Greaves, Bradford D; von Kleeck, Ryan A; An, Hyun Hyung; Powers, Catherine M; Tran, Julie K; Tocker, Aaron M; Jacob, Kimberly D; Davis, Beckley K; Roberts, David M

    2016-06-10

    Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein β-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the β-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate β-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases β-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy. PMID:27068743

  2. Poly(ADP-ribosylation) regulates chromatin organization through histone H3 modification and DNA methylation of the first cell cycle of mouse embryos

    International Nuclear Information System (INIS)

    Highlights: •Histone modification of the mouse pronuclei is regulated by poly(ADP-ribosylation). •Hypermethylation of the mouse female pronuclei is maintained by poly(ADP-ribosylation). •Parp1 is physically interacted with Suz12, which may function in the pronuclei. •Poly(ADP-ribosylation) affects ultrastructure of chromatin of the mouse pronucleus. -- Abstract: We examined the roles of poly(ADP-ribosylation) in chromatin remodeling during the first cell cycle of mouse embryos. Drug-based inhibition of poly(ADP-ribosylation) by a PARP inhibitor, PJ-34, revealed up-regulation of dimethylation of histone H3 at lysine 4 in male pronuclei and down-regulation of dimethylation of histone H3 at lysine 9 (H3K9) and lysine 27 (H3K27). Association of poly(ADP-ribosylation) with histone modification was suggested to be supported by the interaction of Suz12, a histone methyltransferase in the polycomb complex, with Parp1. PARP activity was suggested to be required for a proper localization and maintenance of Suz12 on chromosomes. Notably, DNA methylation level of female pronuclei in one-cell embryos was robustly decreased by PJ-34. Electron microscopic analysis showed a frequent appearance of unusual electron-dense areas within the female pronuclei, implying the disorganized and hypercondensed chromatin ultrastructure. These results show that poly(ADP-ribosylation) is important for the integrity of non-equivalent epigenetic dynamics of pronuclei during the first cell cycle of mouse embryos

  3. Poly(ADP-ribosylation) regulates chromatin organization through histone H3 modification and DNA methylation of the first cell cycle of mouse embryos

    Energy Technology Data Exchange (ETDEWEB)

    Osada, Tomoharu, E-mail: osada.tomoharu@mg.medience.co.jp [Advanced Medical Science Research Department, Mitsubishi Chemical Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaragi 314-0255 (Japan); Department of Regenerative and Developmental Biology, Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida-shi, Tokyo 194-8511 (Japan); Rydén, Anna-Margareta [Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Masutani, Mitsuko, E-mail: mmasutan@ncc.go.jp [Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2013-04-26

    Highlights: •Histone modification of the mouse pronuclei is regulated by poly(ADP-ribosylation). •Hypermethylation of the mouse female pronuclei is maintained by poly(ADP-ribosylation). •Parp1 is physically interacted with Suz12, which may function in the pronuclei. •Poly(ADP-ribosylation) affects ultrastructure of chromatin of the mouse pronucleus. -- Abstract: We examined the roles of poly(ADP-ribosylation) in chromatin remodeling during the first cell cycle of mouse embryos. Drug-based inhibition of poly(ADP-ribosylation) by a PARP inhibitor, PJ-34, revealed up-regulation of dimethylation of histone H3 at lysine 4 in male pronuclei and down-regulation of dimethylation of histone H3 at lysine 9 (H3K9) and lysine 27 (H3K27). Association of poly(ADP-ribosylation) with histone modification was suggested to be supported by the interaction of Suz12, a histone methyltransferase in the polycomb complex, with Parp1. PARP activity was suggested to be required for a proper localization and maintenance of Suz12 on chromosomes. Notably, DNA methylation level of female pronuclei in one-cell embryos was robustly decreased by PJ-34. Electron microscopic analysis showed a frequent appearance of unusual electron-dense areas within the female pronuclei, implying the disorganized and hypercondensed chromatin ultrastructure. These results show that poly(ADP-ribosylation) is important for the integrity of non-equivalent epigenetic dynamics of pronuclei during the first cell cycle of mouse embryos.

  4. Poly(ADP-ribose) polymerase 1 regulates activity of DNA polymerase {beta} in long patch base excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Sukhanova, Maria; Khodyreva, Svetlana [Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk (Russian Federation); Lavrik, Olga, E-mail: lavrik@niboch.nsc.ru [Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk (Russian Federation)

    2010-03-01

    Poly(ADP-ribose)polymerase 1 (PARP1), functioning as DNA nick-sensor, interacts with base excision repair (BER) DNA intermediates containing single-strand breaks. When bound to DNA breaks, PARP1 catalyzes synthesis of poly(ADP-ribose) covalently attached to itself and some nuclear proteins. Autopoly(ADP-ribosyl)ation of PARP1 facilitates its dissociation from DNA breaks and is considered as a factor regulating DNA repair. In the study, using system reconstituted from purified BER proteins, bovine testis nuclear extract and model BER DNA intermediates, we examined the influence of PARP1 and its autopoly(ADP-ribosyl)ation on DNA polymerase {beta} (Pol {beta})-mediated long patch (LP) BER DNA synthesis that is accomplished through a cooperation between Pol {beta} and apurinic/apyrimidinic endonuclease1 (APE1) or flap endonuclease 1 (FEN1) and gap-filling activity of Pol {beta}. PARP1 upon interaction with nicked LP BER DNA intermediated, formed after gap-filling, was shown to suppress the subsequent steps in LP pathway. PARP1 interferes with APE1-dependent stimulation of DNA synthesis by Pol {beta} via strand-displacement mechanism. PARP1 also represses Pol {beta}/FEN1-mediated LP BER DNA synthesis via a 'gap translation' mechanism inhibiting FEN1 activity on the nicked DNA intermediate. Poly(ADP-ribosyl)ation of PARP1 abolishes its inhibitory influence on LP BER DNA synthesis catalyzed by Pol {beta} both via APE1-mediated strand-displacement and FEN1-mediated 'gap translation' mechanism. Thus PARP1 may act as a negative regulator of Pol {beta} activity in LP BER pathway and poly(ADP-ribosyl)ation of PARP1 seems to play a critical role in enablement of Pol {beta}-mediated DNA synthesis in this process. In contrast, interaction of PARP1 with one nucleotide gapped DNA mimicking the intermediate of short patch (SP) BER slightly inhibits the gap-filling activity of Pol {beta} and the overall efficiency of SP BER is practically unaffected by PARP1. Thus

  5. Poly(ADP-ribose) polymerase 1 regulates activity of DNA polymerase β in long patch base excision repair

    International Nuclear Information System (INIS)

    Poly(ADP-ribose)polymerase 1 (PARP1), functioning as DNA nick-sensor, interacts with base excision repair (BER) DNA intermediates containing single-strand breaks. When bound to DNA breaks, PARP1 catalyzes synthesis of poly(ADP-ribose) covalently attached to itself and some nuclear proteins. Autopoly(ADP-ribosyl)ation of PARP1 facilitates its dissociation from DNA breaks and is considered as a factor regulating DNA repair. In the study, using system reconstituted from purified BER proteins, bovine testis nuclear extract and model BER DNA intermediates, we examined the influence of PARP1 and its autopoly(ADP-ribosyl)ation on DNA polymerase β (Pol β)-mediated long patch (LP) BER DNA synthesis that is accomplished through a cooperation between Pol β and apurinic/apyrimidinic endonuclease1 (APE1) or flap endonuclease 1 (FEN1) and gap-filling activity of Pol β. PARP1 upon interaction with nicked LP BER DNA intermediated, formed after gap-filling, was shown to suppress the subsequent steps in LP pathway. PARP1 interferes with APE1-dependent stimulation of DNA synthesis by Pol β via strand-displacement mechanism. PARP1 also represses Pol β/FEN1-mediated LP BER DNA synthesis via a 'gap translation' mechanism inhibiting FEN1 activity on the nicked DNA intermediate. Poly(ADP-ribosyl)ation of PARP1 abolishes its inhibitory influence on LP BER DNA synthesis catalyzed by Pol β both via APE1-mediated strand-displacement and FEN1-mediated 'gap translation' mechanism. Thus PARP1 may act as a negative regulator of Pol β activity in LP BER pathway and poly(ADP-ribosyl)ation of PARP1 seems to play a critical role in enablement of Pol β-mediated DNA synthesis in this process. In contrast, interaction of PARP1 with one nucleotide gapped DNA mimicking the intermediate of short patch (SP) BER slightly inhibits the gap-filling activity of Pol β and the overall efficiency of SP BER is practically unaffected by PARP1. Thus, PARP1 differentially influences DNA synthesis in SP- and

  6. Activation of Reg gene, a gene for insulin-producing β-cell regeneration: Poly(ADP-ribose) polymerase binds Reg promoter and regulates the transcription by autopoly(ADP-ribosyl)ation

    OpenAIRE

    Akiyama, Takako; Takasawa, Shin; Nata, Koji; Kobayashi, Seiichi; Abe, Michiaki; Shervani, Nausheen J.; Ikeda, Takayuki; NAKAGAWA, Kei; Unno, Michiaki; Matsuno, Seiki; Okamoto, Hiroshi

    2000-01-01

    The regeneration of pancreatic islet β cells is important for the prevention and cure of diabetes mellitus. We have demonstrated that the administration of poly(ADP-ribose) synthetase/polymerase (PARP) inhibitors such as nicotinamide to 90% depancreatized rats induces islet regeneration. From the regenerating islet-derived cDNA library, we have isolated Reg (regenerating gene) and demonstrated that Reg protein induces β-cell replication via the Reg receptor and ame...

  7. 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...

  8. 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...

  9. Mice lacking the ADP ribosyl cyclase CD38 exhibit attenuated renal vasoconstriction to angiotensin II, endothelin-1, and norepinephrine

    OpenAIRE

    Thai, Tiffany L.; Arendshorst, William J.

    2009-01-01

    ADP ribosyl (ADPR) cyclases comprise a family of ectoenzymes recently shown to influence cytosolic Ca2+ concentration in a variety of cell types. At least two ADPR cyclase family members have been identified in mammals: CD38 and CD157. We recently found reduced renal vascular reactivity to angiotensin II (ANG II), endothelin-1 (ET-1), and norepinephrine (NE) in the presence of the broad ADPR cyclase inhibitor nicotinamide. We hypothesized that CD38 mediates effects attributed to ADPR cyclase....

  10. Effect of Ad-p16 Combined with CDDP or As2O3 on Human Bladder Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    朱朝辉; 邢诗安; 林晨

    2003-01-01

    Summary: To evaluate the therapeutic efficiency of combined use of p16-expressing adenovirus and chemotherapeutic agents CDDP or As2O3 on human bladder cancer cell line E J, the human bladder cancer cell line EJ were transfected with adenovirus-mediated p16 gene (Ad-p16), with administration of cisplatin (CDDP) or arsenic trioxide (As2O3). The cell growth, morphological changes, cell cycle, apoptosis and molecular changes were measured using cell counting, reverse microscopy, flow cytometry, cloning formation, immunocytochemical assays and in vivo therapy experiments to evaluate the therapeutic efficacy of such combined regimen. Ad-p16 transfer and CDDP or As2O3 administration to EJ cells could exert substantially stronger therapeutic effects than the single agent treatment. Especially in in vivo experiments, combined administration of p16 and CDDP or As2O3 induced almost tumor diminish compared to the partial tumor diminish induced by single agent. Moreover,delivery of Ad-p16, or administration of minimal-dose CDDP or As2O3 or combined regimen could induce massive apoptosis of EJ cell. Cell cycle analysis demonstrated that administration of CDDP or As2O3 remarkably arrested EJ cell in G1 prior to apoptotic cell death. When treated with combined regimen, cells were arrested in G1 to a greater extent prior to apoptotic cell death. It is concluded that after introduction into EJ cell, Ad-p16 shows enhanced therapeutic efficacy for EJ cell when used in combination with CDDP or As2O3.

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

    OpenAIRE

    Cornelia eSpetea; Pfeil, Bernard E.; Benoit eSchoefs

    2012-01-01

    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...

  12. Poly(ADP-Ribose) Polymerase 1 Is Not Strictly Required for Infection of Murine Cells by Retroviruses

    OpenAIRE

    Siva, Amara C; Bushman, Frederic

    2002-01-01

    The DNA-breaking and -joining steps initiating retroviral integration are well understood, but the later steps, thought to be carried out by cellular DNA repair enzymes, have not been fully characterized. Poly(ADP-ribose) polymerase 1 (PARP-1) has been proposed to play a role late during retroviral integration, because infection by human immunodeficiency virus (HIV)-based vectors was reported to be strongly inhibited in PARP-1-deficient fibroblasts. PARP-1, a nuclear enzyme, binds tightly to ...

  13. Inhibition of poly(ADP-ribose) polymerase 1 protects against acute myeloid leukemia by suppressing the myeloproliferative leukemia virus oncogene

    OpenAIRE

    Wang, Lingbo; Cai, Weili; Zhang, Wei; Chen, Xueying; Dong, Wenqian; Tang, Dongqi; Zhang, Yun; Ji, Chunyan; Zhang, Mingxiang

    2015-01-01

    An abnormal expression of poly(ADP-ribose) polymerase 1 (PARP-1) has been described in many tumors. PARP-1 promotes tumorigenesis and cancer progression by acting on different molecular pathways. PARP-1 inhibitors can be used with radiotherapy or chemotherapy to enhance the susceptibility of tumor cells to the treatment. However, the specific mechanism of PARP-1 in acute myeloid leukemia (AML) remains unknown. Our study showed that expression of PARP-1 was upregulated in AML patients. PARP-1 ...

  14. Cisplatin induces primary necrosis through poly(ADP-ribose) polymerase 1 activation in kidney proximal tubular cells

    OpenAIRE

    Park, Seulgee; Yoon, Sang Pil; Kim, Jinu

    2015-01-01

    Treatment with cisplatin for cancer therapy has a major side effect such as nephrotoxicity; however, the role of poly (ADP-ribose) polymerase 1 (PARP1) in necrosis in response to cisplatin nephrotoxicity remains to be defined. Here we report that cisplatin induces primary necrosis through PARP1 activation in kidney proximal tubular cells derived from human, pig and mouse. Treatment with high dose of cisplatin for 4 and 8 hours induced primary necrosis, as represented by the percentage of prop...

  15. Rapid Evolution of PARP Genes Suggests a Broad Role for ADP-Ribosylation in Host-Virus Conflicts

    Science.gov (United States)

    Daugherty, Matthew D.; Young, Janet M.; Kerns, Julie A.; Malik, Harmit S.

    2014-01-01

    Post-translational protein modifications such as phosphorylation and ubiquitinylation are common molecular targets of conflict between viruses and their hosts. However, the role of other post-translational modifications, such as ADP-ribosylation, in host-virus interactions is less well characterized. ADP-ribosylation is carried out by proteins encoded by the PARP (also called ARTD) gene family. The majority of the 17 human PARP genes are poorly characterized. However, one PARP protein, PARP13/ZAP, has broad antiviral activity and has evolved under positive (diversifying) selection in primates. Such evolution is typical of domains that are locked in antagonistic ‘arms races’ with viral factors. To identify additional PARP genes that may be involved in host-virus interactions, we performed evolutionary analyses on all primate PARP genes to search for signatures of rapid evolution. Contrary to expectations that most PARP genes are involved in ‘housekeeping’ functions, we found that nearly one-third of PARP genes are evolving under strong recurrent positive selection. We identified a >300 amino acid disordered region of PARP4, a component of cytoplasmic vault structures, to be rapidly evolving in several mammalian lineages, suggesting this region serves as an important host-pathogen specificity interface. We also found positive selection of PARP9, 14 and 15, the only three human genes that contain both PARP domains and macrodomains. Macrodomains uniquely recognize, and in some cases can reverse, protein mono-ADP-ribosylation, and we observed strong signatures of recurrent positive selection throughout the macro-PARP macrodomains. Furthermore, PARP14 and PARP15 have undergone repeated rounds of gene birth and loss during vertebrate evolution, consistent with recurrent gene innovation. Together with previous studies that implicated several PARPs in immunity, as well as those that demonstrated a role for virally encoded macrodomains in host immune evasion, our

  16. Metabotropic glutamate receptor activation and intracellular cyclic ADP-ribose release Ca2+ from the same store in cultured DRG neurones.

    Science.gov (United States)

    Pollock, J; Crawford, J H; Wootton, J F; Seabrook, G R; Scott, R H

    1999-01-01

    The whole cell patch clamp technique has been used to record Ca(2+)-activated cation and chloride conductances evoked by release of Ca2+ from intracellular stores of cultured neonatal dorsal root ganglion neurones. The aim of this study was to investigate metabotropic glutamate receptor (mGluR) mechanisms and evaluate a possible role for cyclic ADP-ribose as an intracellular signalling molecule. Glutamate and the metabotropic glutamate receptor agonist (1S, 3R)-ACPD-evoked transient depolarizations, Ca(2+)-activated inward currents and rises in intracellular Ca2+. The (1S, 3R)-ACPD-activated currents were insensitive to InsP3 signalling inhibitors, heparin and pentosan polysulphate. Intracellular application of ryanodine alone activated currents in this study and proved a difficult tool to use as a potential inhibitor of cyclic ADP-ribose-mediated responses. However, intracellular dantrolene did attenuate both (1S, 3R)-ACPD and cyclic ADP-ribose responses. Intracellular photo-release of cGMP and cyclic ADP-ribose mimicked the responses to mGluR receptor activation. Intracellular application of nicotinamide and W7 inhibited the responses to photo-released cGMP but did not prevent responses to mGluR activation. The cyclic ADP-ribose receptor antagonist 8-amino cyclic ADP-ribose attenuated responses to (1S, 3R)-ACPD, cGMP and cyclic ADP-ribose, but some Ca(2+)-activated inward currents were still observed in the presence of this antagonist. In conclusion, mGluR receptor activation, cGMP and cyclic ADP-ribose release Ca2+ from intracellular stores. Some evidence suggests that pharmacologically related pathways are involved. PMID:10598278

  17. Identification of an Iron-Sulfur Cluster That Modulates the Enzymatic Activity in NarE, a Neisseria meningitidis ADP-ribosyltransferase*

    OpenAIRE

    Del Vecchio, Mariangela; Pogni, Rebecca; Baratto, Maria Camilla; Nobbs, Angela; Rappuoli, Rino; Pizza, Mariagrazia; Balducci, Enrico

    2009-01-01

    In prokaryotes, mono-ADP-ribose transfer enzymes represent a family of exotoxins that display activity in a variety of bacterial pathogens responsible for causing disease in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report here that NarE, a putative ADP-ribosylating toxin previously identified from Neisseria meningitidis, which shares structural homologies with Escherichia coli heat labile enterotoxin and toxin from Vibrio chole...

  18. Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin.

    OpenAIRE

    Lobet, Y; Cluff, C W; Cieplak, W

    1991-01-01

    Previous studies of the S1 subunit of pertussis toxin, an NAD(+)-dependent ADP-ribosyltransferase, suggested that a small amino-terminal region of amino acid sequence similarity to the active fragments of both cholera toxin and Escherichia coli heat-labile enterotoxin represents a region containing critical active-site residues that might be involved in the binding of the substrate NAD+. Other studies of two other bacterial toxins possessing ADP-ribosyltransferase activity, diphtheria toxin a...

  19. 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.

  20. Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    OpenAIRE

    Guérin Sylvain L; Leclerc Steeve; Desnoyers Serge; Zaniolo Karine

    2007-01-01

    Abstract Background Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to a large array of acceptor proteins, which comprises histones, transcription factors, as well as PARP-1 itself. We have previously demonstrated that transcription of the PARP-1 gene essenti...

  1. Effects of oral contraceptives, or lanosterol, on ADP-induced aggregation and binding of 125I-fibrinogen to rat platelets

    International Nuclear Information System (INIS)

    The aggregation to ADP and the binding of 125I-fibrinogen to platelets from rats treated with oral contraceptives or normal platelets treated in vitro with lanosterol were compared to their respective controls. Both types of platelets showed a significant increase in ADP-induced aggregation and in binding of fibrinogen, indicating that the effect of oral contraceptives could be partly due to increased levels of lanosterol in platelet membrane

  2. The Ratio of ADP- to TRAP-Induced Platelet Aggregation Quantifies P2Y12-Dependent Platelet Inhibition Independently of the Platelet Count

    OpenAIRE

    Olivier, Christoph B.; Meyer, Melanie; Bauer, Hans; Schnabel, Katharina; Weik, Patrick; Zhou, Qian; Bode, Christoph; Moser, Martin; Diehl, Philipp

    2016-01-01

    Objective This study aimed to assess the association of clinical factors with P2Y12-dependent platelet inhibition as monitored by the ratio of ADP- to TRAP-induced platelet aggregation and conventional ADP-induced aggregation, respectively. Background Controversial findings to identify and overcome high platelet reactivity (HPR) after coronary stent-implantation and to improve clinical outcome by tailored anti-platelet therapy exist. Monitoring anti-platelet therapy ex vivo underlies several ...

  3. The ADP-ribose polymerase Tankyrase regulates adult intestinal stem cell proliferation during homeostasis in Drosophila.

    Science.gov (United States)

    Wang, Zhenghan; Tian, Ai; Benchabane, Hassina; Tacchelly-Benites, Ofelia; Yang, Eungi; Nojima, Hisashi; Ahmed, Yashi

    2016-05-15

    Wnt/β-catenin signaling controls intestinal stem cell (ISC) proliferation, and is aberrantly activated in colorectal cancer. Inhibitors of the ADP-ribose polymerase Tankyrase (Tnks) have become lead therapeutic candidates for Wnt-driven cancers, following the recent discovery that Tnks targets Axin, a negative regulator of Wnt signaling, for proteolysis. Initial reports indicated that Tnks is important for Wnt pathway activation in cultured human cell lines. However, the requirement for Tnks in physiological settings has been less clear, as subsequent studies in mice, fish and flies suggested that Tnks was either entirely dispensable for Wnt-dependent processes in vivo, or alternatively, had tissue-specific roles. Here, using null alleles, we demonstrate that the regulation of Axin by the highly conserved Drosophila Tnks homolog is essential for the control of ISC proliferation. Furthermore, in the adult intestine, where activity of the Wingless pathway is graded and peaks at each compartmental boundary, Tnks is dispensable for signaling in regions where pathway activity is high, but essential where pathway activity is relatively low. Finally, as observed previously for Wingless pathway components, Tnks activity in absorptive enterocytes controls the proliferation of neighboring ISCs non-autonomously by regulating JAK/STAT signaling. These findings reveal the requirement for Tnks in the control of ISC proliferation and suggest an essential role in the amplification of Wnt signaling, with relevance for development, homeostasis and cancer. PMID:27190037

  4. Molecular characterization of multiple cDNA clones for ADP-glucose pyrophosphorylase from Arabidopsis thaliana.

    Science.gov (United States)

    Villand, P; Olsen, O A; Kleczkowski, L A

    1993-12-01

    PCR amplification of cDNA prepared from poly(A)+ RNA from aerial parts of Arabidopsis thaliana, using degenerate nucleotide primers based on conserved regions between the large and small subunits of ADP-glucose pyrophosphorylase (AGP), yielded four different cDNAs of ca. 550 nucleotides each. Based on derived amino acid sequences, the identities between the clones varied from 49 to 69%. Sequence comparison to previously published cDNAs for AGP from various species and tissues has revealed that three of the amplified cDNAs (ApL1, ApL2 and ApL3) correspond to the large subunit of AGP, and one cDNA (ApS) encodes the small subunit of AGP. Both ApL1 and ApS were subsequently found to be present in a cDNA library made from Arabidopsis leaves. All four PCR products are encoded by single genes, as found by genomic Southern analysis. PMID:8292792

  5. Poly (ADP-ribose polymerase 1 protein expression in normal and neoplastic prostatic tissue

    Directory of Open Access Journals (Sweden)

    M. Salemi

    2013-04-01

    Full Text Available A genetic background has been implicated in the development of prostate cancer. Protein microarrays have enabled the identification of proteins, some of which associated with apoptosis, that may play a role in the development of such a tumor. Inhibition of apoptosis is a co-factor that contributes to the onset and progression of prostate cancer, though the molecular mechanisms are not entirely understood. Poly (ADP-ribose polymerase 1 (PARP-1 gene is required for translocation of the apoptosis-inducing factor (AIF from the mitochondria to the nucleus. Hence, it is involved in programmed cell death. Different PARP-1 gene expression has been observed in various tumors such as glioblastoma, lung, ovarian, endometrial, and skin cancers. We evaluated the expression of PARP-1 protein in prostatic cancer and normal prostate tissues by immunohistochemistry in 40 men with prostate cancer and in 37 normal men. Positive nuclear PARP-1 staining was found in all samples (normal prostate and prostate cancer tissues. No cytoplasmic staining was observed in any sample. PARP-1-positive cells resulted significantly higher in patients with prostate carcinoma compared with controls (P<0.001. PARP-1 over-expression in prostate cancer tissue compared with normal prostate suggests a greater activity of PARP-1 in these tumors. These findings suggest that PARP-1 expression in prostate cancer is an attempt to trigger apoptosis in this type of tumor similarly to what reported in other cancers.

  6. ADP ribosylation factor like 2 (Arl2 regulates breast tumor aggressivity in immunodeficient mice.

    Directory of Open Access Journals (Sweden)

    Anne Beghin

    Full Text Available We have previously reported that ADP ribosylation factor like 2 (Arl2, a small GTPase, content influences microtubule dynamics and cell cycle distribution in breast tumor cells, as well as the degree and distribution of phosphorylated P53. Here we show, in two different human breast adenocarcinoma models, that Arl2 content has a major impact on breast tumor cell aggressivity both in vitro and in vivo. Cells with reduced content of Arl2 displayed reduced contact inhibition, increased clonogenic or cluster formation as well as a proliferative advantage over control cells in an in vitro competition assay. These cells also caused larger tumors in SCID mice, a phenotype which was mimicked by the in vivo administration of siRNA directed against Arl2. Cells with increased Arl2 content displayed reduced aggressivity, both in vitro and in vivo, with enhanced necrosis and were also found to contain increased PP2A phosphatase activity. A rt-PCR analysis of fresh human tumor breast samples suggested that low Arl2 expression was associated with larger tumor size and greater risk of lymph node involvement at diagnosis. These data underline the role of Arl2, a small GTPase, as an important regulator of breast tumor cell aggressivity, both in vitro and in vivo.

  7. Poly(ADP-ribose) polymerase inhibition reverses vascular dysfunction after γ-irradiation

    International Nuclear Information System (INIS)

    Purpose: The generation of reactive oxygen species during γ-irradiation may induce DNA damage, leading to activation of the nuclear enzyme poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) culminating in endothelial dysfunction. In the present study, we assessed the effect of PARP inhibition on changes in vascular function after acute and short-term irradiation. Methods and Materials: In the acute experiments, aortic rings were exposed to 20 Gy of γ-irradiation. The aortae were harvested after 1 or 7 days. Two additional groups received the ultrapotent PARP inhibitor, INO-1001, for 1 or 7 days after irradiation. The aortic rings were precontracted by phenylephrine and relaxation to acetylcholine and sodium nitroprusside were studied. Results: The vasoconstrictor response to phenylephrine was significantly lower both acutely and 1 and 7 days after irradiation. Vasorelaxation to acetylcholine and sodium nitroprusside was not impaired acutely after irradiation. One and seven days after irradiation, vasorelaxation to acetylcholine and sodium nitroprusside was significantly enhanced. Treatment with INO-1001 reversed vascular dysfunction after irradiation. Conclusion: Vascular dysfunction was observed 1 and 7 days after irradiation, as evidenced by reduced vasoconstriction, coupled with endothelium-dependent and -independent hyperrelaxation. PARP inhibition restored vascular function and may, therefore, be suitable to reverse vascular dysfunction after irradiation

  8. Green Tea Polyphenols Control Dysregulated Glutamate Dehydrogenase in Transgenic Mice by Hijacking the ADP Activation Site

    Energy Technology Data Exchange (ETDEWEB)

    Li, Changhong; Li, Ming; Chen, Pan; Narayan, Srinivas; Matschinsky, Franz M.; Bennett, Michael J.; Stanley, Charles A.; Smith, Thomas J. (CH-PA); (UPENN); (Danforth)

    2012-05-09

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and, in animals, is extensively regulated by a number of metabolites. Gain of function mutations in GDH that abrogate GTP inhibition cause the hyperinsulinism/hyperammonemia syndrome (HHS), resulting in increased pancreatic {beta}-cell responsiveness to leucine and susceptibility to hypoglycemia following high protein meals. We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate that ECG binds to the same site as the allosteric regulator, ADP. Perifusion assays using pancreatic islets from transgenic mice expressing a human HHS form of GDH demonstrate that the hyperresponse to glutamine caused by dysregulated GDH is blocked by the addition of EGCG. As observed in HHS patients, these transgenic mice are hypersensitive to amino acid feeding, and this is abrogated by oral administration of EGCG prior to challenge. Finally, the low basal blood glucose level in the HHS mouse model is improved upon chronic administration of EGCG. These results suggest that this common natural product or some derivative thereof may prove useful in controlling this genetic disorder. Of broader clinical implication is that other groups have shown that restriction of glutamine catabolism via these GDH inhibitors can be useful in treating various tumors. This HHS transgenic mouse model offers a highly useful means to test these agents in vivo.

  9. Sucrose regulation of ADP-glucose pyrophosphorylase subunit genes transcript levels in leaves and fruits

    Science.gov (United States)

    Li, Xiangyang; Xing, Jinpeng; Gianfagna, Thomas J.; Janes, Harry W.

    2002-01-01

    ADP-glucose pyrophosphorylase (AGPase, EC2.7.7.27) is a key regulatory enzyme in starch biosynthesis. The enzyme is a heterotetramer with two S and two B subunits. In tomato, there are three multiple forms of the S subunit gene. Agp S1, S2 and B are highly expressed in fruit from 10 to 25 days after anthesis. Agp S3 is only weakly expressed in fruit. Sucrose significantly elevates expression of Agp S1, S2 and B in both leaves and fruits. Agp S1 exhibits the highest degree of regulation by sucrose. In fact, sucrose may be required for Agp S1 expression. For excised leaves incubated in water, no transcripts for Agp S1 could be detected in the absence of sucrose, whereas it took up to 16 h in water before transcripts were no longer detectable for Agp S2 and B. Neither Agp S3 nor the tubulin gene is affected by sucrose, demonstrating that this response is specifically regulated by a carbohydrate metabolic signal, and is not due to a general increase in metabolism caused by sucrose treatment. Truncated versions of the promoter for Agp S1 indicate that a specific region 1.3-3.0 kb upstream from the transcription site is responsible for sucrose sensitivity. This region of the S1 promoter contains several cis-acting elements present in the promoters of other genes that are also regulated by sucrose. c2002 Elsevier Science Ireland Ltd. All rights reserved.

  10. Investigation of the interaction between the large and small subunits of potato ADP-glucose pyrophosphorylase.

    Directory of Open Access Journals (Sweden)

    Ibrahim Baris

    2009-10-01

    Full Text Available ADP-glucose pyrophosphorylase (AGPase, a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS and small subunits (SS. Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield.

  11. Docking study and binding free energy calculation of poly (ADP-ribose) polymerase inhibitors.

    Science.gov (United States)

    Ohno, Kazuki; Mitsui, Takashi; Tanida, Yoshiaki; Matsuura, Azuma; Fujitani, Hideaki; Niimi, Tatsuya; Orita, Masaya

    2011-02-01

    Recently, the massively parallel computation of absolute binding free energy with a well-equilibrated system (MP-CAFEE) has been developed. The present study aimed to determine whether the MP-CAFEE method is useful for drug discovery research. In the drug discovery process, it is important for computational chemists to predict the binding affinity accurately without detailed structural information for protein/ligand complex. We investigated the absolute binding free energies for Poly (ADP-ribose) polymerase-1 (PARP-1)/inhibitor complexes, using the MP-CAFEE method. Although each docking model was used as an input structure, it was found that the absolute binding free energies calculated by MP-CAFEE are well consistent with the experimental ones. The accuracy of this method is much higher than that using molecular mechanics Poisson-Boltzmann/surface area (MM/PBSA). Although the simulation time is quite extensive, the reliable predictor of binding free energies would be a useful tool for drug discovery projects. PMID:20480380

  12. Mass spectrometry-based functional proteomics of poly(ADP-ribose) polymerase-1.

    Science.gov (United States)

    Pic, Emilie; Gagné, Jean-Philippe; Poirier, Guy G

    2011-12-01

    PARP-1 is an abundant nuclear protein that plays an essential role in the regulation of many genome integrity and chromatin-based processes, such as DNA repair, replication or transcriptional regulation. PARP-1 modulates the function of chromatin and nuclear proteins through several poly(ADP-ribose) (pADPr)-dependent pathways. Aside from the clearly established role of PARP-1 in the maintenance of genome stability, PARP-1 also emerged as an important regulator that links chromatin functions with extranuclear compartments. pADPr signaling has notably been found to be responsible for PARP-1-mediated mitochondrial dysfunction and cell death. Defining the mechanisms that govern the intrinsic functions of PARP-1 is fundamental to the understanding of signaling networks regulated by pADPr. The emergence of mass spectrometry-based proteomics and its broad applications in the study of biological systems represents an outstanding opportunity to widen our knowledge of the functional spectrum of PARP-1. In this article, we summarize various PARP-1 targeted proteomics studies and proteome-wide analyses that shed light on its protein interaction partners, expression levels and post-translational modifications. PMID:22087659

  13. DNA vector-based RNAi approach for stable depletion of poly(ADP-ribose) polymerase-1

    International Nuclear Information System (INIS)

    RNA-mediated interference (RNAi) is a powerful technique that is now being used in mammalian cells to specifically silence a gene. Some recent studies have used this technique to achieve variable extent of depletion of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). These studies reported either transient silencing of PARP-1 using double-stranded RNA or stable silencing of PARP-1 with a DNA vector which was introduced by a viral delivery system. In contrast, here we report that a simple RNAi approach which utilizes a pBS-U6-based DNA vector containing strategically selected PARP-1 targeting sequence, introduced in the cells by conventional CaPO4 protocol, can be used to achieve stable and specific silencing of PARP-1 in different types of cells. We also provide a detailed strategy for selection and cloning of PARP-1-targeting sequences for the DNA vector, and demonstrate that this technique does not affect expression of its closest functional homolog PARP-2

  14. ADP-Ribosylation Factor 1 Regulates Proliferation, Migration, and Fusion in Early Stage of Osteoclast Differentiation

    Directory of Open Access Journals (Sweden)

    Min Jae Kim

    2015-12-01

    Full Text Available Small G-protein adenosine diphosphate (ADP-ribosylation factors (ARFs regulate a variety of cellular functions, including actin cytoskeleton remodeling, plasma membrane reorganization, and vesicular transport. Here, we propose the functional roles of ARF1 in multiple stages of osteoclast differentiation. ARF1 was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL-induced osteoclast differentiation and transiently activated in an initial stage of their differentiation. Differentiation of ARF1-deficient osteoclast precursors into mature osteoclasts temporarily increased in pre-maturation stage of osteoclasts followed by reduced formation of mature osteoclasts, indicating that ARF1 regulates the osteoclastogenic process. ARF1 deficiency resulted in reduced osteoclast precursor proliferation and migration as well as increasing cell-cell fusion. In addition, ARF1 silencing downregulated c-Jun N-terminal kinase (JNK, Akt, osteopontin, and macrophage colony-stimulating factor (M-CSF-receptor c-Fms as well as upregulating several fusion-related genes including CD44, CD47, E-cadherin, and meltrin-α. Collectively, we showed that ARF1 stimulated proliferation and migration of osteoclast precursors while suppressing their fusion, suggesting that ARF1 may be a plausible inter-player that mediates the transition to osteoclast fusion at multiple steps during osteoclast differentiation

  15. 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.

  16. Small G proteins in peroxisome biogenesis: the potential involvement of ADP-ribosylation factor 6

    Directory of Open Access Journals (Sweden)

    Mannaerts Guy P

    2009-08-01

    Full Text Available Abstract Background Peroxisomes execute diverse and vital functions in virtually every eukaryote. New peroxisomes form by budding from pre-existing organelles or de novo by vesiculation of the ER. It has been suggested that ADP-ribosylation factors and COPI coatomer complexes are involved in these processes. Results Here we show that all viable Saccharomyces cerevisiae strains deficient in one of the small GTPases which have an important role in the regulation of vesicular transport contain functional peroxisomes, and that the number of these organelles in oleate-grown cells is significantly upregulated in the arf1 and arf3 null strains compared to the wild-type strain. In addition, we provide evidence that a portion of endogenous Arf6, the mammalian orthologue of yeast Arf3, is associated with the cytoplasmic face of rat liver peroxisomes. Despite this, ablation of Arf6 did neither influence the regulation of peroxisome abundance nor affect the localization of peroxisomal proteins in cultured fetal hepatocytes. However, co-overexpression of wild-type, GTP hydrolysis-defective or (dominant-negative GTP binding-defective forms of Arf1 and Arf6 caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology. Conclusion These observations suggest that Arf6 is a key player in mammalian peroxisome biogenesis. In addition, they also lend strong support to and extend the concept that specific Arf isoform pairs may act in tandem to regulate exclusive trafficking pathways.

  17. Poly (ADP) ribose polymerase inhibition: A potential treatment of malignant peripheral nerve sheath tumor.

    Science.gov (United States)

    Kivlin, Christine M; Watson, Kelsey L; Al Sannaa, Ghadah A; Belousov, Roman; Ingram, Davis R; Huang, Kai-Lieh; May, Caitlin D; Bolshakov, Svetlana; Landers, Sharon M; Kalam, Azad Abul; Slopis, John M; McCutcheon, Ian E; Pollock, Raphael E; Lev, Dina; Lazar, Alexander J; Torres, Keila E

    2016-02-01

    Poly (ADP) ribose polymerase (PARP) inhibitors, first evaluated nearly a decade ago, are primarily used in malignancies with known defects in DNA repair genes, such as alterations in breast cancer, early onset 1/2 (BRCA1/2). While no specific mutations in BRCA1/2 have been reported in malignant peripheral nerve sheath tumors (MPNSTs), MPNST cells could be effectively targeted with a PARP inhibitor to drive cells to synthetic lethality due to their complex karyotype and high level of inherent genomic instability. In this study, we assessed the expression levels of PARP1 and PARP2 in MPNST patient tumor samples and correlated these findings with overall survival. We also determined the level of PARP activity in MPNST cell lines. In addition, we evaluated the efficacy of the PARP inhibitor AZD2281 (Olaparib) in MPNST cell lines. We observed decreased MPNST cell proliferation and enhanced apoptosis in vitro at doses similar to, or less than, the doses used in cell lines with established defective DNA repair genes. Furthermore, AZD2281 significantly reduced local growth of MPNST xenografts, decreased the development of macroscopic lung metastases, and increased survival of mice with metastatic disease. Our results suggest that AZD2281 could be an effective therapeutic option in MPNST and should be further investigated for its potential clinical use in this malignancy. PMID:26650448

  18. Poly (ADP-ribose) polymerases inhibitor, Zj6413, as a potential therapeutic agent against breast cancer.

    Science.gov (United States)

    Zhou, Qin; Ji, Ming; Zhou, Jie; Jin, Jing; Xue, Nina; Chen, Ju; Xu, Bailing; Chen, Xiaoguang

    2016-05-01

    Poly (ADP-ribose) polymerases (PARPs) facilitate repairing of cancer cell DNA damage as a mean to promote cancer proliferation and metastasis. Inhibitors of PARPs which interfering DNA repair, in context of defects in other DNA repair mechanisms, can thus be potentially exploited to inhibit or even kill cancer cells. However, nondiscriminatory inhibition of PARPs, such as PARP2, may lead to undesired consequences. Here, we demonstrated the design and development of the Zj6413 as a potent and selective PARP1 catalytic inhibitor. It trapped PARP1/2 at damaged sites of DNA. As expected, the Zj6413 showed notable anti-tumor activity against breast cancer gene (BRCA) deficient triple negative breast cancers (TNBCs). Zj6413 treated breast cancers (BCs) showed an elevated level of DNA damage evidenced by the accumulation of γ-H2AX foci and DNA damaged related proteins. Zj6413 also induced G2/M arrest and cell death in the MX-1, MDA-MB-453 BC cells, exerted chemo-sensitizing effect on BRCA proficient cancer cells and potentiated Temozolomide (TMZ)'s cytotoxicity in MX-1 xenograft tumors mice. In conclusion, our study provided evidence that a new PARP inhibitor strongly inhibited the catalytic activity of PARPs, trapped them on nicked DNA and damaged the cancer cells, eventually inhibiting the growth of breast tumor cells in vitro and in vivo. PMID:26920250

  19. Non-critical phase-matching fourth harmonic generation of a 1053-nm laser in an ADP crystal.

    Science.gov (United States)

    Ji, Shaohua; Wang, Fang; Zhu, Lili; Xu, Xinguang; Wang, Zhengping; Sun, Xun

    2013-01-01

    In current inertial confinement fusion (ICF) facilities, KDP and DKDP crystals are the second harmonic generation (SHG) and third harmonic generation (THG) materials for the Nd:glass laser (1053 nm). Based on the trend for the development of short wavelengths for ICF driving lasers, technical solutions for fourth harmonic generation (FHG) will undoubtedly attract more and more attention. In this paper, the rapid growth of an ADP crystal and non-critical phase-matching (NCPM) FHG of a 1053-nm laser using an ADP crystal are reported. The NCPM temperature is 33.7°C. The conversion efficiency from 526 to 263 nm is 70%, and the angular acceptance range is 55.4 mrad; these results are superior to those for the DKDP crystals. This research has shown that ADP crystals will be a competitive candidate in future ICF facilities when the utilisation of high-energy, high-efficiency UV lasers at wavelengths shorter than the present 351 nm is of interest. PMID:23549389

  20. Effect of inhibitors of poly(ADP-ribose) polymerase on the heat response of HeLa S3 cells.

    Science.gov (United States)

    Burgman, P; Konings, A W

    1988-12-01

    The purpose of this study was to investigate a possible involvement of poly(ADP-ribosyl)ation reactions in hyperthermic cell killing and hyperthermic DNA strand-break induction and repair in HeLa S3 cells. The inhibitors of poly(ADP-ribose) polymerase, 3-aminobenzamide (3AB) and 4-aminobenzamide (4AB), were used as tools in this study. Both inhibitors could sensitize the cells for hyperthermic cell killing equally well, although 3AB is known to be a more effective enzyme inhibitor. The heat sensitization at the level of cell killing could be reversed when the compounds were still present during a 4-h postincubation at 37 degrees C. More heat-induced DNA strand breaks were formed in the presence of 3AB and 4AB. Repair of strand breaks was inhibited during the postincubation at 37 degrees C. Thus the effect of 3AB and 4AB on DNA strand-break repair was different from the cited effect on cell survival. It is concluded that the sensitizing effect of 3AB and 4AB on hyperthermic cell killing is not caused by inhibition of poly(ADP-ribose) polymerase and is also not related to repair of DNA strand breaks. PMID:3144718

  1. Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis.

    Science.gov (United States)

    Pham, Phuong Anh; Wahl, Vanessa; Tohge, Takayuki; de Souza, Laise Rosado; Zhang, Youjun; Do, Phuc Thi; Olas, Justyna J; Stitt, Mark; Araújo, Wagner L; Fernie, Alisdair R

    2015-11-01

    The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds. PMID:26428915

  2. Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

    Science.gov (United States)

    Min, Wookee; Bruhn, Christopher; Grigaravicius, Paulius; Zhou, Zhong-Wei; Li, Fu; Krüger, Anja; Siddeek, Bénazir; Greulich, Karl-Otto; Popp, Oliver; Meisezahl, Chris; Calkhoven, Cornelis F.; Bürkle, Alexander; Xu, Xingzhi; Wang, Zhao-Qi

    2013-12-01

    Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.

  3. In silico characterization of the family of PARP-like poly(ADP-ribosyltransferases (pARTs

    Directory of Open Access Journals (Sweden)

    Dittmar Katharina

    2005-10-01

    Full Text Available Abstract Background ADP-ribosylation is an enzyme-catalyzed posttranslational protein modification in which mono(ADP-ribosyltransferases (mARTs and poly(ADP-ribosyltransferases (pARTs transfer the ADP-ribose moiety from NAD onto specific amino acid side chains and/or ADP-ribose units on target proteins. Results Using a combination of database search tools we identified the genes encoding recognizable pART domains in the public genome databases. In humans, the pART family encompasses 17 members. For 16 of these genes, an orthologue exists also in the mouse, rat, and pufferfish. Based on the degree of amino acid sequence similarity in the catalytic domain, conserved intron positions, and fused protein domains, pARTs can be divided into five major subgroups. All six members of groups 1 and 2 contain the H-Y-E trias of amino acid residues found also in the active sites of Diphtheria toxin and Pseudomonas exotoxin A, while the eleven members of groups 3 – 5 carry variations of this motif. The pART catalytic domain is found associated in Lego-like fashion with a variety of domains, including nucleic acid-binding, protein-protein interaction, and ubiquitylation domains. Some of these domain associations appear to be very ancient since they are observed also in insects, fungi, amoebae, and plants. The recently completed genome of the pufferfish T. nigroviridis contains recognizable orthologues for all pARTs except for pART7. The nearly completed albeit still fragmentary chicken genome contains recognizable orthologues for twelve pARTs. Simpler eucaryotes generally contain fewer pARTs: two in the fly D. melanogaster, three each in the mosquito A. gambiae, the nematode C. elegans, and the ascomycete microfungus G. zeae, six in the amoeba E. histolytica, nine in the slime mold D. discoideum, and ten in the cress plant A. thaliana. GenBank contains two pART homologues from the large double stranded DNA viruses Chilo iridescent virus and Bacteriophage Aeh1

  4. Sensitization of prostate cancer to ionizing radiation by targeting poly(ADP-robose) polymerase: preclinical studies

    International Nuclear Information System (INIS)

    Poly(ADP-ribose) polymerase (PARP) is a DNA-binding enzyme which plays important roles in the maintenance of genome stability, immediate cellular responses to DNA damage, and apoptosis. A DNA-binding domain of PARP (PARP-DBD) acts as a dominant-negative mutant by binding to DNA strand breaks irreversibly and sensitizing mammalian cells to DNA-damaging agents (1, 2). To direct the expression of human PARP-DBD to prostate we developed recombinant plasmids expressing the PARP-DBD under the control of the 5'-flanking sequences of the human prostate-specific antigen (PSA) gene. In vitro studies revealed that PSA promoter driven expression of the PARP-DBD showed prostate tissue specificity and androgen responsiveness and sensitized LNCaP cells to DNA-damaging agents, such as ionizing radiation and etoposide (3). To assess the efficiency of this strategy in vivo, we developed a cationic liposome-mediated gene delivery of PARP-DBD plasmid in tumor xenografts of PSA producing and androgen sensitive prostate cancer cells (LNCaP and 22Rv1). Tumor bearing mice were treated with intratumoral liposome-complexed PARP-DBD (LE-PARP-DBD), ionizing radiation (IR) or a combination of LE-PARP-DBD and IR. Control groups received blank liposomes or were left untreated. Administration of LE-PARP-DBD resulted in expression of dominant-negative mutant of PARP in tumor cells and enhanced radiation-induced inhibition of tumor growth. These results provide a proof-of- principle for a novel therapeutic strategy to control prostate cancer. The study was supported in part by grants from the U.S. Army Medical Research and Development Command DAMD 17-00-1-0019 and DAMD 17-00-1-0276 (to V.S.). (1) J.Biol.Chem., 265:18721-18724, 1990; (2) Cancer Research, 58: 3495-3498, 1998; (3) Cancer Research, 62: 6879-6883, 2002

  5. 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.

  6. Poly(ADP-ribose) polymerase-1 protects from oxidative stress induced endothelial dysfunction

    International Nuclear Information System (INIS)

    Highlights: ► The nuclear enzyme PARP-1 is a downstream effector of oxidative stress. ► PARP-1 protects from oxidative stress induced endothelial dysfunction. ► This effect is mediated through inhibition of vasoconstrictor prostanoid production. ► Thus, PARP-1 may play a protective role as antioxidant defense mechanism. -- Abstract: Background: Generation of reactive oxygen species (ROS) is a key feature of vascular disease. Activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1) is a downstream effector of oxidative stress. Methods: PARP-1(−/−) and PARP-1(+/+) mice were injected with paraquat (PQ; 10 mg/kg i.p.) to induce intracellular oxidative stress. Aortic rings were suspended in organ chambers for isometric tension recording to analyze vascular function. Results: PQ treatment markedly impaired endothelium-dependent relaxations to acetylcholine in PARP-1(−/−), but not PARP-1(+/+) mice (p < 0.0001). Maximal relaxation was 45% in PQ treated PARP-1(−/−) mice compared to 79% in PARP-1(+/+) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (SNP) were not altered. After PQ treatment, L-NAME enhanced contractions to norepinephrine by 2.0-fold in PARP-1(−/−) mice, and those to acetylcholine by 3.3-fold, respectively, as compared to PARP-1(+/+) mice. PEG-superoxide dismutase (SOD) and PEG-catalase prevented the effect of PQ on endothelium-dependent relaxations to acetylcholine in PARP-1(−/−) mice (p < 0.001 vs. PQ treated PARP-1(+/+) mice. Indomethacin restored endothelium-dependent relaxations to acetylcholine in PQ treated PARP-1(−/−) mice (p < 0.05 vs. PQ treated PARP-1(+/+). Conclusion: PARP-1 protects from acute intracellular oxidative stress induced endothelial dysfunction by inhibiting ROS induced production of vasoconstrictor prostanoids.

  7. Poly(ADP-ribose) polymerase-1 protects from oxidative stress induced endothelial dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Gebhard, Catherine; Staehli, Barbara E. [Cardiovascular Research, Physiology Institute, University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Cardiology, Cardiovascular Center, University Hospital Zurich, Raemistrasse 100, 8091 Zurich (Switzerland); Shi, Yi; Camici, Giovanni G.; Akhmedov, Alexander; Hoegger, Lisa; Lohmann, Christine [Cardiovascular Research, Physiology Institute, University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Matter, Christian M. [Cardiovascular Research, Physiology Institute, University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Cardiology, Cardiovascular Center, University Hospital Zurich, Raemistrasse 100, 8091 Zurich (Switzerland); Hassa, Paul O.; Hottiger, Michael O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Malinski, Tadeusz [Department of Chemistry and Biochemistry, Ohio University, Athens, OH (United States); Luescher, Thomas F. [Cardiovascular Research, Physiology Institute, University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Cardiology, Cardiovascular Center, University Hospital Zurich, Raemistrasse 100, 8091 Zurich (Switzerland); and others

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer The nuclear enzyme PARP-1 is a downstream effector of oxidative stress. Black-Right-Pointing-Pointer PARP-1 protects from oxidative stress induced endothelial dysfunction. Black-Right-Pointing-Pointer This effect is mediated through inhibition of vasoconstrictor prostanoid production. Black-Right-Pointing-Pointer Thus, PARP-1 may play a protective role as antioxidant defense mechanism. -- Abstract: Background: Generation of reactive oxygen species (ROS) is a key feature of vascular disease. Activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1) is a downstream effector of oxidative stress. Methods: PARP-1(-/-) and PARP-1(+/+) mice were injected with paraquat (PQ; 10 mg/kg i.p.) to induce intracellular oxidative stress. Aortic rings were suspended in organ chambers for isometric tension recording to analyze vascular function. Results: PQ treatment markedly impaired endothelium-dependent relaxations to acetylcholine in PARP-1(-/-), but not PARP-1(+/+) mice (p < 0.0001). Maximal relaxation was 45% in PQ treated PARP-1(-/-) mice compared to 79% in PARP-1(+/+) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (SNP) were not altered. After PQ treatment, L-NAME enhanced contractions to norepinephrine by 2.0-fold in PARP-1(-/-) mice, and those to acetylcholine by 3.3-fold, respectively, as compared to PARP-1(+/+) mice. PEG-superoxide dismutase (SOD) and PEG-catalase prevented the effect of PQ on endothelium-dependent relaxations to acetylcholine in PARP-1(-/-) mice (p < 0.001 vs. PQ treated PARP-1(+/+) mice. Indomethacin restored endothelium-dependent relaxations to acetylcholine in PQ treated PARP-1(-/-) mice (p < 0.05 vs. PQ treated PARP-1(+/+). Conclusion: PARP-1 protects from acute intracellular oxidative stress induced endothelial dysfunction by inhibiting ROS induced production of vasoconstrictor prostanoids.

  8. 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.

  9. The different large subunit isoforms of Arabidopsis thaliana ADP-glucose pyrophosphorylase confer distinct kinetic and regulatory properties to the heterotetrameric enzyme.

    Science.gov (United States)

    Crevillén, Pedro; Ballicora, Miguel A; Mérida, Angel; Preiss, Jack; Romero, José M

    2003-08-01

    ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis and is allosterically regulated by the levels of 3-phosphoglycerate and phosphate in plants. ADP-glucose pyrophosphorylases from plants are heterotetramers composed of two types of subunits (small and large). In this study, the six Arabidopsis thaliana genes coding for ADP-glucose pyrophosphorylase isoforms (two small and four large subunits) have been cloned and expressed in an Escherichia coli mutant deficient in ADP-glucose pyrophosphorylase activity. The co-expression of the small subunit APS1 with the different Arabidopsis large subunits (APL1, APL2, APL3, and APL4) resulted in heterotetramers with different regulatory and kinetic properties. Heterotetramers composed of APS1 and APL1 showed the highest sensitivity to the allosteric effectors as well as the highest apparent affinity for the substrates (glucose-1-phosphate and ATP), whereas heterotetramers formed by APS1 and APL2 showed the lower response to allosteric effectors and the lower affinity for the substrates. No activity was detected for the second gene coding for a small subunit isoform (APS2) annotated in the Arabidopsis genome. This lack of activity is possibly due to the absence of essential amino acids involved in catalysis and/or in the binding of glucose-1-phosphate and 3-phosphoglycerate. Kinetic and regulatory properties of the different heterotetramers, together with sequence analysis has allowed us to make a distinction between sink and source enzymes, because the combination of different large subunits would provide a high plasticity to ADP-glucose pyrophosphorylase activity and regulation. This is the first experimental data concerning the role that all the ADP-glucose pyrophosphorylase isoforms play in a single plant species. This phenomenon could have an important role in vivo, because different large subunits would confer distinct regulatory properties to ADP-glucose pyrophosphorylase according

  10. 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

    2013-10-01

    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. PMID:24153004

  11. Crosstalk between SET7/9-dependent methylation and ARTD1-mediated ADP-ribosylation of histone H1.4

    Directory of Open Access Journals (Sweden)

    Kassner Ingrid

    2013-01-01

    Full Text Available Abstract Background Different histone post-translational modifications (PTMs fine-tune and integrate different cellular signaling pathways at the chromatin level. ADP-ribose modification of histones by cellular ADP-ribosyltransferases such as ARTD1 (PARP1 is one of the many elements of the histone code. All 5 histone proteins were described to be ADP-ribosylated in vitro and in vivo. However, the crosstalk between ADP-ribosylation and other modifications is little understood. Results In experiments with isolated histones, it was found that ADP-ribosylation of H3 by ARTD1 prevents H3 methylation by SET7/9. However, poly(ADP-ribosylation (PARylation of histone H3 surprisingly allowed subsequent methylation of H1 by SET7/9. Histone H1 was thus identified as a new target for SET7/9. The SET7/9 methylation sites in H1.4 were pinpointed to the last lysine residues of the six KAK motifs in the C-terminal domain (K121, K129, K159, K171, K177 and K192. Interestingly, H1 and the known SET7/9 target protein H3 competed with each other for SET7/9-dependent methylation. Conclusions The results presented here identify H1.4 as a novel SET7/9 target protein, and document an intricate crosstalk between H3 and H1 methylation and PARylation, thus implying substrate competition as a regulatory mechanism. Thereby, these results underline the role of ADP-ribosylation as an element of the histone code.

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

    International Nuclear Information System (INIS)

    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

  13. 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.

  14. 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

    Science.gov (United States)

    Fedor, Marián; Kovář, František; Galajda, Peter; Bolek, Tomáš; Stančiaková, Lucia; Fedorová, Jana; Staško, Ján; Kubisz, Peter; Mokáň, Marián

    2016-01-01

    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. PMID:27493970

  15. Carcinogen-inflicted DNA damage causes a dramatic increase in the degradation of chromatin-bound poly(ADP-ribose) in mammalian cells

    International Nuclear Information System (INIS)

    A characteristic response of eukaryotic cells to treatment with carcinogens is de novo poly(ADP-ribosylation) of chromatin proteins, a reaction which acts to modulate subsequent DNA excision repair by a hitherto unidentified molecular mechanism. DNA strand breaks represent the molecular signal which activates the chromatin enzyme poly(ADP-ribose) polymerase and thus stimulates poly(ADP-ribose) biosynthesis. They have now observed that carcinogen-inflicted DNA damage may also cause a more than 600-fold stimulation of the degradation of protein-bound poly(ADP-ribose) in chromatin of rat hepatocytes in primary culture. As a consequence, the metabolic half-life of the polymer decreases from 7.7 h in undamaged control cells to 5.5 min and 2.5 min following damage of cells with 45 and 150 J/m2 of UV light of 254 nm, respectively. Similarly, damage of hepatocellular DNA inflicted with either 20, 50 or 200 μM N-methyl-N'-nitro-N-nitrosoguanidine, a monofunctional alkylating agent, caused a dramatic decrease in the polymer half-life to 5.1 min, 2.3 min, and 41 sec, respectively. Therefore, their results suggest that the dynamic removal of polymeric ADP-ribose residues from their chromatin acceptors represents an obligatory postincisional event in DNA excision repair of mammalian cells

  16. Significant decrease of ADP release rate underlies the potent activity of dimethylenastron to inhibit mitotic kinesin Eg5 and cancer cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Linlin [Lung Cancer Institute, Tianjin Medical University General Hospital, 154 Anshan Road, Heping District, Tianjin 300052 (China); Sun, Xiaodong; Xie, Songbo; Yu, Haiyang [Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071 (China); Zhong, Diansheng, E-mail: Zhongdsh@hotmail.com [Lung Cancer Institute, Tianjin Medical University General Hospital, 154 Anshan Road, Heping District, Tianjin 300052 (China); Department of Oncology, Tianjin Medical University General Hospital, 154 Anshan Road, Heping District, Tianjin 300052 (China)

    2014-05-09

    Highlights: • DIMEN displays higher anti-proliferative activity than enastron. • DIMEN induced mitotic arrest and apoptosis more significantly than enastron. • DIMEN blocked the conformational change of ADP-binding pocket more effectively. • DIMEN hindered ADP release more potently than enastron. - Abstract: Eg5 is a mitotic kinesin that plays a crucial role in the formation of bipolar mitotic spindles, by hydrolyzing ATP to push apart anti-parallel microtubules. Dimethylenastron is potent specific small molecule inhibitor of Eg5. The mechanism by which dimethylenastron inhibits Eg5 function remains unclear. By comparing with enastron, here we report that dimethylenastron prevents the growth of pancreatic and lung cancer cells more effectively, by halting mitotic progression and triggering apoptosis. We analyze their interactions with ADP-bound Eg5 crystal structure, and find that dimethylenastron binds Eg5 motor domain with higher affinity. In addition, dimethylenastron allosterically blocks the conformational change of the “sandwich”-like ADP-binding pocket more effectively. We subsequently use biochemical approach to reveal that dimethylenastron slows ADP release more significantly than enastron. These data thus provide biological, structural and mechanistic insights into the potent inhibitory activity of dimethylenastron.

  17. The role of PaAAC1 encoding a mitochondrial ADP/ATP carrier in the biosynthesis of extracellular glycolipids, mannosylerythritol lipids, in the basidiomycetous yeast Pseudozyma antarctica.

    Science.gov (United States)

    Morita, Tomotake; Ito, Emi; Fukuoka, Tokuma; Imura, Tomohiro; Kitamoto, Dai

    2010-07-01

    Pseudozyma antarctica produces large amounts of the glycolipid biosurfactants known as mannosylerythritol lipids (MEL), which show not only excellent surface-active properties but also versatile biochemical actions. A gene homologous with a mitochondrial ADP/ATP carrier was dominantly expressed in P. antarctica under MEL-producing conditions on the basis of previous gene expression analysis. The gene encoding the mitochondrial ADP/ATP carrier of P. antarctica (PaAAC1) contained a putative open reading frame of 954 bp and encodes a polypeptide of 317 amino acids. The deduced translation product shared high identity of 66%, 70%, 69%, 74%, 75% and 52% with the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae (AAC1), S. cerevisiae (AAC2), S. cerevisiae (AAC3), Kluyveromyces lactis (KlAAC), Neurospora crassa (NcAAC) and human (ANT1), respectively, and conserved the consensus sequences of all ADP/ATP carrier proteins. The gene expression by introducing a plasmid pUXV1-PaAAC1 into the yeast cells increased the MEL production. In addition, the expression of PaAAC1 in which the conserved arginine and leucine required for ATP transport activity were replaced with isoleucine and serine, respectively, failed to increase MEL production. Accordingly, these results suggest that PaAAC1 encoding a mitochondrial ADP/ATP carrier should be involved in MEL biosynthesis in the yeast. PMID:20146402

  18. 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)

    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 N2-(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 N2 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

  19. TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase

    OpenAIRE

    Fonfria, Elena; Marshall, Ian C B; Benham, Christopher D; Boyfield, Izzy; Brown, Jason D; Hill, Kerstin; Hughes, Jane P; Skaper, Stephen D.; McNulty, Shaun

    2004-01-01

    TRPM2 (melastatin-like transient receptor potential 2 channel) is a nonselective cation channel that is activated under conditions of oxidative stress leading to an increase in intracellular free Ca2+ concentration ([Ca2+]i) and cell death. We investigated the role of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) on hydrogen peroxide (H2O2)-mediated TRPM2 activation using a tetracycline-inducible TRPM2-expressing cell line.In whole-cell patch-clamp recordings, intracellular adenine...

  20. Dabigatran and rivaroxaban do not affect AA- and ADP-induced platelet aggregation in patients receiving concomitant platelet inhibitors.

    Science.gov (United States)

    Olivier, Christoph B; Weik, Patrick; Meyer, Melanie; Weber, Susanne; Diehl, Philipp; Bode, Christoph; Moser, Martin; Zhou, Qian

    2016-08-01

    Dabigatran and rivaroxaban are novel, vitamin K-independent oral anticoagulants (NOACs) and act via antagonism of the coagulation factor (F) IIa (dabigatran) or FXa (rivaroxaban), respectively. Compared to vitamin-K-antagonists, NOACs have shown non-inferiority of risk and benefit in patients with non valvular atrial fibrillation (AF). In clinical practice there is increasing use of NOACs combined with platelet inhibitors in patients with AF and coronary artery disease. However, whether NOACs affect the function of platelet inhibitors remains incompletely known. This observational study aimed to assess the platelet function in patients receiving dabigatran or rivaroxaban and concomitant platelet inhibitors. A single centre observational study was performed analysing the platelet aggregation of patients treated with dabigatran or rivaroxaban with or without concomitant platelet inhibitors. Measurements before the initiation of NOAC therapy served as the respective control group. Platelet aggregation was measured by multiple electrode aggregometry and was induced with adenosine diphosphate (ADP, 6.5 µM) and arachidonic acid (AA, 0.5 mM), respectively. In order to evaluate whether NOACs interact with platelet inhibition by ASA or the P2Y12-antagonist clopidogrel, 87 patients were grouped according to their concomitant antiplatelet medication. Comparing the ADP- and AA-induced platelet aggregation in patients without concomitant platelet inhibitors (n = 45) no significant differences under therapy with dabigatran (d) or rivaroxaban (r) compared to the control group (c) were observed. In patients taking clopidogrel as a concomitant platelet inhibitor (n = 21), neither dabigatran nor rivaroxaban affected the ADP-induced platelet aggregation (c 20 ± 11, d 21 ± 14, r 18 ± 8 AU*min, p = 0.200). Patients receiving dabigatran or rivaroxaban in combination with ASA (n = 42; 21 ASA only, 21 ASA + clopidogrel) showed no significant differences of the AA

  1. TNP-8N3-ADP photoaffinity labeling of two Na,K-ATPase sequences under separate Na+ plus K+ control.

    Science.gov (United States)

    Ward, Douglas G; Taylor, Mark; Lilley, Kathryn S; Cavieres, José D

    2006-03-14

    ATP has high- and low-affinity effects on the sodium pump and other P-type ATPases. We have approached this question by using 2',3'-O-(trinitrophenyl)-8-azidoadenosine 5'-diphosphate (TNP-8N(3)-ADP) to photoinactivate and label Na,K-ATPase, both in its native state and after covalent FITC block of its high-affinity ATP site. With the native enzyme, the photoinactivation rate constant increases hyperbolically with a K(D(TNP-8N)3(-)(ADP)) of 0.11 microM; TNP-ATP and ATP protect the site with high affinities. The inactivation does not require Na(+), but K(+) inhibits with a K(K)' of 12 microM; Na(+) reverses this effect, with a K(Na) of 0.17 mM. This pattern suggests that Na(+) and K(+) are binding at sites in their "intracellular" conformation. It was known that FITC did not abolish the reverse phosphorylation by P(i), or the K(+)-phosphatase activity, and that TNP-8N(3)-ADP could subsequently photoinactivate the latter with >100-fold lower affinity; in that case, the cation sites acted as if facing outward [Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284, 33759-33765]. Native and FITC-modified enzymes have now been photolabeled with TNP-8N(3)-[alpha-(32)P]ADP and alpha-chain soluble tryptic peptides separated by reverse-phase HPLC. With native Na,K-ATPase, three labeled peaks lead to the unique sequence alpha-(470)Ile-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-X-Tyr-Gln-Leu-Ser-Ile-His-Lys(487), the dropped residue being alphaLys480. With the FITC enzyme, instead, two independent labeling and purification cycles return the sequence alpha-(721)Ala-Asp-Ile-Gly-Val-Ala-Met-Gly-Ile-Ala-Gly-Ser-Asp-Val-Ser-Lys(736). These results suggest that Na,K-ATPase also has a low-affinity nucleotide binding region, one that is under distinctive allosteric control by Na(+) and K(+). Moreover, the cation effects seem compatible with a slow, passive Na(+)/K(+) carrier behavior of the FITC-modified sodium pump. PMID:16519541

  2. Non-critical phase-matching fourth harmonic generation of a 1053-nm laser in an ADP crystal

    OpenAIRE

    Shaohua Ji; Fang Wang; Lili Zhu; Xinguang Xu; Zhengping Wang; Xun Sun

    2013-01-01

    In current inertial confinement fusion (ICF) facilities, KDP and DKDP crystals are the second harmonic generation (SHG) and third harmonic generation (THG) materials for the Nd:glass laser (1053 nm). Based on the trend for the development of short wavelengths for ICF driving lasers, technical solutions for fourth harmonic generation (FHG) will undoubtedly attract more and more attention. In this paper, the rapid growth of an ADP crystal and non-critical phase-matching (NCPM) FHG of a 1053-nm ...

  3. ADP-ribosylation factor and Rho proteins mediate fMLP-dependent activation of phospholipase D in human neutrophils

    OpenAIRE

    Fensome, A.; J. Whatmore; De Morgan, C.; Jones, D.; Cockcroft, S

    1998-01-01

    Activation of intact human neutrophils by fMLP stimulates phospholipase D (PLD) by an unknown signaling pathway. The small GTPase, ADP-ribosylation factor (ARF), and Rho proteins regulate the activity of PLD1 directly. Cell permeabilization with streptolysin O leads Do loss of cytosolic proteins including ARF but not Rho proteins from the human neutrophils, PLD activation by fMLP is refractory in these cytosol-depleted cells. Readdition of myr-ARF1 but not non-myr-ARF1 restores fMLP-stimulate...

  4. ADP-abhängige Acyl-CoA Synthetasen in Archaea, Bacteria und Eukarya: Charakterisierung, Funktion und Phylogenie

    OpenAIRE

    Schmidt, Marcel Clemens

    2013-01-01

    ADP-bildende Acetyl-CoA Synthetasen (ACD) gehören wie die Succinyl-CoA Synthetasen (SCS) zur Proteinfamilie der NDP-bildenden Acyl-CoA Synthetasen. Beide Enzyme sind ubiquitär in allen drei Domänen des Lebens vorhanden und katalysieren die NDP- und Pi-abhängige Umsetzung von Acyl-CoA Estern (Acetyl-CoA bzw. Succinyl-CoA) zu den kor-respondierenden Säuren unter der Bildung von NTP über Substratstufenphosphorylierung. Die ACD-Reaktion wurde erstmals in dem Acetat bildenden Protisten Entamo...

  5. Poly(ADP-ribose) Polymerase-1 (PARP-1) Inhibition Improves Coronary Arteriole Function in Type 2 Diabetes

    OpenAIRE

    Choi, Soo-Kyoung; Galán, Maria; Kassan, Modar; Partyka, Megan; Trebak, Mohamed; Matrougui, Khalid

    2012-01-01

    Type 2 diabetes (T2D) is associated with microvascular dysfunction. We hypothesized that increased Poly - (ADP-ribose) polymerase-1 (PARP-1) activity contributes to microvascular dysfunction in T2D. T2D (db-/db-) and non-diabetic control (db-/db+) mice were treated with two different PARP-1 inhibitors (INO-1001, 5 mg/Kg/day and ABT-888, 15 mg/Kg/day) for two weeks. Isolated coronary arterioles (CA) were mounted in an arteriograph. Pressure-induced myogenic tone (MT) was significantly potentia...

  6. SIRT1 promotes cell survival under stress by deacetylation-dependent deactivation of poly(ADP-ribose) polymerase 1

    OpenAIRE

    Rajamohan, S B; V. B. Pillai; Gupta, M.; Sundaresan, N R; Birukov, K G; Samant, S.; Hottiger, M O; Gupta, M P

    2009-01-01

    Poly(ADP-ribose) polymerase 1 (PARP1) and SIRT1 deacetylase are two NAD-dependent enzymes which play major roles in the decision of a cell to live or to die in a stress situation. Because of the dependence of both enzymes on NAD, cross talk between them has been suggested. Here, we show that PARP1 is acetylated after stress of cardiomyocytes, resulting in the activation of PARP1, which is independent of DNA damage. SIRT1 physically binds to and deacetylates PARP1. Increased acetylation of PAR...

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

    OpenAIRE

    Fernet Marie; Giocanti Nicole; Noël Georges; Mégnin-Chanet Frédérique; Favaudon Vincent

    2003-01-01

    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...

  8. PIASy Mediates SUMO-2/3 Conjugation of Poly(ADP-ribose) Polymerase 1 (PARP1) on Mitotic Chromosomes*

    OpenAIRE

    Ryu, Hyunju; Al-Ani, Gada; Deckert, Katelyn; Kirkpatrick, Donald; Gygi, Steven P.; Dasso, Mary; Azuma, Yoshiaki

    2010-01-01

    PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstitu...

  9. Direct phosphorylation and regulation of poly(ADP-ribose) polymerase-1 by extracellular signal-regulated kinases 1/2

    OpenAIRE

    Kauppinen, Tiina M; Chan, Wai Y.; Suh, Sang Won; Wiggins, Amanda K.; Eric J. Huang; Swanson, Raymond A.

    2006-01-01

    Sustained activation of poly(ADP-ribose) polymerase-1 (PARP-1) and extracellular signal-regulated kinases 1/2 (ERK1/2) both promote neuronal death. Here we identify a direct link between these two cell death pathways. In a rat model of hypoglycemic brain injury, neuronal PARP-1 activation and subsequent neuronal death were blocked by the ERK1/2 inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059). In neuron cultures, PARP-1-mediated neuronal death induced by N-methyl-d-aspart...

  10. p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates

    OpenAIRE

    Dutto, Ilaria; Sukhanova, Maria; Tillhon, Micol; Cazzalini, Ornella; Stivala, Lucia A.; Scovassi, A. Ivana; Lavrik, Olga; Prosperi, Ennio

    2016-01-01

    The cell cycle inhibitor p21CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates contain...

  11. Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair, chromosomal alterations, and mutations.

    Science.gov (United States)

    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, we have measured the influence of 3AB on DNA repair 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. Both acentric fragments and exchanges increase indicating that the presence of 3AB slows down the repair of DNA strand breaks (probably DNA double strand breaks), thus making breaks available for interaction with each other to give rise to exchanges. 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. Most 3AB induced SCEs occur during the second cell cycle, in which DNA containing bromouridine (BU) is used as template for replication. BU containing DNA appears to be prone to errors during replication. The extent of increase in the frequencies of SCEs by 3AB is correlated with the amount of BU incorporated in the DNA of the cells. The frequencies of spontaneously occurring DNA single strand breaks in cells grown in BrdUrd containing medium are higher than in the cells grown in normal medium and this increase depends on the amount of BU incorporated in the DNA of these cells. We have studied the extent of increase in the frequencies of SCEs due to 1 mM 3AB in several human cell lines, 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

  12. 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

  13. 聚(ADP-核糖)聚合酶-1在糖尿病神经病变中的作用%Role of poly(ADP-ribose) polymerase-1 in diabetic neuropathy

    Institute of Scientific and Technical Information of China (English)

    项舟弘; 苏青

    2010-01-01

    聚(ADP-核糖)聚合酶(PARP)-1是一类具有重要生理功能的核酶,它在糖尿病神经病变的发病中发挥重要作用.PARP-1活化导致NAD~+耗竭、能量衰竭、转录调控和基因表达发生改变、3-磷酸甘油醛脱氧酶受抑制,从而参与糖尿病神经病变的发生、发展.多个动物实验显示PARP-1抑制剂对糖尿病神经病变有改善作用,为治疗糖尿病神经病变的新药研发指明了方向.%Poly(ADP-ribose) polymerase (PARP)-1 is a nuclear enzyme with multiple physiologi-cal functions,emerging as a fundamental mechanism in the pathogenesis of diabetic neuropathy.PARP-1 acti-vation results in NAD~+ depletion, energy failure, changes of transcriptional regulation and gene expression,in-hibition of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase,which towards the pathways im-plicated in diabetic neuropathy.Several animal tests showed that treatment with PARP-1 inhibitor could im-prove diabetic neuropathy, so it may be a new spot in new drug research.

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

    International Nuclear Information System (INIS)

    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

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

    International Nuclear Information System (INIS)

    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

  16. The structure of Aquifex aeolicus FtsH in the ADP-bound state reveals a C2-symmetric hexamer.

    Science.gov (United States)

    Vostrukhina, Marina; Popov, Alexander; Brunstein, Elena; Lanz, Martin A; Baumgartner, Renato; Bieniossek, Christoph; Schacherl, Magdalena; Baumann, Ulrich

    2015-06-01

    The crystal structure of a truncated, soluble quadruple mutant of FtsH from Aquifex aeolicus comprising the AAA and protease domains has been determined at 2.96 Å resolution in space group I222. The protein crystallizes as a hexamer, with the protease domain forming layers in the ab plane. Contacts between these layers are mediated by the AAA domains. These are highly disordered in one crystal form, but are clearly visible in a related form with a shorter c axis. Here, adenosine diphosphate (ADP) is bound to each subunit and the AAA ring exhibits twofold symmetry. The arrangement is different from the ADP-bound state of an analogously truncated, soluble FtsH construct from Thermotoga maritima. The pore is completely closed and the phenylalanine residues in the pore line a contiguous path. The protease hexamer is very similar to those described for other FtsH structures. To resolve certain open issues regarding a conserved glycine in the linker between the AAA and protease domains, as well as the active-site switch β-strand, mutations have been introduced in the full-length membrane-bound protein. Activity analysis of these point mutants reveals the crucial importance of these residues for proteolytic activity and is in accord with previous interpretation of the active-site switch and the importance of the linker glycine residue. PMID:26057670

  17. 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.

  18. [Effect of ADP and GroES on interaction of molecular chaperonin GroEL with non-native lysozyme].

    Science.gov (United States)

    Marchenko, N Iu; Marchenkov, V V; Kotova, N V; Semisotnov, G V; Bulankina, N I; Kaliman, P A

    2003-01-01

    The interaction of the molecular chaperonin GroEL with fluorescein-labeled lysozyme in the presence of high concentrations of thiol reagent--dithiothreitol (DTT) has been studied. In case of high concentrations of DTT lysozyme loses the native conformation due to the disruption of the intramolecular disulfide bonds stabilizing its structure and effectively aggregates. It has been shown that in the presence of high concentrations of DTT and two-fold molar excess of GroEL the lysozyme tightly interacts with GroEL that essentially decreases the efficiency of its aggregation. The addition of ADP to the complex of GroEL with nonnative lysozyme noticeably decreases the interaction of the chaperonin with nonnative protein target resulting in some increase of the efficiency of its aggregation. However, the addition of the co-chaperonin GroES together with ADP (i.e. the formation of the complex of GroEL with GroES) leads to drastic weakness of the interaction of GroEL with nonnative lysozyme and the efficiency of its aggregation becomes comparable with that in the absence of GroEL. PMID:14577157

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Ya-Chen; Hsu, Chiao-Yu [Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan (China); Yao, Ya-Li [Department of Biotechnology, Asia University, Taichung 41354, Taiwan (China); Yang, Wen-Ming, E-mail: yangwm@nchu.edu.tw [Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan (China)

    2013-02-01

    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.

  1. Poly-ADP-ribosylation-mediated degradation of ARTD1 by the NLRP3 inflammasome is a prerequisite for osteoclast maturation

    Science.gov (United States)

    Wang, C; Qu, C; Alippe, Y; Bonar, S L; Civitelli, R; Abu-Amer, Y; Hottiger, M O; Mbalaviele, G

    2016-01-01

    Evidence implicates ARTD1 in cell differentiation, but its role in skeletal metabolism remains unknown. Osteoclasts (OC), the bone-resorbing cells, differentiate from macrophages under the influence of macrophage colony-stimulating factor (M-CSF) and receptor-activator of NF-κB ligand (RANKL). We found that M-CSF induced ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1) auto-ADP-ribosylation in macrophages, a modification that marked ARTD1 for cleavage, and subsequently, for degradation upon RANKL exposure. We established that ARTD1 proteolysis was NLRP3 inflammasome-dependent, and occurred via the proteasome pathway. Since ARTD1 is cleaved at aspartate214, we studied the impact of ARTD1 rendered uncleavable by D214N substitution (ARTD1D214N) on skeletal homeostasis. ARTD1D214N, unlike wild-type ARTD1, was resistant to cleavage and degradation during osteoclastogenesis. As a result, ARTD1D214N altered histone modification and promoted the abundance of the repressors of osteoclastogenesis by interfering with the expression of B lymphocyte-induced maturation protein 1 (Blimp1), the master regulator of anti-osteoclastogenic transcription factors. Importantly, ARTD1D214N-expressing mice exhibited higher bone mass compared with controls, owing to decreased osteoclastogenesis while bone formation was unaffected. Thus, unless it is degraded, ARTD1 represses OC development through transcriptional regulation. PMID:27010854

  2. Growth of ADP-KDP mixed crystal and its optical, mechanical, dielectric, piezoelectric and laser damage threshold studies

    Science.gov (United States)

    Rajesh, P.; Ramasamy, P.; Bhagavannarayana, G.

    2013-01-01

    Good quality ADP-KDP mixed crystal (90:10) is grown by slow cooling method. The size of the grown crystal is 80×10×10 mm3. The mounted seed size was 5×10×10 mm3 and the crystal was grown along the 'c' axis. HRXRD studies have been done in the near and far regions of the seed crystal. The FWHM of these diffraction curves are 28 and 29 arcsec, which are almost the same. The close values of FWHM of both the specimens indicate that the quality of the crystal remains nearly the same throughout the crystal. 80% of transparency is observed from the UV-vis studies in the entire visible region. Vickers hardness studies indicate that the mixed crystal is mechanically more stable compared to the ADP. Higher piezoelectric coefficient is observed in mixed crystals. Dielectric measurements are carried out. From the laser damage threshold studies, it is observed that higher energy is required to damage the mixed crystal and it indicates that the laser stability of the mixed crystal is high.

  3. Poly(ADP-ribose) polymerase activation induces high mobility group box 1 release from proximal tubular cells during cisplatin nephrotoxicity.

    Science.gov (United States)

    Kim, J

    2016-06-20

    Cisplatin is one of the most potent chemotherapy drugs against cancer, but its major side effect such as nephrotoxicity limits its use. Inhibition of poly(ADP-ribose) polymerase (PARP) protects against various renal diseases via gene transactivation and/or ADP-ribosylation. However, the role of PARP in necrotic cell death during cisplatin nephrotoxicity remains an open question. Here we demonstrated that pharmacological inhibition of PARP by postconditioning dose-dependently prevented tubular injury and renal dysfunction following cisplatin administration in mice. PARP inhibition by postconditioning also attenuated ATP depletion during cisplatin nephrotoxicity. Systemic release of high mobility group box 1 (HMGB1) protein in plasma induced by cisplatin administration was significantly diminished by PARP inhibition by postconditioning. In in vitro kidney proximal tubular cell lines, PARP inhibition by postconditioning also diminished HMGB1 release from cells. These data demonstrate that cisplatin-induced PARP1 activation contributes to HMGB1 release from kidney proximal tubular cells, resulting in the promotion of inflammation during cisplatin nephrotoxicity. PMID:26447520

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Vladimir N. Anisimov

    2008-04-01

    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.; P< .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.

  6. Poly(ADP-Ribose) Polymerase 1 and Ste20-Like Kinase hKFC Act as Transcriptional Repressors for Gamma-2 Herpesvirus Lytic Replication

    OpenAIRE

    Gwack, Yousang; Nakamura, Hiroyuki; Lee, Sun Hwa; Souvlis, John; Yustein, Jason T.; Gygi, Steve; Kung, Hsing-Jien; Jung, Jae U.

    2003-01-01

    The replication and transcription activator (RTA) of gamma-2 herpesvirus is sufficient to drive the entire virus lytic cycle. Hence, the control of RTA activity should play an important role in the maintenance of viral latency. Here, we demonstrate that cellular poly(ADP-ribose) polymerase 1 (PARP-1) and Ste20-like kinase hKFC interact with the serine/threonine-rich region of gamma-2 herpesvirus RTA and that these interactions efficiently transfer poly(ADP-ribose) and phosphate units to RTA. ...

  7. Identification and characterization of xcpR encoding a subunit of the general secretory pathway necessary for dodecane degradation in Acinetobacter calcoaceticus ADP1.

    OpenAIRE

    Parche, S; Geissdörfer, W.; Hillen, W

    1997-01-01

    A mutant of Acinetobacter calcoaceticus ADP1 unable to grow on alkanes was complemented for growth on hexadecane with a DNA fragment encoding a protein with homology to XcpR, a subunit of the general secretion pathway for exoproteins in Pseudomonas aeruginosa. Insertional inactivation of xcpR in A. calcoaceticus ADP1 by transcriptional fusion to lacZ abolishes secretion of lipase and esterase and leads to lack of growth on dodecane and slower growth on hexadecane. We, therefore, propose the p...

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

    International Nuclear Information System (INIS)

    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.)

  9. Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene

    Directory of Open Access Journals (Sweden)

    Kirschner Ralf D

    2008-10-01

    Full Text Available Abstract Background Mono-ADP-ribosyltransferase (ART 1 belongs to a family of mammalian ectoenzymes that catalyze the transfer of ADP-ribose from NAD+ to a target protein. ART1 is predominantly expressed in skeletal and cardiac muscle. It ADP-ribosylates α7-integrin which together with β1-integrin forms a dimer and binds to laminin, a protein of the extracellular matrix involved in cell adhesion. This posttranslational modification leads to an increased laminin binding affinity. Results Using C2C12 and C3H-10T 1/2 cells as models of myogenesis, we found that ART1 expression was restricted to myotube formation. We identified a fragment spanning the gene 1.3 kb upstream of the transcriptional start site as the functional promoter of the ART1 gene. This region contains an E box and an A/T-rich element, two conserved binding sites for transcription factors found in the promoters of most skeletal muscle specific genes. Mutating the DNA consensus sequence of either the E box or the A/T-rich element resulted in a nearly complete loss of ART1 promoter inducibility, indicating a cooperative role of the transcription factors binding to those sites. Gel mobility shift analyses carried out with nuclear extracts from C2C12 and C3H-10T 1/2 cells revealed binding of myogenin to the E box and MEF-2 to the A/T-rich element, the binding being restricted to C2C12 and C3H-10T 1/2 myotubes. Conclusion Here we describe the molecular mechanism underlying the regulation of the ART1 gene expression in skeletal muscle cells. The differentiation-dependent upregulation of ART1 mRNA is induced by the binding of myogenin to an E box and of MEF-2 to an A/T-rich element in the proximal promoter region of the ART1 gene. Thus the transcriptional regulation involves molecular mechanisms similar to those used to activate muscle-specific genes.

  10. Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure

    International Nuclear Information System (INIS)

    Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21WAF1/CIP1 expression, normally a block to cell cycle progression after DNA damage, is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 μM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 μM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.

  11. A specific isoform of poly(ADP-ribose) glycohydrolase is targeted to the mitochondrial matrix by a N-terminal mitochondrial targeting sequence

    International Nuclear Information System (INIS)

    Poly(ADP-ribose) polymerases (PARPs) convert NAD to polymers of ADP-ribose that are converted to free ADP-ribose by poly(ADP-ribose) glycohydrolase (PARG). The activation of the nuclear enzyme PARP-1 following genotoxic stress has been linked to release of apoptosis inducing factor from the mitochondria, but the mechanisms by which signals are transmitted between nuclear and mitochondrial compartments are not well understood. The study reported here has examined the relationship between PARG and mitochondria in HeLa cells. Endogenous PARG associated with the mitochondrial fraction migrated in the range of 60 kDa. Transient transfection of cells with PARG expression constructs with amino acids encoded by exon 4 at the N-terminus was targeted to the mitochondria as demonstrated by subcellular fractionation and immunofluorescence microscopy of whole cells. Deletion and missense mutants allowed identification of a canonical N-terminal mitochondrial targeting sequence consisting of the first 16 amino acids encoded by PARG exon 4. Sub-mitochondrial localization experiments indicate that this mitochondrial PARG isoform is targeted to the mitochondrial matrix. The identification of a PARG isoform as a component of the mitochondrial matrix raises several interesting possibilities concerning mechanisms of nuclear-mitochondrial cross talk involved in regulation of cell death pathways.

  12. Pre-exposure to heat shock inhibits peroxynitrite-induced activation of poly(ADP) ribosyltransferase and protects against peroxynitrite cytotoxicity in J774 macrophages.

    Science.gov (United States)

    Szabó, C; Wong, H R; Salzman, A L

    1996-11-14

    The reaction of nitric oxide (NO) with superoxide yields the cytotoxic oxidant peroxynitrite, produced during inflammation and shock. A novel pathway of peroxynitrite cytotoxicity involves activation of the nuclear enzyme poly(ADP) ribosyltransferase, and concomitant ADP-ribosylation. NAD+ consumption and exhaustion of intracellular energy stores. In the present report we provide evidence that pre-exposure of J774 macrophages to heat shock reduces peroxynitrite-induced activation of poly(ADP) ribosyltransferase and protects against the peroxynitrite-induced suppression of mitochondrial respiration. The protection was significant at 8 h after heat shock, but was absent at 24 h after heat shock. Thus, the protection showed a temporal correlation with the expression of heat shock protein 70, the expression of which was maximal at 8 h. Exposure to heat shock did not alter the expression of poly(ADP) ribosyltransferase over 24 h. In summary, the heat shock phenotype or heat shock proteins may protect against peroxynitrite induced cytotoxicity. PMID:8960887

  13. 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.; Costelloe, T.; Romeijn, R.J.; Pastink, A.; van Attikum, H.; Marteijn, J.A.; Vermeulen, W.; Sroczynski, Nicholas; Mailand, N.

    2013-01-01

    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...

  14. Current Status of Poly(ADP-ribose Polymerase Inhibitors as Novel Therapeutic Agents for Triple-Negative Breast Cancer

    Directory of Open Access Journals (Sweden)

    David J. Hiller

    2012-01-01

    Full Text Available Triple-negative breast cancer (TNBC is an aggressive type of breast cancer that is clinically defined as lacking estrogen and progesterone receptors, as well as being ERBB2 (HER-2 negative. Without specific therapeutic targets, TNBC carries a worse prognosis than other types of breast cancer in the absence of therapy. Research has now further differentiated breast cancer into subtypes based on genetic expression patterns. One of these subtypes, basal-like, frequently overlaps with the clinical picture of TNBC. Additionally, both TNBC and basal-like breast cancer link to BRCA mutations. Recent pharmaceutical advances have created a class of drugs, poly(ADP-ribose polymerase (PARP inhibitors, which are showing potential to effectively treat these patients. The aim of this paper is to summarize the basis behind PARP inhibitors and update the current status of their development in clinical trials for the treatment of TNBC.

  15. ADP-glucose pyrophosphorylase is localized to both the cytoplasm and plastids in developing pericarp of tomato fruit

    Science.gov (United States)

    Chen, B. Y.; Wang, Y.; Janes, H. W.

    1998-01-01

    The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.

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

    International Nuclear Information System (INIS)

    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. 31P 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. ADP-glucose pyrophosphorylase gene plays a key role in the quality of corm and yield of cormels in gladiolus.

    Science.gov (United States)

    Seng, Shanshan; Wu, Jian; Sui, Juanjuan; Wu, Chenyu; Zhong, Xionghui; Liu, Chen; Liu, Chao; Gong, Benhe; Zhang, Fengqin; He, Junna; Yi, Mingfang

    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. PMID:27107698

  18. 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

  19. 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.

  20. Transcriptional Reprogramming and Resistance to Colonic Mucosal Injury in Poly(ADP-ribose) Polymerase 1 (PARP1)-deficient Mice.

    Science.gov (United States)

    Larmonier, Claire B; Shehab, Kareem W; Laubitz, Daniel; Jamwal, Deepa R; Ghishan, Fayez K; Kiela, Pawel R

    2016-04-22

    Poly(ADP-ribose) polymerases (PARPs) synthesize and bind branched polymers of ADP-ribose to acceptor proteins using NAD as a substrate and participate in the control of gene transcription and DNA repair. PARP1, the most abundant isoform, regulates the expression of proinflammatory mediator cytokines, chemokines, and adhesion molecules, and inhibition of PARP1 enzymatic activity reduced or ameliorated autoimmune diseases in several experimental models, including colitis. However, the mechanism(s) underlying the protective effects of PARP1 inhibition in colitis and the cell types in which Parp1 deletion has the most significant impact are unknown. The objective of the current study was to determine the impact of Parp1 deletion on the innate immune response to mucosal injury and on the gut microbiome composition. Parp1 deficiency was evaluated in DSS-induced colitis in WT, Parp1(-/-), Rag2(-/-), and Rag2(-/-)×Parp1(-/-) double knock-out mice. Genome-wide analysis of the colonic transcriptome and fecal 16S amplicon profiling was performed. Compared with WT, we demonstrated that Parp1(-/-) were protected from dextran-sulfate sodium-induced colitis and that this protection was associated with a dramatic transcriptional reprogramming in the colon. PARP1 deficiency was also associated with a modulation of the colonic microbiota (increases relative abundance of Clostridia clusters IV and XIVa) and a concomitant increase in the frequency of mucosal CD4(+)CD25(+) Foxp3(+) regulatory T cells. The protective effects conferred by Parp1 deletion were lost in Rag2(-/-) × Parp1(-/-) mice, highlighting the role of the adaptive immune system for full protection. PMID:26912654

  1. Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin.

    Science.gov (United States)

    Lobet, Y; Cluff, C W; Cieplak, W

    1991-01-01

    Previous studies of the S1 subunit of pertussis toxin, an NAD(+)-dependent ADP-ribosyltransferase, suggested that a small amino-terminal region of amino acid sequence similarity to the active fragments of both cholera toxin and Escherichia coli heat-labile enterotoxin represents a region containing critical active-site residues that might be involved in the binding of the substrate NAD+. Other studies of two other bacterial toxins possessing ADP-ribosyltransferase activity, diphtheria toxin and Pseudomonas exotoxin A, have revealed the presence of essential glutamic acid residues vicinal to the active site. To help determine the relevance of these observations to activities of the enterotoxins, the A-subunit gene of the E. coli heat-labile enterotoxin was subjected to site-specific mutagenesis in the region encoding the amino-terminal region of similarity to the S1 subunit of pertussis toxin delineated by residues 6 through 17 and at two glutamic acid residues, 110 and 112, that are conserved in the active domains of all of the heat-labile enterotoxin variants and in cholera toxin. Mutant proteins in which arginine 7 was either deleted or replaced with lysine exhibited undetectable levels of ADP-ribosyltransferase activity. However, limited trypsinolysis of the arginine 7 mutants yielded fragmentation kinetics that were different from that yielded by the wild-type recombinant subunit or the authentic A subunit. In contrast, mutant proteins in which glutamic acid residues at either position 110 or 112 were replaced with aspartic acid responded like the wild-type subunit upon limited trypsinolysis, while exhibiting severely depressed, but detectable, ADP-ribosyltransferase activity. The latter results may indicate that either glutamic acid 110 or glutamic acid 112 of the A subunit of heat-labile enterotoxin is analogous to those active-site glutamic acids identified in several other ADP-ribosylating toxins. Images PMID:1908825

  2. Gi- and Gq-coupled ADP (P2Y receptors act in opposition to modulate nociceptive signaling and inflammatory pain behavior

    Directory of Open Access Journals (Sweden)

    Molliver Derek C

    2010-04-01

    Full Text Available Abstract Background Investigations of nucleotide signaling in nociception to date have focused on actions of adenosine triphosphate (ATP. Both ATP-gated ion channels (P2X receptors and G protein-coupled (P2Y receptors contribute to nociceptive signaling in peripheral sensory neurons. In addition, several studies have implicated the Gq-coupled adenosine diphosphate (ADP receptor P2Y1 in sensory transduction. In this study, we examined the expression and function of P2Y1 and the Gi-coupled receptors P2Y12, P2Y13 and P2Y14 in sensory neurons to determine their contribution to nociception. Results We detected mRNA and protein for ADP receptors P2Y12 and P2Y13 in mouse dorsal root ganglia (DRG. P2Y14, a homologous Gi-coupled nucleotide receptor, is also expressed in DRG. Immunohistochemical analysis of receptor distribution indicated that these receptors are widely expressed in nociceptive neurons. Using ratiometric calcium imaging, we found that ADP evokes increases in intracellular calcium in isolated DRG neurons and also produces a pertussis toxin-sensitive inhibition of depolarization-evoked calcium transients. The inhibitory effect of ADP was unaltered in the presence of the selective P2Y1 antagonist MRS2179 and in neurons isolated from P2Y1 knockout mice, whereas ADP-evoked calcium transients were greatly reduced. Analysis of behavioral responses to noxious heat before and after inflammatory injury (injection of complete Freund's adjuvant into the hindpaw revealed that P2Y1 is required for the full expression of inflammatory hyperalgesia, whereas local injection of agonists for Gi-coupled P2Y receptors reduced hyperalgesia. Conclusions We report that Gi-coupled P2Y receptors are widely expressed in peripheral sensory neurons. Agonists for these receptors inhibit nociceptive signaling in isolated neurons and reduce behavioral hyperalgesia in vivo. Anti-nociceptive actions of these receptors appear to be antagonized by the Gq-coupled ADP receptor

  3. 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)

    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 370C at an enzyme:substrate ratio of 1:100 in buffer solution containing 20 mM Tris-HCl, pH 7.6, 10 mM KCl, 1 mM MgCl2, 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-14C] nicotinaide adenine dinucleotide ([14C]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 [14C] ADP-ribosylated components, the gels were dried and exposed with RM-V x-ray film

  4. Effect and Mechanism of Radiosensitization of Poly (ADP-Ribose Polymerase 
Inhibitor on Lewis Cells and Xenografts

    Directory of Open Access Journals (Sweden)

    Wei WANG

    2016-01-01

    Full Text Available Background and objective The DNA damage of the irradiated tumor cells is mainly single strand breaks (SSBs and double strand breaks (DSBs, in which the frequency of occurrence of SSBs is dozens of times than DSBs. However, most of the SSBs could be repaired by the Poly (ADP-Ribose Polymerase (PARP and other related factors. Recently listed drug-Olaparib (PARP1/PARP2/PARP3 inhibitor could target the repair pathways of single strand breaks, and recent clinical trials of PARP inhibitors combined with chemotherapy obtained encouraging results. The aim of this study is to investigate the effect and potential mechanism of radiosensitization of Poly (ADP-Ribose polymerase inhibitor-Olaparib on lewis cells and xenografts. Methods The inhibition concentration 10% inhibitory concentration (IC10 of Olaparib to Lewis cells was detected by methyl thiazolyltetrazolium (MTT assay. The radiosensitization effect of Olaparib on Lewis cells was determined by classical colony forming assay. Lewis xenografts models were established, and the mice were randomly divided into four groups: Control group, Olaparib group, Radiotherapy group (RT, 2 Gy×5 d, Olaparib combined with RT group. Xenograft volume was measured during the treatment. Flow cytometry was used to analyze the apoptosis rate of the Lewis cells in each group, and the apoptosis of xenograft tissues was observed by TUNEL stain. The ralative protein levels of γH2AX (associated with DNA strand breaks repair, Bax/Bcl-2, Caspase-3 (apoptosis-associated protein were detected by Western blot in vitro and in vivo. Results The IC10 value of Olaparib was 4.4 µmol/L. The radio-sensitivity enhancement ratio (SER of Olaparib combined with RT was 1.211 in vitro. Compared with RT (2 Gy×5 d alone, the combination of Olaparib with fractionated radiotherapy significantly increased the growth delay of Lewis xenografts (P<0.001. Flow cytometry and TUNEL analysis indicated that the apoptosis rate in the combination group was

  5. Possible Association of HLA-DRB1 Gene with the Autoantibody against Myocardial Mitochondria ADP/ATP Carrier in Dilated Cardiomyopathy

    Institute of Scientific and Technical Information of China (English)

    王秋芬; 廖玉华; 龚非力; 毛焕元; 张金枝

    2002-01-01

    Summary: To probe the genetic background and immunopathogenesis of dilated cardiomyopathy (DCM) 77 patients with DCM, HLA-DRB1 gene polymorphism were analyzed by using the polymerase chain reaction /sequence specific primer (PCR/SSP) technique and autoantibody against myocardial mitochondria ADP/ATP carrier were examined by using the Immunoblot analysis. The frequency of HLA-DRB1 * 0901 allele was significantly higher in DCM patients in which autoantibody against ADP/ATP carrier of myocardial mitochondria is positive in contrast with those in which the autoantibody is negative (25.46 % vs 3.45 %, P<0.05), the relative risk (RR) being 9.56. The other frequencies of HLA-DRB1 alleles have no significant difference in the antibody positive group and negative group. It is possible that a subset of DCM patients may exist in which autoimmunity is associated with genetic factors.

  6. Experimental and numerical investigation of ADP square crystal with large aperture in the new Final Optics Assembly under the non-critical phase matching

    Science.gov (United States)

    Sun, Fuzhong; Zhang, Peng; Bai, Qingshun; Lu, Lihua; Xiang, Yong

    2016-04-01

    This paper presented a new Final Optics Assembly (FOA) of ammonium dihydrogen phosphate (ADP) square crystal with large aperture under the non-critical phase matching (NCPM), which controlled by the constant temperature water, and the temperature distribution was analyzed by simulation and experiment. Firstly, thermal analysis was carried out, as well as the temperature distribution of the cavity only heated under different velocities was analyzed. Then, the temperature distributions of ADP square crystal in the cavity were achieved using the Finite Volume Method (FVM), and this prediction was validated by the experiment results when the velocity is 0.1 m/s and 0.5 m/s. Finally, the optimal FHG conversion efficiency was obtained and the comparison of different heating methods was also highlighted.

  7. Increased poly(ADP-ribosyl)ation in skeletal muscle tissue of pediatric patients with severe burn injury: prevention by propranolol treatment

    OpenAIRE

    Oláh, Gábor; Finnerty, Celeste; Sbrana, Elena; Elijah, Itoro; Gerö, Domokos; Herndon, David; Szabó, Csaba

    2011-01-01

    Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) has been shown to promote cellular energetic collapse and cellular necrosis in various forms of critical illness. Most of the evidence implicating the PARP pathway in disease processes is derived from preclinical studies. With respect to PARP and burns, studies in rodent and large animal models of burn injury have demonstrated the activation of PARP in various tissues and the beneficial effect of its pharmacological inhibiti...

  8. An ADP-Ribosylation Factor GTPase-activating Protein Git2-short/KIAA0148 Is Involved in Subcellular Localization of Paxillin and Actin Cytoskeletal Organization

    OpenAIRE

    Mazaki, Yuichi; Hashimoto, Shigeru; Okawa, Katsuya; Tsubouchi, Asako; Nakamura, Kuniaki; Yagi, Ryohei; Yano, Hajime; Kondo, Akiko; Iwamatsu, Akihiro; Mizoguchi, Akira; Sabe, Hisataka

    2001-01-01

    Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin...

  9. Respiration of mitochondria in living organisms is controlled by the ATP/ADP ratio and phosphorylation pattern of cytochrome c oxidase

    OpenAIRE

    Ramzan, Rabia

    2010-01-01

    The "second mechanism of respiratory control" (allosteric ATP-inhibition of cytochrome c oxidase (CcO)) is demonstrated for the first time in intact isolated rat liver and heart mitochondria. The problems of measuring the kinetics of allosteric ATP-inhibition in isolated mitochondria were investigated. And it was found that only at very high ATP/ADP ratios, this inhibition is obtained and requires an ATP-regenerating system consis...

  10. Poly(ADP-ribose) Polymerase 1 Is Indispensable for Transforming Growth Factor-β Induced Smad3 Activation in Vascular Smooth Muscle Cell

    OpenAIRE

    Dan Huang; Yan Wang; Lin Wang; Fengxiao Zhang; Shan Deng; Rui Wang; Yun Zhang; Kai Huang

    2011-01-01

    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 trans...

  11. BIG2, A Guanine Nucleotide Exchange Factor for ADP-Ribosylation Factors: Its Localization to Recycling Endosomes and Implication in the Endosome IntegrityD⃞

    OpenAIRE

    Shin, Hye-Won; Morinaga, Naoko; NODA, Masatoshi; Nakayama, Kazuhisa

    2004-01-01

    Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is a...

  12. Modulation of Poly(ADP-Ribose) Polymerase-1 (PARP-1)-Mediated Oxidative Cell Injury by Ring Finger Protein 146 (RNF146) in Cardiac Myocytes

    OpenAIRE

    Gerö, Domokos; Szoleczky, Petra; Chatzianastasiou, Athanasia; Papapetropoulos, Andreas; Szabo, Csaba

    2014-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) activation is a hallmark of oxidative stress–induced cellular injury that can lead to energetic failure and necrotic cell death via depleting the cellular nicotinamide adenine dinucleotide (NAD+) and ATP pools. Pharmacological PARP-1 inhibition or genetic PARP-1 deficiency exert protective effects in multiple models of cardiomyocyte injury. However, the connection between nuclear PARP-1 activation and depletion of the cytoplasmic and mitochondrial energy...

  13. Transcription regulation of TNF-α-early response genes by poly(ADP-ribose) polymerase-1 in murine heart endothelial cells

    OpenAIRE

    Carrillo, Ana; Monreal, Yolanda; Ramírez, Pablo; Marín, Luis; Parrilla, Pascual; Oliver, Francisco Javier; Yélamos, José

    2004-01-01

    [EN] Poly(ADP-ribose) polymerase-1 (PARP-1) has been involved in endothelial cell dysfunction associated with various pathophysiological conditions. The intrinsic mechanism of PARP-1-mediated endothelial cell dysfunction could be related to PARP-1 overactivation, NAD+ consumption and ATP depletion. An alternative way could involve transcription regulation. By using high-density microarrays, we examined early tumor necrosis factor α(TNF- α)- stimulated gene expressio...

  14. 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...

  15. NF-κB transcriptional activation by TNFα requires phospholipase C, extracellular signal-regulated kinase 2 and poly(ADP-ribose) polymerase-1

    OpenAIRE

    Vuong, Billy; Hogan-Cann, Adam D. J.; Alano, Conrad C.; Stevenson, Mackenzie; Chan, Wai Yee; Anderson, Christopher M.; Swanson, Raymond A.; Kauppinen, Tiina M

    2015-01-01

    Background The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is required for pro-inflammatory effects of TNFα. Our previous studies demonstrated that PARP-1 mediates TNFα-induced NF-κB activation in glia. Here, we evaluated the mechanisms by which TNFα activates PARP-1 and PARP-1 mediates NF-κB activation. Methods Primary cultures of mouse cortical astrocytes and microglia were treated with TNFα and suitable signaling pathway modulators (pharmacological and molecular). Outcome measure...

  16. Insights into Glycogen Metabolism in Chemolithoautotrophic Bacteria from Distinctive Kinetic and Regulatory Properties of ADP-Glucose Pyrophosphorylase from Nitrosomonas europaea

    OpenAIRE

    Machtey, Matías; Kuhn, Misty L.; Flasch, Diane A; Aleanzi, Mabel; Ballicora, Miguel A; Iglesias, Alberto A.

    2012-01-01

    Nitrosomonas europaea is a chemolithoautotroph that obtains energy by oxidizing ammonia in the presence of oxygen and fixes CO2 via the Benson-Calvin cycle. Despite its environmental and evolutionary importance, very little is known about the regulation and metabolism of glycogen, a source of carbon and energy storage. Here, we cloned and heterologously expressed the genes coding for two major putative enzymes of the glycogen synthetic pathway in N. europaea, ADP-glucose pyrophosphorylase and...

  17. Vault Poly(ADP-Ribose) Polymerase Is Associated with Mammalian Telomerase and Is Dispensable for Telomerase Function and Vault Structure In Vivo

    OpenAIRE

    Liu, Yie; Snow, Bryan E.; Kickhoefer, Valerie A; Erdmann, Natalie; Zhou, Wen; Wakeham, Andrew; Gomez, Marla; Rome, Leonard H.; Harrington, Lea

    2004-01-01

    Vault poly(ADP-ribose) polymerase (VPARP) was originally identified as a minor protein component of the vault ribonucleoprotein particle, which may be involved in molecular assembly or subcellular transport. In addition to the association of VPARP with the cytoplasmic vault particle, subpopulations of VPARP localize to the nucleus and the mitotic spindle, indicating that VPARP may have other cellular functions. We found that VPARP was associated with telomerase activity and interacted with ex...

  18. Poly(ADP-ribose) polymerase 1 inhibition protects human aortic endothelial cells against LPS-induced inflammation response

    Institute of Scientific and Technical Information of China (English)

    Xiaonu Peng; Wenjun Li; Wei Zhang

    2012-01-01

    Atherosclerosis is a chronic inflammatory disease.Tolllike receptor 4 (TLR4) is an important signaling receptor and plays a critical role in the inflammatory response.Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that can regulate the expression of various inflammatory genes.In this study,we investigated the role and the underlying mechanisms of PARP1 on lipopolysaccharide (LPS)-induced inflammation in human aortic endothelial cells.Compared with the control,LPS stimulation increased the protein expression of TLR4 and PARP1.TLR4 inhibition reduced LPS-induced upregulation of inducible nitric oxide synthase (iNOS) and ICAM-1 as well as PARP1. Nuclear factor κB (NF-κB) inhibition decreased ICAM-1 and iNOS expression.Inhibition of PARP1 decreased protein expression of inflammatory cytokines induced by LPS stimulation,probably through preventing NF-KB nuclear translocation. Our study demonstrated that LPS increased ICAM-1 and iNOS expression via TLR4/PARP1/NF-KB pathway.PARP1 might be an indispensable factor in TLR4-mediated inflammation after LPS stimulation.PARP1 inhibition might shed light on the treatment of LPS-induced inflammatory cytokines expression during atherosclerosis.

  19. The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity.

    Science.gov (United States)

    Iordanov, Iordan; Mihályi, Csaba; Tóth, Balázs; Csanády, László

    2016-01-01

    Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel essential for immunocyte activation, insulin secretion, and postischemic cell death. TRPM2 is activated by ADP ribose (ADPR) binding to its C-terminal cytosolic NUDT9-homology (NUDT9H) domain, homologous to the soluble mitochondrial ADPR pyrophosphatase (ADPRase) NUDT9. Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme, but insolubility of isolated NUDT9H hampered further investigations. Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9. When expressed in E.coli, chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified. In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity (kcat~4-9s(-1)), that of NUDT9H did not support catalysis. Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating (K1/2~1-5 μM), confirming functionality of chimeric domains. Thus, TRPM2 is not a 'chanzyme'. Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies. PMID:27383051

  20. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: Two guanine nucleotide-dependent activators of cholera toxin

    International Nuclear Information System (INIS)

    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

  1. Continuous inhibition of poly(ADP-ribose) polymerase does not reduce reperfusion injury in isolated rat heart.

    Science.gov (United States)

    Nishizawa, Kenya; Yanagida, Shigeki; Yamagishi, Tadashi; Takayama, Eiichi; Bessho, Motoaki; Kusuhara, Masatoshi; Adachi, Takeshi; Ohsuzu, Fumitaka

    2013-07-01

    Poly(ADP-ribose) polymerase (PARP), an enzyme that is important to the regulation of nuclear function, is activated by DNA strand breakage. In massive DNA damage, PARP is overactivated, exhausting nicotinamide adenine dinucleotide and leading to cell death. Recent studies have succeeded in reducing cellular damage in ischemia/reperfusion by inhibiting PARP. However, PARP plays an important part in the DNA repair system, and its inhibition may be hazardous in certain situations. We compared the short-time inhibition of PARP against continuous inhibition during ischemia/reperfusion using isolated rat hearts. The hearts were reperfused after 21 minutes of ischemia with a bolus injection of 3-aminobenzamide (3-AB) (10 mg/kg) followed by continuous 3-AB infusion (50 μM) for the whole reperfusion period or for the first 6 minutes or without 3-AB. At the end of reperfusion, contractile function, high-energy phosphate content, nicotinamide adenine dinucleotide content, and infarcted area were significantly preserved in the 3-AB 6-minute group. In the 3-AB continuous group, these advantages were not apparent. At the end of reperfusion, PARP cleavage had significantly proceeded in the 3-AB continuous group, indicating initiation of the apoptotic cascade. Thus, continuous PARP inhibition by 3-AB does not reduce reperfusion injury in the isolated rat heart, which may be because of acceleration of apoptosis. PMID:23846805

  2. ADP-ribosylation Factor-related Protein 1 Interacts with NS5A and Regulates Hepatitis C Virus Propagation.

    Science.gov (United States)

    Lim, Yun-Sook; Ngo, Huong T T; Lee, Jihye; Son, Kidong; Park, Eun-Mee; Hwang, Soon B

    2016-01-01

    The life cycle of hepatitis C virus (HCV) is tightly coupled to the lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have previously screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet (LD) formation using cell culture-grown HCV (HCVcc)-infected cells. In this study, we selected and characterized the gene encoding ADP-ribosylation factor-related protein 1 (ARFRP1). ARFRP1 is essential for LD growth and is involved in the regulation of lipolysis. siRNA-mediated knockdown of ARFRP1 significantly inhibited HCV replication in both subgenomic replicon cells and HCVcc-infected cells. ARFRP1 interacted with NS5A and NS5A partially colocalized with LD. Silencing of ARFRP1 abrogated HCV-induced LD growth and viral protein expressions. Moreover, ARFRP1 recruited synaptosomal-associated protein 23 (SNAP23) to sites in close proximity to LDs in HCV-infected cells. Silencing of ARFRP1 ablated relocalization of SNAP23 to LD. These data indicate that HCV regulates ARFRP1 for LD growth to facilitate viral propagation and thus ARFRP1 may be a potential target for antiviral therapy. PMID:27550144

  3. 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.

  4. Effect of meta-iodo-benzylguanine, inhibitor of mono (ADP-ribosylation), on X-irradiated L5178Y cells

    International Nuclear Information System (INIS)

    We examined the effect of meta-iodo-benzylguanine (MIBG), an inhibitor of mono (ADP-ribosylation), on growth and clonogenic ability of X-irradiated L5178Y-R (LY-R, radiation resistant) and L5178Y-S (LY-S, radiation sensitive) murine lymphoma cells. We also measured free Ca2+ concentration in LY cells and in human lymphocytes, with the fluorescent calcium chelator, Fura-2. We found no radiosensitization by MIBG in either LY sublines, whereas free Ca2+ concentration increased in MIBG-treated and X-irradiated LY-S cells, as compared to X-irradiated only; in X-irradiated LY-R cells MIGB did not modify the concentration of Ca2+. We conclude that there is no direct relation between the level of free Ca2+, as determined by fluorescent chelator, and the lethal effect of X-irradiation. We also discuss possible artifacts as reasons of incorrect estimation of intracellular Ca2+ concentration. (author). 36 refs, 3 figs, 1 tab

  5. 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. PMID:24576433

  6. A high-throughput screening-compatible homogeneous time-resolved fluorescence assay measuring the glycohydrolase activity of human poly(ADP-ribose) glycohydrolase.

    Science.gov (United States)

    Stowell, Alexandra I J; James, Dominic I; Waddell, Ian D; Bennett, Neil; Truman, Caroline; Hardern, Ian M; Ogilvie, Donald J

    2016-06-15

    Poly(ADP-ribose) (PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed and, from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition. PMID:27036617

  7. Crystallization and preliminary X-ray analysis of molecular chaperone-like diol dehydratase-reactivating factor in ADP-bound and nucleotide-free forms

    International Nuclear Information System (INIS)

    The molecular chaperone-like reactivating factor for adenosylcobalamin (coenzyme B12) dependent diol dehydratase was crystallized in ADP-bound and nucleotide-free forms. Preliminary X-ray analysis indicated that crystals are orthorhombic and diffract to 2.0 Å. Adenosylcobalamin (coenzyme B12) dependent diol dehydratase (EC 4.2.1.28) catalyzes the conversion of 1,2-diols and glycerol to the corresponding aldehydes. It undergoes mechanism-based inactivation by glycerol. The diol dehydratase-reactivating factor (DDR) reactivates the inactivated holoenzymes in the presence of adenosylcobalamin, ATP and Mg2+ by mediating the release of a damaged cofactor. This molecular chaperone-like factor was overexpressed in Escherichia coli, purified and crystallized in the ADP-bound and nucleotide-free forms by the sandwich-drop vapour-diffusion method. The crystals of the ADP-bound form belong to the orthorhombic system, with space group P212121 and unit-cell parameters a = 83.26, b = 84.60, c = 280.09 Å, and diffract to 2.0 Å. In the absence of nucleotide, DDR crystals were orthorhombic, with space group P212121 and unit-cell parameters a = 81.92, b = 85.37, c = 296.99 Å and diffract to 3.0 Å. Crystals of both forms were suitable for structural analysis

  8. Poly(ADP-ribosylation acts in the DNA demethylation of mouse primordial germ cells also with DNA damage-independent roles.

    Directory of Open Access Journals (Sweden)

    Fabio Ciccarone

    Full Text Available Poly(ADP-ribosylation regulates chromatin structure and transcription driving epigenetic events. In particular, Parp1 is able to directly influence DNA methylation patterns controlling transcription and activity of Dnmt1. Here, we show that ADP-ribose polymer levels and Parp1 expression are noticeably high in mouse primordial germ cells (PGCs when the bulk of DNA demethylation occurs during germline epigenetic reprogramming in the embryo. Notably, Parp1 activity is stimulated in PGCs even before its participation in the DNA damage response associated with active DNA demethylation. We demonstrate that PARP inhibition impairs both genome-wide and locus-specific DNA methylation erasure in PGCs. Moreover, we evidence that impairment of PARP activity causes a significant reduction of expression of the gene coding for Tet1 hydroxylases involved in active DNA demethylation. Taken together these results demonstrate new and adjuvant roles of poly(ADP-ribosylation during germline DNA demethylation and suggest its possible more general involvement in genome reprogramming.

  9. Prokaryotic expression and subcellular localization of ADP/ATP carrier protein in microsporidian Nosema bombycis%家蚕微孢子虫ADP/ATP转运蛋白的原核表达及间接免疫荧光定位分析

    Institute of Scientific and Technical Information of China (English)

    党晓群; 林立鹏; 李春峰; 潘国庆; 李田; 龙梦娴; 周泽扬

    2014-01-01

    [目的]家蚕微孢子虫Nosema bombycis ADP/ATP转运蛋白可能参与搬运宿主细胞的能量.本研究克隆家蚕微孢子虫ADP/ATP转运蛋白基因,并进行原核表达、抗体制备及间接免疫荧光定位,为控制和防治家蚕微粒子病提供理论基础.[方法]通过同源序列比对鉴定家蚕微孢子虫N.bombycis ADP/ATP转运蛋白序列,采用生物合成的方法将编码3段面向膜内侧肽段的核酸序列拼接合成,在其两端引入Bglll和SalⅠ酶切位点,克隆至pUC57载体并测序,再亚克隆至含有二氢叶酸还原酶(dihydrofolate reductase,DHFR)标签的表达载体pQE40中,然后利用BamH Ⅰ和Sal Ⅰ酶切获得含有DHFR标签的重组序列,并连接至pET30a(+)载体中进行诱导表达.通过SDSPAGE、镍柱亲和层析和免疫印迹法鉴定表达蛋白,利用间接免疫荧光对ADP/ATP转运蛋白的分布进行检测.[结果]家蚕微孢子虫的ADP/ATP转运蛋白编码序列(GenBank登录号为EOB13854.1)全长1 524 bp,编码蛋白含有507个氨基酸残基,预测分子质量为59 kDa,等电点为9.35.具有12个跨膜结构域和TLC结构域,其中TLC结构域含有4个功能保守位点.与蜜蜂微孢子虫的ADP/ATP转运蛋白比较,氨基酸序列一致性达30%.系统进化分析表明微孢子虫ADP/ATP转运蛋白聚为一类,具有共同的起源.成功构建了NbADP/ATP-△TM-DHFR-pET30a原核表达重组质粒,目的基因获得表达,其融合蛋白分子量约为37 kDa,纯化重组蛋白并制备了多克隆抗体.免疫印迹分析表明,成熟微孢子虫中表达ADP/ATP转运蛋白;间接免疫荧光定位结果显示,家蚕微孢子虫孢子ADP/ATP转运蛋白定位于孢子质膜上.[结论]本研究将为阻断微孢子虫能量来源,达到控制和防治家蚕微粒子病提供新的思路.

  10. High residual platelet reactivity (HRPR) for adenosine diphosphate (ADP) stimuli is a determinant factor for long-term outcomes in acute ischemic stroke with anti-platelet agents: The meaning of HRPR after ADP might be more prominent in large atherosclerotic infarction than other subtypes of AIS.

    Science.gov (United States)

    Cha, Jae-Kwan; Park, Hyun-Seok; Nah, Hyun-Wook; Kim, Dae-Hyun; Kang, Myong-Jin; Choi, Jae-Hyung; Huh, Jae-Taeck; Suh, Hyun-Kyung

    2016-07-01

    High residual platelet activation (HRPA) after ADP stimuli has associated with recurrent vascular events in acute atherothrombosis with the use of antiplatelet agents (APAs). However, there has been little evidence supporting this association in acute ischemic stroke (AIS). In this study, we evaluated the influences of HRPR after ADP stimuli on the 1-year incidence of recurrent cardiovascular events and mortality in AIS with APAs. We conducted an observational, referral center cohort study on 968 AIS patients with APAs from January 2010 to December 2013 who were evaluated using optical platelet aggregometry (OPA). All patients received the dual APA combination of aspirin and clopidogrel or aspirin alone. We evaluated their platelet function 5 days after hospital admission using OPA. HRPR after ADP stimuli was defined as platelet aggregation of 70 % or greater according to OPA after 10 µM ADP stimuli. The primary endpoint was a composite of all causes of death, myocardial infarction, and stroke at the 1-year follow-up. The secondary endpoints were each component of the primary endpoint. The event rate of primary endpoint was 11.3 % (109/968). Its rate was significantly higher in the patients with HRPR (16.7 %) than in those without (9.7 %). HPRP was independently associated with the primary endpoint (OR = 1.97, CI 1.22-3.18, p < 0.01). According to the AIS subtype, the presence of HRPR was independently significant for the occurrence of the primary endpoint in the large artery atherosclerosis (LAA) subtype only (OR = 2.26, CI 1.15-4.45, p = 0.02). In this study, the presence of HRPR after ADP stimuli is associated with a poor long-term outcome after acute ischemic stroke. In particular, the influence of this factor might be more prominent in LAA compared with other types of AIS. PMID:26680778

  11. Latonduine Analogs Restore F508del-Cystic Fibrosis Transmembrane Conductance Regulator Trafficking through the Modulation of Poly-ADP Ribose Polymerase 3 and Poly-ADP Ribose Polymerase 16 Activity.

    Science.gov (United States)

    Carlile, Graeme W; Robert, Renaud; Matthes, Elizabeth; Yang, Qi; Solari, Roberto; Hatley, Richard; Edge, Colin M; Hanrahan, John W; Andersen, Raymond; Thomas, David Y; Birault, Véronique

    2016-08-01

    Cystic fibrosis (CF) is a major lethal genetic disease caused by mutations in the CF transmembrane conductance regulator gene (CFTR). This encodes a chloride ion channel on the apical surface of epithelial cells. The most common mutation in CFTR (F508del-CFTR) generates a protein that is misfolded and retained in the endoplasmic reticulum. Identifying small molecules that correct this CFTR trafficking defect is a promising approach in CF therapy. However, to date only modest efficacy has been reported for correctors in clinical trials. We identified the marine sponge metabolite latonduine as a corrector. We have now developed a series of latonduine derivatives that are more potent F508del-CFTR correctors with one (MCG315 [2,3-dihydro-1H-2-benzazepin-1-one]) having 10-fold increased corrector activity and an EC50 of 72.25 nM. We show that the latonduine analogs inhibit poly-ADP ribose polymerase (PARP) isozymes 1, 3, and 16. Further our molecular modeling studies point to the latonduine analogs binding to the PARP nicotinamide-binding domain. We established the relationship between the ability of the latonduine analogs to inhibit PARP-16 and their ability to correct F508del-CFTR trafficking. We show that latonduine can inhibit both PARP-3 and -16 and that this is necessary for CFTR correction. We demonstrate that latonduine triggers correction by regulating the activity of the unfolded protein response activator inositol-requiring enzyme (IRE-1) via modulation of the level of its ribosylation by PARP-16. These results establish latonduines novel site of action as well as its proteostatic mechanism of action. PMID:27193581

  12. 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. PMID:20435371

  13. Structure of spinach acetohydroxyacid isomeroreductase complexed with its reaction product dihydroxymethylvalerate, manganese and (phospho)-ADP-ribose.

    Science.gov (United States)

    Thomazeau, K; Dumas, R; Halgand, F; Forest, E; Douce, R; Biou, V

    2000-04-01

    Acetohydroxyacid isomeroreductase catalyses a two-step reaction composed of an alkyl migration followed by an NADPH-dependent reduction. Both steps require a divalent cation and the first step has a strong preference for magnesium. Manganese ions are highly unfavourable to the reaction: only 3% residual activity is observed in the presence of this cation. Acetohydroxyacid isomeroreductase has been crystallized with its substrate, 2-aceto-2-hydroxybutyrate (AHB), Mn(2+) and NADPH. The 1.6 A resolution electron-density map showed the reaction product (2,3-dihydroxy-3-methylvalerate, DHMV) and a density corresponding to (phospho)-ADP-ribose instead of the whole NADP(+). This is one of the few structures of an enzyme complexed with its reaction product. The structure of this complex was refined to an R factor of 19.3% and an R(free) of 22.5%. The overall structure of the enzyme is very similar to that of the complex with the reaction-intermediate analogue IpOHA [N-hydroxy-N-isopropyloxamate; Biou et al. (1997), EMBO J. 16, 3405-3415]. However, the active site shows some differences: the nicotinamide is cleaved and the surrounding amino acids have rearranged accordingly. Comparison between the structures corresponding to the reaction intermediate and to the end of the reaction allowed the proposal of a reaction scheme. Taking this result into account, the enzyme was crystallized with Ni(2+) and Zn(2+), for which only 0.02% residual activity were measured; however, the crystals of AHB/Zn/NADPH and of AHB/Ni/NADPH also contain the reaction product. Moreover, mass-spectrometry measurements confirmed the -cleavage of nicotinamide. PMID:10739911

  14. 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.

  15. 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.

  16. 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.

  17. Effect of ADP on the orientation of spin-labeled myosin heads in muscle fibers: A high-resolution study with deuterated spin labels

    International Nuclear Information System (INIS)

    We have used electron paramagnetic resonance (EPR) to determine the effects of ADP on the orientational distribution of nitroxide spin labels attached to myosin heads in skinned rabbit psoas muscle fibers. To maximize the specificity of labeling, we spin-labeled isolated myosin heads (subfragment 1) on a single reactive thiol (SH1) and diffused them into unlabeled muscle fibers. To maximize spectral and orientational resolution, we used perdeuterated spin labels, 2H-MSL and 2H-IASL, eliminating superhyperfine broadening and thus narrowing the line widths. Two different spin labels were used, with different orientation relative to the myosin head, to ensure that the results are not affected by unfavorable probe orientation. In rigor, a very narrow three-line spectrum was observed for both spin labels, indicating a narrow orientational distribution, as reported previously. ADP induced very slight changes in the spectrum, corresponding to very slight (but significant) changes in the orientational distribution. These changes were quantified by a digital analysis of the spectra, using a two-step simplex fitting procedure. First, the magnetic tensor values and line widths were determined by fitting the spectrum of a randomly oriented sample. Then the spectrum of oriented fibers was fit to a model by assuming a Gaussian distribution of the tilt angle (theta) and twist angle (phi) of the nitroxide principal axes relative to the fiber axis. A single-Gaussian distribution resulted in inadequate fits, but a two-component model gave excellent results. ADP induces a small (less than 5 degrees) rotation of the major components for both spin labels, along with a similarly small increase of disorder about the average positions

  18. Identification and localization of two brefeldin A-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors in a macromolecular complex

    OpenAIRE

    Yamaji, Ryoichi; Adamik, Ronald; Takeda, Kazuyo; Togawa, Akira; Pacheco-Rodriguez, Gustavo; Ferrans, Victor J.; Moss, Joel; Vaughan, Martha

    2000-01-01

    Two brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors, 200-kDa BIG1 and 190-kDa BIG2, were copurified from bovine brain cytosol associated with >670-kDa macromolecular complexes. When observed by immunofluorescence in HeLa S3 and HepG2 cells, endogenous BIG1 and coexpressed BIG2 were distributed in a punctate pattern throughout the cytosol, and also concentrated in the perinuclear region, where endogenous BIG1 and BIG2 each partially colocalized wit...

  19. 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.; Rycovska, A.; Cellengova, G.; Kolarov, J.

    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...

  20. 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...

  1. Preparation of /sup 14/C-labelled AMP, ADP and ATP from adenine-8-/sup 14/C by using Brevibacterium ammoniagenes

    Energy Technology Data Exchange (ETDEWEB)

    Pande, V.N. (Bhabha Atomic Research Centre, Bombay (India). Biochemistry and Food Technology Div.)

    1985-04-01

    High radiochemical yields of /sup 14/C-labelled adenine nucleotides (AMP, 4.6%, ADP, 15.5% and ATP 59.5%) could be obtained by growing the cells of Brevibacterium ammoniagenes in the presence of /sup 14/C-adenine. The specific radioactivity of the adenine nucleotides almost reached that of /sup 14/C-adenine indicating negligible dilution of the label. The procedure is convenient and especially suited for commercial preparation of the radiolabelled nucleotides directly from labelled adenine. Preliminary results indicate that the organism could also be used for the preparation of radiolabelled guanine nucleotides.

  2. Identification of Poly (ADP-ribose) Polymerase-1 (PARP-1) as a Novel Krüppel-like Factor 8-interacting and -regulating Protein*

    OpenAIRE

    Lu, Heng; Wang, Xianhui; Li, Tianshu; Urvalek, Alison M.; Yu, Lin; Li, Jieli; Zhu, Jinghua; Lin, Qishan; Peng, Xu; Zhao, Jihe

    2011-01-01

    Krüppel-like factor 8 (KLF8) regulates critical gene transcription and cellular events associated with cancer. However, KLF8-interacting proteins remain largely unidentified. Using co-immunoprecipitation (co-IP), mass spectrometry, and GST pulldown assays, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel KLF8-interacting protein. Co-IP and Western blotting indicated that KLF8 is also a PARP-1 substrate. Mutation of the cysteines in the zinc finger domain of KLF8 abolished PARP-...

  3. Normal telomere length and chromosomal end capping in poly(ADP-ribose) polymerase–deficient mice and primary cells despite increased chromosomal instability

    OpenAIRE

    Samper, Enrique; Goytisolo, Fermín A.; Murcia, Josiane Ménissier-de; González-Suárez, Eva; Cigudosa, Juan C.; de Murcia, Gilbert; Blasco, María A

    2001-01-01

    Poly(ADP-ribose) polymerase (PARP)-1, a detector of single-strand breaks, plays a key role in the cellular response to DNA damage. PARP-1–deficient mice are hypersensitive to genotoxic agents and display genomic instability due to a DNA repair defect in the base excision repair pathway. A previous report suggested that PARP-1–deficient mice also had a severe telomeric dysfunction consisting of telomere shortening and increased end-to-end fusions (d'Adda di Fagagna, F., M.P. Hande, W.-M. Tong,...

  4. Apurinic/apyrimidinic (AP) site recognition by the 5′-dRP/AP lyase in poly(ADP-ribose) polymerase-1 (PARP-1)

    OpenAIRE

    Khodyreva, S. N.; Prasad, R; Ilina, E. S.; Sukhanova, M. V.; Kutuzov, M. M.; Liu, Y.; Hou, E. W.; Wilson, S H; Lavrik, O. I.

    2010-01-01

    The capacity of human poly(ADP-ribose) polymerase-1 (PARP-1) to interact with intact apurinic/apyrimidinic (AP) sites in DNA has been demonstrated. In cell extracts, sodium borohydride reduction of the PARP-1/AP site DNA complex resulted in covalent cross-linking of PARP-1 to DNA; the identity of cross-linked PARP-1 was confirmed by mass spectrometry. Using purified human PARP-1, the specificity of PARP-1 binding to AP site-containing DNA was confirmed in competition binding experiments. PARP...

  5. Genetic Cooperation between the Werner Syndrome Protein and Poly(ADP-Ribose) Polymerase-1 in Preventing Chromatid Breaks, Complex Chromosomal Rearrangements, and Cancer in Mice

    OpenAIRE

    Lebel, Michel; Lavoie, Josée; Gaudreault, Isabelle; Bronsard, Marc; Drouin, Régen

    2003-01-01

    Werner syndrome is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for Werner syndrome encodes a DNA helicase/exonuclease protein. Participation in a replication complex is among the several functions postulated for the WRN protein. The poly(ADP-ribose) polymerase-1 (PARP-1) enzyme, which is known to bind to DNA strand breaks, is also associated with the DNA replication complex. To determine whether Wrn and PARP-1 enzymes act in c...

  6. Acetylation of poly(ADP-ribose) polymerase-1 by p300/CREB-binding protein regulates coactivation of NF-kappaB-dependent transcription

    OpenAIRE

    Hassa, P O; et al

    2005-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) and nuclear factor kappaB (NF-kappaB) have both been demonstrated to play a pathophysiological role in a number of inflammatory disorders. We recently presented evidence that PARP-1 can act as a promoter-specific coactivator of NF-kappaB in vivo independent of its enzymatic activity. PARP-1 directly interacts with p300 and both subunits of NF-kappaB (p65 and p50) and synergistically coactivates NF-kappaB-dependent transcription. Here we show that PARP-1 ...

  7. Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes.

    OpenAIRE

    Müller-Röber, B; Sonnewald, U.; Willmitzer, L.

    1992-01-01

    Transgenic potato plants were created in which the expression of ADP-glucose pyrophosphorylase (AGPase) was inhibited by introducing a chimeric gene containing the coding region of one of the subunits of the AGPase linked in an antisense orientation to the CaMV 35S promoter. Partial inhibition of the AGPase enzyme was achieved in leaves and almost complete inhibition in tubers. This resulted in the abolition of starch formation in tubers, thus proving that AGPase has a unique role in starch b...

  8. Carbon Dynamics, Development and Stress Responses in Arabidopsis: Involvement of the APL4 Subunit of ADP-Glucose Pyrophosphorylase (Starch Synthesis)

    OpenAIRE

    Gouesbet, Gwenola; Ramel, Fanny; Cabello-Hurtado, Francisco; Penno, Christophe; Bechtold, Nicole; Couée, Ivan; El Amrani, Abdelhak

    2011-01-01

    An Arabidopsis thaliana T-DNA insertional mutant was identified and characterized for enhanced tolerance to the singlet-oxygen-generating herbicide atrazine in comparison to wild-type. This enhanced atrazine tolerance mutant was shown to be affected in the promoter structure and in the regulation of expression of the APL4 isoform of ADP-glucose pyrophosphorylase, a key enzyme of the starch biosynthesis pathway, thus resulting in decrease of APL4 mRNA levels. The impact of this regulatory muta...

  9. Sugars and light/dark exposure trigger differential regulation of ADP-glucose pyrophosphorylase genes in Arabidopsis thaliana (thale cress).

    Science.gov (United States)

    Sokolov, L N; Déjardin, A; Kleczkowski, L A

    1998-12-15

    Expression of four Arabidopsis (thale cress) genes corresponding to the small (ApS) and large subunits (ApL1, ApL2, ApL3) of ADP-glucose pyrophosphorylase (AGPase), a key enzyme of starch biosynthesis, was found to be profoundly and differentially regulated by sugar and light/dark exposures. Transcript levels of both ApL2 and ApL3, and to a lesser extent ApS, increased severalfold upon feeding sucrose or glucose to the detached leaves in the dark, whereas the mRNA content for ApL1 decreased under the same conditions. Glucose was, in general, less effective than sucrose in inducing regulation of AGPase genes, possibly due to observed limitations in its uptake when compared with sucrose uptake by detached leaves. Osmotic agents [sorbitol, poly(ethylene glycol)] had no effect on ApS, ApL2 and ApL3 transcript level, but they did mimic the effect of sucrose on ApL1 gene, suggesting that the latter is regulated by osmotic pressure rather than any particular sugar. For all the genes the sugar effect was closely mimicked by an exposure of the dark-pre-adapted leaves to the light. Under both dark and light conditions, sucrose fed to the detached leaves was found to be rapidly metabolized to hexoses and, to some extent, starch. Starch production reflected most probably an increase in substrate availability for AGPase reaction rather than being due to changes in AGPase protein content, since both the sugar feeding and light exposure had little or no effect on the activity of AGPase or on the levels of its small and large subunit proteins in leaf extracts. The data suggest tight translational or post-translational control, but they may also reflect spatial control of AGPase gene expression within a leaf. The sugar/light-dependent regulation of AGPase gene expression may represent a part of a general cellular response to the availability/allocation of carbohydrates during photosynthesis. PMID:9841881

  10. Analysis of ADP-glucose pyrophosphorylase expression during turion formation induced by abscisic acid in Spirodela polyrhiza (greater duckweed

    Directory of Open Access Journals (Sweden)

    Wang Wenqin

    2012-01-01

    Full Text Available Abstract Background Aquatic plants differ in their development from terrestrial plants in their morphology and physiology, but little is known about the molecular basis of the major phases of their life cycle. Interestingly, in place of seeds of terrestrial plants their dormant phase is represented by turions, which circumvents sexual reproduction. However, like seeds turions provide energy storage for starting the next growing season. Results To begin a characterization of the transition from the growth to the dormant phase we used abscisic acid (ABA, a plant hormone, to induce controlled turion formation in Spirodela polyrhiza and investigated their differentiation from fronds, representing their growth phase, into turions with respect to morphological, ultra-structural characteristics, and starch content. Turions were rich in anthocyanin pigmentation and had a density that submerged them to the bottom of liquid medium. Transmission electron microscopy (TEM of turions showed in comparison to fronds shrunken vacuoles, smaller intercellular space, and abundant starch granules surrounded by thylakoid membranes. Turions accumulated more than 60% starch in dry mass after two weeks of ABA treatment. To further understand the mechanism of the developmental switch from fronds to turions, we cloned and sequenced the genes of three large-subunit ADP-glucose pyrophosphorylases (APLs. All three putative protein and exon sequences were conserved, but the corresponding genomic sequences were extremely variable mainly due to the invasion of miniature inverted-repeat transposable elements (MITEs into introns. A molecular three-dimensional model of the SpAPLs was consistent with their regulatory mechanism in the interaction with the substrate (ATP and allosteric activator (3-PGA to permit conformational changes of its structure. Gene expression analysis revealed that each gene was associated with distinct temporal expression during turion formation. APL2 and

  11. Evaluation of poly (ADP-ribose) polymerase inhibitor ABT-888 combined with radiotherapy and temozolomide in glioblastoma

    International Nuclear Information System (INIS)

    The cytotoxicity of radiotherapy and chemotherapy can be enhanced by modulating DNA repair. PARP is a family of enzymes required for an efficient base-excision repair of DNA single-strand breaks and inhibition of PARP can prevent the repair of these lesions. The current study investigates the trimodal combination of ABT-888, a potent inhibitor of PARP1-2, ionizing radiation and temozolomide(TMZ)-based chemotherapy in glioblastoma (GBM) cells. Four human GBM cell lines were treated for 5 h with 5 μM ABT-888 before being exposed to X-rays concurrently with TMZ at doses of 5 or 10 μM for 2 h. ABT-888′s PARP inhibition was measured using immunodetection of poly(ADP-ribose) (pADPr). Cell survival and the different cell death pathways were examined via clonogenic assay and morphological characterization of the cell and cell nucleus. Combining ABT-888 with radiation yielded enhanced cell killing in all four cell lines, as demonstrated by a sensitizer enhancement ratio at 50% survival (SER50) ranging between 1.12 and 1.37. Radio- and chemo-sensitization was further enhanced when ABT-888 was combined with both X-rays and TMZ in the O6-methylguanine-DNA-methyltransferase (MGMT)-methylated cell lines with a SER50 up to 1.44. This effect was also measured in one of the MGMT-unmethylated cell lines with a SER50 value of 1.30. Apoptosis induction by ABT-888, TMZ and X-rays was also considered and the effect of ABT-888 on the number of apoptotic cells was noticeable at later time points. In addition, this work showed that ABT-888 mediated sensitization is replication dependent, thus demonstrating that this effect might be more pronounced in tumour cells in which endogenous replication lesions are present in a larger proportion than in normal cells. This study suggests that ABT-888 has the clinical potential to enhance the current standard treatment for GBM, in combination with conventional chemo-radiotherapy. Interestingly, our results suggest that the use of PARP inhibitors

  12. Pharmacological inhibition of poly(ADP-ribose) polymerase-1 modulates resistance of human glioblastoma stem cells to temozolomide

    International Nuclear Information System (INIS)

    Chemoresistance of glioblastoma multiforme (GBM) has been attributed to the presence within the tumor of cancer stem cells (GSCs). The standard therapy for GBM consists of surgery followed by radiotherapy and the chemotherapeutic agent temozolomide (TMZ). However, TMZ efficacy is limited by O6-methylguanine-DNA-methyltransferase (MGMT) and Mismatch Repair (MMR) functions. Strategies to counteract TMZ resistance include its combination with poly(ADP-ribose) polymerase inhibitors (PARPi), which hamper the repair of N-methylpurines. PARPi are also investigated as monotherapy for tumors with deficiency of homologous recombination (HR). We have investigated whether PARPi may restore GSC sensitivity to TMZ or may be effective as monotherapy. Ten human GSC lines were assayed for MMR proteins, MGMT and PARP-1 expression/activity, MGMT promoter methylation and sensitivity to TMZ or PARPi, alone and in combination. Since PTEN defects are frequently detected in GBM and may cause HR dysfunction, PTEN expression was also analyzed. The statistical analysis of the differences in drug sensitivity among the cell lines was performed using the ANOVA and Bonferroni’s post-test or the non-parametric Kruskal-Wallis analysis and Dunn’s post-test for multiple comparisons. Synergism between TMZ and PARPi was analyzed by the median-effect method of Chou and Talalay. Correlation analyses were done using the Spearman’s rank test. All GSCs were MMR-proficient and resistance to TMZ was mainly associated with high MGMT activity or low proliferation rate. MGMT promoter hypermethylation of GSCs correlated both with low MGMT activity/expression (Spearman’s test, P = 0.004 and P = 0.01) and with longer overall survival of GBM patients (P = 0.02). Sensitivity of each GSC line to PARPi as single agent did not correlate with PARP-1 or PTEN expression. Notably, PARPi and TMZ combination exerted synergistic antitumor effects in eight out of ten GSC lines and the TMZ dose reduction achieved

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

    International Nuclear Information System (INIS)

    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 32P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingested ethanol in a liquid diet. 32P-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

  14. Effect of cobalt and DL-malic acid on the growth rate, crystalline perfection, optical, mechanical, dielectric, piezoelectric properties and SHG efficiency of ADP single crystals

    Energy Technology Data Exchange (ETDEWEB)

    Rajesh, P. [Centre for Crystal Growth, SSN College of Engineering, Kalavakkam 603110, Chennai, Tamilnadu 603110 (India); Ramasamy, P., E-mail: ramasamyp@ssn.edu.i [Centre for Crystal Growth, SSN College of Engineering, Kalavakkam 603110, Chennai, Tamilnadu 603110 (India); Kumar, Binay [Department of Physics and Astrophysics, University of Delhi, Delhi 110007 (India); Bhagavannarayana, G. [Materials characterization Division, National Physical Laboratory, New Delhi 110 012 (India)

    2010-05-15

    Effects of the additions of cobalt (II) acetate hexahydrate and DL-malic acid on the growth and various properties of ammonium dihydrogen orthophosphate single crystals grown by slow evaporation method have been studied. The grown crystals were subjected to UV-vis, microhardness, dielectric, piezoelectric, high resolution X-ray diffraction and SHG studies. UV spectra show good transparency in the entire visible region which is an essential requirement for a nonlinear optical crystal. Vickers hardness study carried out on (1 0 0) face at room temperature shows increased hardness of the crystals added with DL-malic acid compared to the pure and cobalt (II) acetate hexahydrate added crystals. Dielectric constant and dielectric loss were measured for the grown crystals for different frequencies and temperatures. It reveals that the DL-malic acid added ADP crystals have low dielectric loss. Crystalline perfection of the grown crystals was analyzed using HRXRD. Good piezoelectric behaviour was observed for all the crystals. Preliminary measurements indicate that the second harmonic generation efficiency of the DL-malic acid doped crystals is greater than pure and cobalt (II) acetate hexahydrate added ADP.

  15. The level of Ets-1 protein is regulated by poly(ADP-ribose polymerase-1 (PARP-1 in cancer cells to prevent DNA damage.

    Directory of Open Access Journals (Sweden)

    Arnaud J Legrand

    Full Text Available Ets-1 is a transcription factor that regulates many genes involved in cancer progression and in tumour invasion. It is a poor prognostic marker for breast, lung, colorectal and ovary carcinomas. Here, we identified poly(ADP-ribose polymerase-1 (PARP-1 as a novel interaction partner of Ets-1. We show that Ets-1 activates, by direct interaction, the catalytic activity of PARP-1 and is then poly(ADP-ribosylated in a DNA-independent manner. The catalytic inhibition of PARP-1 enhanced Ets-1 transcriptional activity and caused its massive accumulation in cell nuclei. Ets-1 expression was correlated with an increase in DNA damage when PARP-1 was inhibited, leading to cancer cell death. Moreover, PARP-1 inhibitors caused only Ets-1-expressing cells to accumulate DNA damage. These results provide new insight into Ets-1 regulation in cancer cells and its link with DNA repair proteins. Furthermore, our findings suggest that PARP-1 inhibitors would be useful in a new therapeutic strategy that specifically targets Ets-1-expressing tumours.

  16. The human apurinic/apyrimidinic endonuclease-1 suppresses activation of poly(adp-ribose) polymerase-1 induced by DNA single strand breaks

    International Nuclear Information System (INIS)

    DNA single-strand breaks (SSB) activate poly (ADP-ribose) polymerase 1 (PARP1), which then polymerizes ADP-ribosyl groups on various nuclear proteins, consuming cellular energy. Although PARP1 has a role in repairing SSB, activation of PARP1 also causes necrosis and inflammation due to depletion of cellular energy. Here we show that the major mammalian apurinic/apyrimidinic (AP) endonuclease-1 (APE1), an essential DNA repair protein, binds to SSB and suppresses the activation of PARP1. APE1's high affinity for SSB requires Arg177, which is unique in mammalian APEs. PARP1's binding to the cleaved DNA was inhibited, and PARP1 activation was suppressed by the wild-type APE1, but not by the R177A mutant APE1 protein. Cells transiently transfected with the wild-type APE1 decreased the PARP1 activation after H2O2 treatment, while such suppression did not occur with the expression of the R177A APE1 mutant. These results suggest that APE1 suppresses the activation of PARP1 during the repair process of the DNA damage generated by oxidative stress, which may have an important implication for cells to avoid necrosis due to energy depletion

  17. Reaction Mechanism and Structural Model of ADP-forming Acetyl-CoA Synthetase from the Hyperthermophilic Archaeon Pyrococcus furiosus: EVIDENCE FOR A SECOND ACTIVE SITE HISTIDINE RESIDUE*S⃞

    OpenAIRE

    Bräsen, Christopher; Schmidt, Marcel; Grötzinger, Joachim; Schönheit, Peter

    2008-01-01

    In Archaea, acetate formation and ATP synthesis from acetyl-CoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + Pi ⇆ acetate + ATP + HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain orga...

  18. 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.

  19. 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

  20. 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.

  1. Online cleanup of accelerated solvent extractions for determination of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in royal jelly using high-performance liquid chromatography.

    Science.gov (United States)

    Xue, Xiaofeng; Wang, Feng; Zhou, Jinhui; Chen, Fang; Li, Yi; Zhao, Jing

    2009-06-10

    Determination of the levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in royal jelly is important for the study of its pharmacological activities, health benefits, and adenosine phosphate degradation. In this study was developed a novel method to determine ATP, ADP, and AMP levels in royal jelly using accelerated solvent extraction (ASE) followed by online cleanup and high-performance liquid chromatography (HPLC) with diode array detection (DAD). The optimum extraction conditions were obtained using an 11 mL ASE cell, ethanol/water (5:5 v/v) as the extraction solvent, 1500 psi, 80 degrees C, a 5 min static time, and a 60% flush volume. Optimum separation of the three compounds was achieved in extraction procedures developed here were compared with the classical adenosine phosphate extraction procedures (perchloric acid). The results indicate that the two techniques are similar in terms of recovery and reproducibility, but when other factors such as extraction time, environmental protection, and worker's health are considered, ASE is preferable to the classical extraction method. With this ASE-HPLC method, a minisurvey of ATP, ADP, and AMP levels in 15 samples of royal jelly of different origins was performed. Sample results indicated that the AMP concentration was 24.2-2214.4 mg kg(-1), whereas ATP and ADP were not detectable or present only at low levels. PMID:19435312

  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. PMID:25049175

  3. Enhancement of the radiosensitivity of cultured lymphoma cells by 6(5H)-phenanthridinone, a novel poly (ADP-ribose) polymerase inhibitor

    International Nuclear Information System (INIS)

    Poly(ADP-ribose)polymerase (PARP, EC 2.4.2.30) is a chromatin-bound enzyme involved in a number of cellular processes linked to DNA metabolism such as proliferation, differentiation, DNA repair and apoptosis. Therefore, its inhibition, by preventing DNA repair to occur, has been proposed to potentiate radiotherapy or chemotherapy of tumors. However, the inhibitors used so far, mostly benzamide derivatives, gave un-conclusive results due to their weak inhibition potency and to their poor specificity. Recently, a new generation of PARP inhibitors has emerged, much more specific and active. We previously characterized one of them, namely 6(5H)-phenanthridinone (Phen), and showed its ability to potentiate the effects of anticancer drugs upon cultured tumor cells. These results prompted us to investigate whether Phen could also potentiate the effects of γ-radiations. (authors)

  4. Recovery of human lymphocytes damaged with. gamma. -radiation or enzymatically produced oxygen radicals: different effects of poly(ADP-ribosyl)polymerase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Marini, M.; Zunica, G. (Ist. di Istologia ed Embriologia Generale, Bologna (Italy)); Tamba, M. (Consiglio Nazionale delle Ricerche, Bologna (Italy). Lab. di Fotochimica e Radiazioni d' Alta Energia); Cossarizza, A.; Monti, D.; Franceschi, C. (Ist. di Patologia Generale, Modena (Italy))

    1990-08-01

    Quiescent human lymphocytes were damaged in two different ways, both producing toxic oxygen radicals: xanthine oxidase plus hypoxanthine (XOD/HYP), or {gamma}-rays. Under conditions where DNA synthesis was reduced to 10-20% of control, inhibitors of poly(ADP-ribosyl)polymerase (ADPRP, an enzyme that becomes activated in the presence of DNA strand breaks) allowed lymphocytes to recover completely when the damage was caused by XOD/HYP, but they did not affect DNA synthesis of irradiated cells. However, a protective effect of ADPRP inhibitors was observed with irradiated lymphocytes receiving doses {ge}50Gy. Unscheduled DNA synthesis was significantly increased when lymphocytes were damaged by high radiation doses in the presence of ADPRP inhibitors. (author).

  5. Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca²⁺ signalling in human platelets.

    Science.gov (United States)

    Lever, Robert A; Hussain, Azhar; Sun, Benjamin B; Sage, Stewart O; Harper, Alan G S

    2015-12-01

    Rises in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca(2+)]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca(2+)]cyt (Ca(2+) buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca(2+) or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca(2+) signalling, we here monitor Ca(2+) flux around the platelet by measuring net Ca(2+) fluxes to or from the extracellular space and the intracellular Ca(2+) stores, which act as the major sources and sinks for Ca(2+) influx into and efflux from the cytosol, as well as monitoring the cytosolic Na(+) concentration ([Na(+)]cyt), which influences platelet Ca(2+) fluxes via Na(+)/Ca(2+) exchange. The intracellular store Ca(2+) concentration ([Ca(2+)]st) was monitored using Fluo-5N, the extracellular Ca(2+) concentration ([Ca(2+)]ext) was monitored using Fluo-4 whilst [Ca(2+)]cyt and [Na(+)]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca(2+)]cyt in the absence of extracellular Ca(2+). PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca(2+) release and Ca(2+) removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca(2+)]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na(+)]cyt which would be expected to reduce Ca(2+) removal via the Na(+)/Ca(2+) exchanger (NCX). Thrombin-evoked rises in [Na(+)]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non

  6. Increased poly(ADP-ribosyl)ation in skeletal muscle tissue of pediatric patients with severe burn injury: prevention by propranolol treatment

    Science.gov (United States)

    Oláh, Gábor; Finnerty, Celeste; Sbrana, Elena; Elijah, Itoro; Gerö, Domokos; Herndon, David; Szabó, Csaba

    2011-01-01

    Summary Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) has been shown to promote cellular energetic collapse and cellular necrosis in various forms of critical illness. Most of the evidence implicating the PARP pathway in disease processes is derived from preclinical studies. With respect to PARP and burns, studies in rodent and large animal models of burn injury have demonstrated the activation of PARP in various tissues and the beneficial effect of its pharmacological inhibition. The aim of the current study was to measure the activation of PARP in human skeletal muscle biopsies at various stages of severe pediatric burn injury and to identify the cell types where this activation may occur. Another aim of the study was to test the effect of propranolol (an effective treatment of patients with burns), on the activation of PARP in skeletal muscle biopsies. PARP activation was measured by Western blotting for its product, poly(ADP-ribose) (PAR). The localization of PARP activation was determined by PAR immunohistochemistry. The results showed that PARP becomes activated in the skeletal muscle tissue after burns, with the peak of the activation occurring in the middle stage of the disease (13–18 days after burns). Even at the late stage of the disease (69–369 days post-burn) an elevated degree of PARP activation persisted in some of the patients. Immunohistochemical studies localized the staining of PAR primarily to vascular endothelial cells, and occasionally to resident mononuclear cells. There was a marked suppression of PARP activation in the skeletal muscle biopsies of patients who received propranolol treatment. We conclude that human burn injury is associated with the activation of PARP. We hypothesize that this response may contribute to the inflammatory responses and cell dysfunction in burns. Some of the clinical benefit of propranolol in burns may be related to its inhibitory effect on PARP activation. PMID:21368715

  7. 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.

  8. Does phosphate release limit the ATPases of soleus myofibrils? Evidence that (A)M. ADP.Pi states predominate on the cross-bridge cycle.

    Science.gov (United States)

    Iorga, Bogdan; Candau, Robin; Travers, Franck; Barman, Tom; Lionne, Corinne

    2004-01-01

    The ATPases (+/-Ca2+) of myofibrils from rabbit soleus (a slow muscle) and psoas (a fast muscle) have different Ea: -Ca2+, 78 and 60 kJ/mol and +Ca2+, 155 and 71 kJ/mol, respectively. At physiological temperatures, the two types of myofibrillar ATPase are very similar and yet the mechanical properties of the muscles are different (Candau et al. (2003) Biophys J 85: 3132-3141). Muscle contraction relies on specific interactions of the different chemical states on the myosin head ATPase pathway with the thin filament. An explanation for the Ea data is that different states populate the pathways of the two types of myofibril because the rate limiting steps are different. Here, we put this to the test by a comparison of the transient kinetics of the initial steps of the ATPases of the two types of myofibril at 4 degrees C. We used two methods: rapid flow quench ('cold ATP chase': titration of active sites, ATP binding kinetics, k(cat); 'Pi burst': ATP cleavage kinetics) and fluorescence stopped-flow (MDCC-phosphate binding protein for free Pi; myofibrillar tryptophan fluorescence for myosin head-thin filament detachment and ATP cleavage kinetics). We find that, as with psoas myofibrils, the most populated state on the cross-bridge cycle of soleus myofibrils, whether relaxed or activated, is (A)M.ADP.Pi. We propose a reaction pathway that includes several (A)M.ADP.Pi sub-states that are either 'weak' or 'strong', depending on the mechanical condition. PMID:15548866

  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)

    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)

  10. The therapy and mechanisms of replication-deficient recombinant adenovirus Ad-p14ARF in hepatocellular carcinoma%复制缺陷型腺病毒Ad-p14ARF治疗肝细胞癌的实验研究及作用机制的探讨

    Institute of Scientific and Technical Information of China (English)

    Haili Qian; Haifeng Song; Xueyan Zhang; Xiao Liang; Ming Fu; Chen Lin

    2007-01-01

    Objective: To study the therapeutic effect and mechanisms of recombinant adenovirus Ad-p14ARF in hepatocellular carcinoma cell lines. Methods: Morphology and trypan blue assay were adopted to evaluate the proliferation of different liver cancer cells after Ad-p14ARF infection. Cell apoptosis was confirmed by detecting phosphatidylserine (PS) externalization with Annexin V/PI double staining. Western blotting assay analyzed the expression of related proteins. Subcutaneous tumor model of BEL7402 was established to evaluate the therapeutic ability of Ad-p14ARF. Results: Ad-p14ARF suppressed cell growth, proliferation and promoted cell apoptosis of cancer cell lines with different genetic background. Ad-p14ARF inhibited growth of liver cancer cells (HepG2, BEL7402) in a dose-dependent manner. Ad-p14ARF leaded to overexpression of Bax and p21, which were the downstream regulating genes of p53. Ad-p14ARF suppressed tumor growth significantly in the experimental therapy in nude mice bearing subcutaneous tumor of BEL7402. Conclusion: P14ARF gene is a powerful tumor suppressor gene to be used in cancer gene therapy. It may play an important role in gene therapy against the malignancies in the future.

  11. Structure Function Relationships of ADP-Glucose Pyrophosphorylase and Branching Enzyme: Manipulation of Their Genes for Alteration of Starch Quanlity and Quantity

    Energy Technology Data Exchange (ETDEWEB)

    Jack Preiss

    2006-02-16

    Conversion of the Potato tuber ADP-glucose Pyrophopshorylase Regulatory Subunit into a Catalytic Subunit. ADP-glucose synthesis, a rate-limiting reaction in starch synthesis, is catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). The enzyme in plants is allosterically activated by 3-phosphoglycerate (3PGA) and inhibited by inorganic phosphate (Pi) and is composed of two subunits as a heterotetramer, a2b2. Subunit a is the catalytic subunit and subunit b is designated as the regulatory subunit.The b subunit increases the affinty of the activator for the catalytic subunit. Recent results have shown that the subunits are derived from the same ancestor subunit as the regulatory subunit can be converted to a catalytically subunit via mutation of just two amino acids. Lys44 and Thr54 in the large subunit from potato tuber were converted to the homologous catalytic subunit residues, Arg33 and Lys43. The activity of the large subunit mutants cannot be readily tested with a co-expressed wild-type small (catalytic) subunit because of the intrinsic activity of the latter. We co-expressed the regulatory-subunit mutants with SmallD145N, an inactive S subunit in which the catalytic Asp145 was mutated. The activity of the small (catalytic) subunit was reduced more than three orders of magnitude. Coexpression of the L subunit double mutant LargeK44R/T54K with SmallD145N generated an enzyme with considerable activity, 10% and 18% of the wildtype enzyme, in the ADP-glucose synthetic and pyrophosphorolytic direction, respectively. Replacement of those two residues in the small subunit by the homologous amino acids in the L subunits (mutations R33K and K43T) decreased the activity one and two orders of magnitude. The wild-type enzyme and SmallD145NLargeK44R/T54K had very similar kinetic properties indicating that the substrate site has been conserved. The fact that only two mutations in the L subunit restored enzyme activity is very strong evidence that the large subunit is

  12. Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences.

    Science.gov (United States)

    Mangerich, Aswin; Debiak, Malgorzata; Birtel, Matthias; Ponath, Viviane; Balszuweit, Frank; Lex, Kirsten; Martello, Rita; Burckhardt-Boer, Waltraud; Strobelt, Romano; Siegert, Markus; Thiermann, Horst; Steinritz, Dirk; Schmidt, Annette; Bürkle, Alexander

    2016-02-26

    Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of

  13. 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

  14. 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)

    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 C4 (LTC4) 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. LTC4 (1 nM) significantly elevated the mRNA levels of PARP-1 by 2.38-fold in VICs. Taken together, these data suggest that valvular

  15. Cyclic ADP-Ribose and Heat Regulate Oxytocin Release via CD38 and TRPM2 in the Hypothalamus during Social or Psychological Stress in Mice

    Science.gov (United States)

    Zhong, Jing; Amina, Sarwat; Liang, Mingkun; Akther, Shirin; Yuhi, Teruko; Nishimura, Tomoko; Tsuji, Chiharu; Tsuji, Takahiro; Liu, Hong-Xiang; Hashii, Minako; Furuhara, Kazumi; Yokoyama, Shigeru; Yamamoto, Yasuhiko; Okamoto, Hiroshi; Zhao, Yong Juan; Lee, Hon Cheung; Tominaga, Makoto; Lopatina, Olga; Higashida, Haruhiro

    2016-01-01

    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 β-NAD+, cADPR, and ADP in mouse oxytocinergic neurons. As these β-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 min 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°C) were higher than those

  16. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

    International Nuclear Information System (INIS)

    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

  17. 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

  18. Binding of 2'(3')-O-(2,4-6-trinitrophenyl) ADP to soluble alpha beta protomers of Na, K-ATPase modified with fluorescein isothiocyanate. Evidence for two distinct nucleotide sites.

    Science.gov (United States)

    Ward, D G; Cavieres, J D

    1996-05-24

    The overall reaction of well-defined solubilized protomers of Na,K-ATPase (one alpha plus one beta subunit) retains the dual ATP dependence observed with the membrane-bound enzyme, with distinctive ATP effects in the submicromolar and submillimolar ranges (Ward, D. G., and Cavieres, J. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5332-5336). We have now found that the K+/-phosphatase activity of the alpha beta protomers is still inhibited by 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP). What is most significant is that the TNP-ADP effect can be observed clearly with protomeric enzyme whose high affinity ATP site has been blocked covalently with fluorescein isothiocyanate. We conclude that nucleotides can bind at two discrete sites in each protomeric unit of Na,K-ATPase. PMID:8647832

  19. Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates the cytotoxic activity of temozolomide in leukemia cells: influence of mismatch repair status and O6-methylguanine-DNA methyltransferase activity

    OpenAIRE

    Horton, Terzah M.; Jenkins, Gaye; Pati, Debananda; Zhang, Linna; Dolan, M. Eileen; Ribes-Zamora, Albert; Bertuch, Alison A.; Blaney, Susan M.; Delaney, Shannon L.; Hegde, Madhuri; Berg, Stacey L.

    2009-01-01

    The poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 potentiates the antitumor activity of temozolomide (TMZ). TMZ resistance results from increased O6-methylguanine-DNA methyltransferase (MGMT) activity and from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP activity in ABT-888 potentiation of TMZ. MMR-proficient and MMR-deficient leukemia cells with varying MGMT activity, as well a...

  20. Decreased expression of plastidial adenylate kinase in potato tubers results in an enhanced rate of respiration and a stimulation of starch synthesis that is attributable to post-translational redox-activation of ADP-glucose pyrophosphorylase

    OpenAIRE

    Oliver, S.; Tiessen, A.; Fernie, A.; Geigenberger, P.

    2008-01-01

    Adenine nucleotides are of general importance for many aspects of cell function, but their role in the regulation of biosynthetic processes is still unclear. It was previously reported that decreased expression of plastidial adenylate kinase, catalysing the interconversion of ATP and AMP to ADP, leads to increased adenylate pools and starch content in transgenic potato tubers. However, the underlying mechanisms were not elucidated. Here, it is shown that decreased expression of plastidial ade...

  1. Affinity labeling of two nucleotide sites on Na,K-ATPase using 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-[alpha-32P]diphosphate (TNP-8N3-[alpha-32P]ADP) as a photoactivatable probe. Label incorporation before and after blocking the high affinity ATP site with fluorescein isothiocyanate.

    Science.gov (United States)

    Ward, D G; Cavieres, J D

    1998-12-11

    ATP and its analogues act on the minimal functional unit of Na, K-ATPase, the alpha beta protomer, with high and low affinity effects. Fluorescein isothiocyanate (FITC) irreversibly blocks the high affinity, or catalytic, ATP site, and yet the surviving K+-phosphatase activity of soluble FITC-modified alphabeta protomers can be photoinactivated by 2'(3')-O-trinitrophenyl (TNP)-8N3-ADP (Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284). We have now used TNP-8N3-[alpha-32P]ADP as a photoaffinity label for Na,K-ATPase. The native enzyme can be photolabeled at 5 microM TNP-8N3-[alpha-32P]ADP, and ATP or FITC treatment prevents labeling of the alpha chain. At 25 microM, however, TNP-8N3-[alpha-32P]ADP can be incorporated in the FITC-modified alpha chain, concurrently with the inactivation of the K+-phosphatase activity, to an extrapolated level of 0.5-1.2 mol of 32P-probe per mol of alpha chain. Photoinactivation and labeling are prevented by TNP-ADP, vanadate, or strophanthidin and are promoted by Na+ or Mg2+, but not K+. The cation effects suggest that the fluorescein-modified enzyme incorporates the TNP-8N3-[alpha-32P]ADP. Mg complex preferentially, and the free probe when in the E1 enzyme form and after occupation of a low-affinity Na+ site. Partial trypsinolysis reveals that the point of TNP-8N3-[alpha-32P]ADP attachment is on the C-terminal 58-kDa fragment of the FITC-modified alpha chain. The affinity labeling of the fluorescein enzyme by TNP-8N3-[alpha-32P]ADP endorses the view that two nucleotide sites can be occupied simultaneously in each alpha subunit of Na,K-ATPase. PMID:9837964

  2. Field observations of SPM using ADV, ADP, and OBS in a shallow estuarine system with low SPM concentration—Vitória Bay, SE Brazil

    Science.gov (United States)

    Moura, Marcel G.; Quaresma, Valeria S.; Bastos, Alex C.; Veronez, Paulo

    2011-03-01

    The present study investigated direct and indirect methods using optical and acoustic instruments for the acquisition of information required to estimate the concentration of suspended particulate matter (SPM) in Vitória Bay, a shallow estuarine system in SE Brazil. The aim was to calibrate and compare the use of different instruments (OBS, ADP, and ADV) to estimate SPM concentration in the water column and near the bed. Concentration was determined by correlating filtered water samples with the optical and acoustic measurements. In general, the methodology proved tenable for the chosen shallow estuarine environment with low SPM concentration (<60 mg/L). Pearson's coefficient ranged from 0.75 to 0.85, when correlating measurements taken at three sampled depths. Differences in the correlation coefficient values showed that calibration at three depths (near the bed, mid-water column, and near the surface) was more effective than for surface samples alone, even in shallow (˜3 m deep) water. When calibration was attempted for concentration in the entire water column with samples at just one elevation, the correlation value was very low, thus increasing the error in estimating the SPM concentration.

  3. Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase.

    Science.gov (United States)

    Wang, Zhenghan; Tacchelly-Benites, Ofelia; Yang, Eungi; Thorne, Curtis A; Nojima, Hisashi; Lee, Ethan; Ahmed, Yashi

    2016-05-01

    Wnt/β-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multiprotein "destruction complex," assembled by the scaffold protein Axin, targets the key transcriptional activator β-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. Here, we use an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. We find that for nearly all Wingless-driven developmental processes, a three- to fourfold increase in Axin is insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, we find that both the tumor suppressor adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and we define separable domains in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation. PMID:26975665

  4. Induction of Poly(ADP-ribose) Polymerase in Mouse Bone Marrow Stromal Cells Exposed to 900 MHz Radiofrequency Fields: Preliminary Observations

    Science.gov (United States)

    He, Qina; Sun, Yulong; Zong, Lin; Tong, Jian; Cao, Yi

    2016-01-01

    Background. Several investigators have reported increased levels of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme which plays an important role in the repair of damaged DNA, in cells exposed to extremely low dose ionizing radiation which does not cause measurable DNA damage. Objective. To examine whether exposure of the cells to nonionizing radiofrequency fields (RF) is capable of increasing messenger RNA of PARP-1 and its protein levels in mouse bone marrow stromal cells (BMSCs). Methods. BMSCs were exposed to 900 MHz RF at 120 μW/cm2 power intensity for 3 hours/day for 5 days. PARP-1 mRNA and its protein levels were examined at 0, 0.5, 1, 2, 4, 6, 8, and 10 hours after exposure using RT-PCR and Western blot analyses. Sham-exposed (SH) cells and those exposed to ionizing radiation were used as unexposed and positive control cells. Results. BMSCs exposed to RF showed significantly increased expression of PARP-1 mRNA and its protein levels after exposure to RF while such changes were not observed in SH-exposed cells. Conclusion. Nonionizing RF exposure is capable of inducing PARP-1.

  5. Effects of 3-aminobenzamide on poly(ADP-ribose)polymerase expression,apoptosis and cell cycle progression of HeLa cells after X-ray irradiation

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The aim of this paper is to study the changes of apoptosis and cell cycle progression in HeLa cells after the poly(ADP-ribose)polymerase(PARP)was inhibited by its inhibitor 3-aminobenzamide(3-AB)and the mechanisms of PARP action on HeLa cells damaged by irradiation.Flow cytometry(FCM)was used to examine the PARP expression and the percentage of apoptotic cells and cell cycle progression.The percentage of HeLa cells with positive expression of PARP protein 2,4,8 and 12 h after administrated with 3-AB was significantly lower than that of the control(P<0.01).The percentages of apoptotic cells in the 3-AB plus irradiation group at the time points of 2,8,12 and 24 h after 2 Gy irradiation were higher than that in the irradiation group(P<0.01 or P<0.05)and the percentage of G2 cells decreased significantly(P<0.01 or P<0.05).It indicates that 3-AB can rapidly inhibit PARP expression of HeLa cells,promote cell apoptosis and block G2 arrest induced by irradiation.

  6. Common and unique genetic interactions of the poly(ADP-ribose) polymerases PARP1 and PARP2 with DNA double-strand break repair pathways.

    Science.gov (United States)

    Ghosh, Rajib; Roy, Sanchita; Kamyab, Johan; Dantzer, Francoise; Franco, Sonia

    2016-09-01

    In mammalian cells, chromatin poly(ADP-ribos)ylation (PARylation) at sites of DNA Double-Strand Breaks (DSBs) is mediated by two highly related enzymes, PARP1 and PARP2. However, enzyme-specific genetic interactions with other DSB repair factors remain largely undefined. In this context, it was previously shown that mice lacking PARP1 and H2AX, a histone variant that promotes DSB repair throughout the cell cycle, or the core nonhomologous end-joining (NHEJ) factor Ku80 are not viable, while mice lacking PARP1 and the noncore NHEJ factor DNA-PKcs are severely growth retarded and markedly lymphoma-prone. Here, we have examined the requirement for PARP2 in these backgrounds. We find that, like PARP1, PARP2 is essential for viability in mice lacking H2AX. Moreover, treatment of H2AX-deficient primary fibroblasts or B lymphocytes with PARP inhibitors leads to activation of the G2/M checkpoint and accumulation of chromatid-type breaks in a lineage- and gene-dose dependent manner. In marked contrast to PARP1, loss of PARP2 does not result in additional phenotypes in growth, development or tumorigenesis in mice lacking either Ku80 or DNA-PKcs. Altogether these findings highlight specific nonoverlapping functions of PARP1 and PARP2 at H2AX-deficient chromatin during replicative phases of the cell cycle and uncover a unique requirement for PARP1 in NHEJ-deficient cells. PMID:27373144

  7. Human nuclear NAD+ ADP-ribosyltransferase: Localization of the gene on chromosome 1q41-q42 and expression of an active human enzyme in Escherichia coli

    International Nuclear Information System (INIS)

    The gene for human nuclear NAD+ ADP-ribosyltransferase was localized to chromosome 1 at q41-q42 by in situ hybridization with a pADPRT-specific cDNA probe. Expression of a pAD-PRT cDNA under control of the lac promoter in Escherichia coli induces the synthesis of a group of related proteins that were immunoreactive with pADPRT antibody and that had catalytic properties very similar to those of the human enzyme. Purification of this enzymatic activity was performed essentially as described for the human enzyme. The Km, pH optimum, optimal reaction temperature, and inhibition by 3-aminobenzamide and 3-methoxybenzamide were found to be similar for the recombinant and the human enzymes. The purified recombinant enzyme consists of two major proteins of Mr 99,000 and Mr 89,000. Both proteins show pADPRT activity in activity gel analysis with [32P]NAD+ as substrate. Microsequencing of these two proteins isolated by denaturing gel electrophoresis and deletion mutagenesis of the pADPRT expression plasmid shows that the Mr 99,000 and Mr 89,000 proteins derive from initiation of translation at interval translational start signals located within the pADPRT cDNA

  8. Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose) polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes.

    Science.gov (United States)

    Cuvillier, O; Rosenthal, D S; Smulson, M E; Spiegel, S

    1998-01-30

    Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7. PMID:9446602

  9. Diminishing impairments in glucose uptake, mitochondrial content, and ADP-stimulated oxygen flux by mesenchymal stem cell therapy in the infarcted heart.

    Science.gov (United States)

    Hughey, Curtis C; James, Freyja D; Ma, Lianli; Bracy, Deanna P; Wang, Zhizhang; Wasserman, David H; Rottman, Jeffrey N; Shearer, Jane

    2014-01-01

    A constant provision of ATP is of necessity for cardiac contraction. As the heart progresses toward failure following a myocardial infarction (MI), it undergoes metabolic alterations that have the potential to compromise the ability to meet energetic demands. This study evaluated the efficacy of mesenchymal stem cell (MSC) transplantation into the infarcted heart to minimize impairments in the metabolic processes that contribute to energy provision. Seven and twenty-eight days following the MI and MSC transplantation, MSC administration minimized cardiac systolic dysfunction. Hyperinsulinemic-euglycemic clamps, coupled with 2-[(14)C]deoxyglucose administration, were employed to assess systemic insulin sensitivity and tissue-specific, insulin-mediated glucose uptake 36 days following the MI in the conscious, unrestrained, C57BL/6 mouse. The improved systolic performance in MSC-treated mice was associated with a preservation of in vivo insulin-stimulated cardiac glucose uptake. Conserved glucose uptake in the heart was linked to the ability of the MSC treatment to diminish the decline in insulin signaling as assessed by Akt phosphorylation. The MSC treatment also sustained mitochondrial content, ADP-stimulated oxygen flux, and mitochondrial oxidative phosphorylation efficiency in the heart. Maintenance of mitochondrial function and density was accompanied by preserved peroxisome proliferator-activated receptor-γ coactivator-1α, a master regulator of mitochondrial biogenesis. These studies provide insight into mechanisms of action that lead to an enhanced energetic state in the infarcted heart following MSC transplantation that may assist in energy provision and dampen cardiac dysfunction. PMID:24196528

  10. 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.

  11. 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)

    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

  12. Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging.

    Science.gov (United States)

    Sukhanova, Maria V; Abrakhi, Sanae; Joshi, Vandana; Pastre, David; Kutuzov, Mikhail M; Anarbaev, Rashid O; Curmi, Patrick A; Hamon, Loic; Lavrik, Olga I

    2016-04-01

    PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages. PMID:26673720

  13. Two-color fluorescence detection of Poly (ADP-Ribose Polymerase-1 (PARP-1 cleavage and DNA strand breaks in etoposide-induced apoptotic cells

    Directory of Open Access Journals (Sweden)

    C Soldani

    2009-12-01

    Full Text Available During apoptosis, the nuclear enzyme Poly(ADPRibose Polymerase-1 (PARP-1 catalyzes the rapid and transient synthesis of poly(ADP-ribose from NAD+ and becomes inactive when cleaved by caspases. The regulation of these two opposite roles of PARP-1 is still unknown. We have recently investigated PARP-1 activation/degradation in Hep-2 cells driven to apoptosis by actinomycin D. In the present work, we have extended our analysis to the effect of the DNA damaging agent etoposide, and paid attention to the relationship between PARP-1 cleavage and DNA fragmentation. An original fluorescent procedure was developed to simultaneously identify in situ the p89 proteolytic fragment of PARP-1 (by immunolabeling and DNA degradation (by the TUNEL assay. The presence of p89 was observed both in cells with advanced signs of apoptosis (where the PARP-1 fragment is extruded from the nucleus into the cytoplasm and in TUNEL-negative cells, with only incipient signs of chromatin condensation; this evidence indicates that PARP-1 degradation in etoposide-treated apoptotic cells may precede DNA cleavage.

  14. Isolation and characterization of cDNAs and genomic DNAs encoding ADP-glucose pyrophosphorylase large and small subunits from sweet potato.

    Science.gov (United States)

    Zhou, Yu-Xi; Chen, Yu-Xiang; Tao, Xiang; Cheng, Xiao-Jie; Wang, Hai-Yan

    2016-04-01

    Sweet potato [Ipomoea batatas (L.) Lam.], the world's seventh most important food crop, is also a major industrial raw material for starch and ethanol production. In the plant starch biosynthesis pathway, ADP-glucose pyrophosphorylase (AGPase) catalyzes the first, rate-limiting step and plays a pivotal role in regulating this process. In spite of the importance of sweet potato as a starch source, only a few studies have focused on the molecular aspects of starch biosynthesis in sweet potato and almost no intensive research has been carried out on the AGPase gene family in this species. In this study, cDNAs encoding two small subunits (SSs) and four large subunits (LSs) of AGPase isoforms were cloned from sweet potato and the genomic organizations of the corresponding AGPase genes were elucidated. Expression pattern analysis revealed that the two SSs were constitutively expressed, whereas the four LSs displayed differential expression patterns in various tissues and at different developmental stages. Co-expression of SSs with different LSs in Escherichia coli yielded eight heterotetramers showing different catalytic activities. Interactions between different SSs and LSs were confirmed by a yeast two-hybrid experiment. Our findings provide comprehensive information about AGPase gene sequences, structures, expression profiles, and subunit interactions in sweet potato. The results can serve as a foundation for elucidation of molecular mechanisms of starch synthesis in tuberous roots, and should contribute to future regulation of starch biosynthesis to improve sweet potato starch yield. PMID:26499957

  15. Biodistribution of 3,4-dihydro-5-[11c]methoxy-1(2h)-isoquinolinone, a potential PET tracer for poly(adp-ribose) synthetase

    International Nuclear Information System (INIS)

    Poly(adenosine diphosphate-ribose) synthetase (PARS) is a nuclear enzyme that is activated by deoxyribonucleic acid (DNA) strand breaks and participates in DNA repair. Excessive PARS activation, however, leads to cell death due to depletion of adenosine triphosphate (ATP). To evaluate whether it is possible to detect excessive activation of PARS with positron emission tomography (PET), we examined the pharmacokinetics of 3,4-dihydro-5-[11C]methoxy-1(2H)-isoquinolinone ([11C]MIQO), a potent poly(ADP-ribose) synthetase inhibitor, in the brain of rats and monkeys. Although the uptake of [11C]MIQO in the brain of normal rats was low, [11C]MIQO was rapidly incorporated into and then quickly washed out from the brain. The uptake of the radiotracer in the brain of normal monkeys was also low; however, [11C]MIQO gave a distribution image that differed from that of cerebral blood flow obtained by [15O]water-PET. No localization of [11C]MIQO in the brain of normal monkeys was observed. Low accumulation of some radioactivity was also observed in muscles surrounding the brain of monkeys, but did not seem to interfere with measurement of [11C]MIQO uptake in the brain with PET. Thus, detection of [11C]MIQO uptake with PET may be useful for detecting PARS activity in ischemic injury

  16. Zinc carnosine protects against hydrogen peroxide-induced DNA damage in WIL2-NS lymphoblastoid cell line independent of poly (ADP-Ribose) polymerase expression.

    Science.gov (United States)

    Ooi, Theng Choon; Mohammad, Nur Hafiza; Sharif, Razinah

    2014-12-01

    The aim of this study is to investigate the ability of zinc carnosine to protect the human lymphoblastoid (WIL2-NS) cell line from hydrogen peroxide-induced DNA damage. Cells were cultured with medium containing zinc carnosine at the concentrations of 0.4, 4, 16 and 32 μM for 9 days prior to treatment with 30 μM of hydrogen peroxide (30 min). Zinc carnosine at the concentration 16 μM was optimal in protecting cells from hydrogen peroxide-induced cytotoxicity and gave the lowest percentage of apoptotic and necrotic cells. Results showed that zinc carnosine was able to induce glutathione production and protect cells from hydrogen peroxide-induced oxidative stress at all concentration and the highest protection was observed at 32-μM zinc carnosine culture. Cytokinesis-block micronucleus cytome assay showed that cells cultured with 4-32 μM of zinc carnosine showed significant reduction in micronuclei formation, nucleoplasmic bridges and nuclear bud frequencies (p carnosines possess antioxidant properties and are able to reduce hydrogen peroxide-induced DNA damage in vitro independent of poly(ADP-ribose) polymerase. Further studies are warranted to understand the mechanism of protection of zinc carnosine against hydrogen peroxide-induced damage. PMID:25326781

  17. Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo.

    Science.gov (United States)

    Liu, Yie; Snow, Bryan E; Kickhoefer, Valerie A; Erdmann, Natalie; Zhou, Wen; Wakeham, Andrew; Gomez, Marla; Rome, Leonard H; Harrington, Lea

    2004-06-01

    Vault poly(ADP-ribose) polymerase (VPARP) was originally identified as a minor protein component of the vault ribonucleoprotein particle, which may be involved in molecular assembly or subcellular transport. In addition to the association of VPARP with the cytoplasmic vault particle, subpopulations of VPARP localize to the nucleus and the mitotic spindle, indicating that VPARP may have other cellular functions. We found that VPARP was associated with telomerase activity and interacted with exogenously expressed telomerase-associated protein 1 (TEP1) in human cells. To study the possible role of VPARP in telomerase and vault complexes in vivo, mVparp-deficient mice were generated. Mice deficient in mVparp were viable and fertile for up to five generations, with no apparent changes in telomerase activity or telomere length. Vaults purified from mVparp-deficient mouse liver appeared intact, and no defect in association with other vault components was observed. Mice deficient in mTep1, whose disruption alone does not affect telomere function but does affect the stability of vault RNA, showed no additional telomerase or telomere-related phenotypes when the mTep1 deficiency was combined with an mVparp deficiency. These data suggest that murine mTep1 and mVparp, alone or in combination, are dispensable for normal development, telomerase catalysis, telomere length maintenance, and vault structure in vivo. PMID:15169895

  18. Structural Requirements of Some 2-(1-Propylpiperidin-4-yl)-1H-benzimidazole-4-carboxamide Derivatives as Poly (ADP-Ribose) Polymerase (PARP) for the Treatment of Cancer: QSAR Approach.

    Science.gov (United States)

    Sharma, Mukesh C

    2016-03-01

    The present study is aimed to elucidate the structural features of substituted 2-(1-propylpiperidin-4-yl)-1H-benzimidazole-4-carboxamide required for poly (ADP-ribose) polymerase inhibition and to obtain predictive 2D QSAR models to guide the rational synthesis of novel poly (ADP-ribose) polymerase inhibitors. The statistical analysis has shown that excellent results are obtained by using partial least regression based on simulated annealing method. The best model was selected based on the highest correlation coefficient r (2) = 0.8590, and cross-validated squared correlation coefficient q (2) = 0.7875 with external predictive ability of [Formula: see text] was developed by stepwise PLS method with the descriptors like T_N_F_1, SdsCHcount, and Rotatable Bond Count. The generated models provide insight into the influence of various interactive fields on the activity and, thus, can help in designing and forecasting the inhibition activity of novel (ADP-ribose) polymerase molecules. PMID:26205198

  19. cDNA sequence of a human skeletal muscle ADP/ATP translocator: lack of a leader peptide, divergence from a fibroblast translocator cDNA, and coevolution with mitochondrial DNA genes

    International Nuclear Information System (INIS)

    The authors have characterized a 1400-nucleotide cDNA for the human skeletal muscle ADP/ATP translocator. The deduced amino acid sequence is 94% homologous to the beef heart ADP/ATP translocator protein and contains only a single additional amino-terminal methionine. This implies that the human translocator lacks an amino-terminal targeting peptide, a conclusion substantiated by measuring the molecular weight of the protein synthesized in vitro. A 1400-nucleotide transcript encoding the skeletal muscle translocator was detected on blots of total RNA from human heart, kidney, skeletal muscle, and HeLa cells by hybridization with oligonucleotide probes homologous to the coding region and 3' noncoding region of the cDNA. However, the level of this mRNA varied substantially among tissues. Comparison of our skeletal muscle translocator sequence with that of a recently published human fibroblast translocator cognate revealed that the two proteins are 88% identical and diverged about 275 million years ago. Hence, tissues vary both in the level of expression of individual translocator genes and in differential expression of cognate translocator genes. Comparison of the base substitution rates of the ADP/ATP translocator and the oxidative phosphorylation genes encoded by mitochondrial DNA revealed that the mitochondrial DNA genes fix 10 times more synonymous substitutions and 12 times more replacement substitutions; yet, these nuclear and cytoplasmic respiration genes experience comparable evolutionary constraints. This suggest that the mitochondrial DNA genes are highly prone to deleterious mutations

  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.