Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

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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.

Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

International Nuclear Information System (INIS)

Aoyagi-Scharber, Mika; Gardberg, Anna S.; Yip, Bryan K.; Wang, Bing; Shen, Yuqiao; Fitzpatrick, Paul A.

2014-01-01

BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity. Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers. In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity

Imidazoquinolinone, imidazopyridine, and isoquinolindione derivatives as novel and potent inhibitors of the poly(ADP-ribose) polymerase (PARP): a comparison with standard PARP inhibitors.

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Eltze, Tobias; Boer, Rainer; Wagner, Thomas; Weinbrenner, Steffen; McDonald, Michelle C; Thiemermann, Christoph; Bürkle, Alexander; Klein, Thomas

2008-12-01

We have identified three novel structures for inhibitors of the poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA and implicated in DNA repair, apoptosis, organ dysfunction or necrosis. 2-[4-(5-Methyl-1H-imidazol-4-yl)-piperidin-1-yl]-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK49187), 2-(4-pyridin-2-yl-phenyl)-4,5-dihydro-imidazo[4,5,1-i,j]quinolin-6-one (BYK236864), 6-chloro-8-hydroxy-2,3-dimethyl-imidazo-[1,2-alpha]-pyridine (BYK20370), and 4-(1-methyl-1H-pyrrol-2-ylmethylene)-4H-isoquinolin-1,3-dione (BYK204165) inhibited cell-free recombinant human PARP-1 with pIC(50) values of 8.36, 7.81, 6.40, and 7.35 (pK(i) 7.97, 7.43, 5.90, and 7.05), and murine PARP-2 with pIC(50) values of 7.50, 7.55, 5.71, and 5.38, respectively. BYK49187, BYK236864, and BYK20370 displayed no selectivity for PARP-1/2, whereas BYK204165 displayed 100-fold selectivity for PARP-1. The IC(50) values for inhibition of poly(ADP-ribose) synthesis in human lung epithelial A549 and cervical carcinoma C4I cells as well in rat cardiac myoblast H9c2 cells after PARP activation by H(2)O(2) were highly significantly correlated with those at cell-free PARP-1 (r(2) = 0.89-0.96, P < 0.001) but less with those at PARP-2 (r(2) = 0.78-0.84, P < 0.01). The infarct size caused by coronary artery occlusion and reperfusion in the anesthetized rat was reduced by 22% (P < 0.05) by treatment with BYK49187 (3 mg/kg i.v. bolus and 3 mg/kg/h i.v. during 2-h reperfusion), whereas the weaker PARP inhibitors, BYK236864 and BYK20370, were not cardioprotective. In conclusion, the imidazoquinolinone BYK49187 is a potent inhibitor of human PARP-1 activity in cell-free and cellular assays in vitro and reduces myocardial infarct size in vivo. The isoquinolindione BYK204165 was found to be 100-fold more selective for PARP-1. Thus, both compounds might be novel and valuable tools for investigating PARP-1-mediated effects.

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

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Maria Rosaria Faraone Mennella

2018-01-01

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

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

International Nuclear Information System (INIS)

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

1979-01-01

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

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

NARCIS (Netherlands)

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

2012-01-01

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

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

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Prashanth Komirishetty

2016-01-01

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

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

Science.gov (United States)

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

2016-10-01

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

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.

Sensitization to radiation and alkylating agents by inhibitors of poly(ADP-ribose) polymerase is enhanced in cells deficient in DNA double-strand break repair.

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Löser, Dana A; Shibata, Atsushi; Shibata, Akiko K; Woodbine, Lisa J; Jeggo, Penny A; Chalmers, Anthony J

2010-06-01

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents.

ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib.

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Wang, Chen; Jette, Nicholas; Moussienko, Daniel; Bebb, D Gwyn; Lees-Miller, Susan P

2017-04-01

The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

NARCIS (Netherlands)

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

1993-01-01

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

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

Science.gov (United States)

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

2018-03-23

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

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

International Nuclear Information System (INIS)

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

1981-01-01

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

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

NARCIS (Netherlands)

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

2008-01-01

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

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

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Tkachenko A. S.

2017-12-01

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

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

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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.

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

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Kara A Boltz

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

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

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Townley, Jennifer M.; Shippen, Dorothy E.

2014-01-01

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

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

National Research Council Canada - National Science Library

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

2004-01-01

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

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

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

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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

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

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

2011-01-01

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

Poly(ADP-ribose polymerase 1 (PARP1 overexpression in human breast cancer stem cells and resistance to olaparib.

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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.

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

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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.

Targeting poly(ADP-ribose)polymerase1 in neurological diseases: A promising trove for new pharmacological interventions to enter clinical translation.

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Sriram, Chandra Shekhar; Jangra, Ashok; Kasala, Eshvendar Reddy; Bodduluru, Lakshmi Narendra; Bezbaruah, Babul Kumar

2014-10-01

The highly conserved abundant nuclear protein poly(ADP-ribose)polymerase1 (PARP1) functions at the center of cellular stress response and is mainly implied in DNA damage repair mechanism. Apart from its involvement in DNA damage repair, it does sway multiple vital cellular processes such as cell death pathways, cell aging, insulator function, chromatin modification, transcription and mitotic apparatus function. Since brain is the principal organ vulnerable to oxidative stress and inflammatory responses, upon stress encounters robust DNA damage can occur and intense PARP1 activation may result that will lead to various CNS diseases. In the context of soaring interest towards PARP1 as a therapeutic target for newer pharmacological interventions, here in the present review, we are attempting to give a silhouette of the role of PARP1 in the neurological diseases and the potential of its inhibitors to enter clinical translation, along with its structural and functional aspects. Copyright © 2014 Elsevier Ltd. All rights reserved.

Design, Synthesis and Evaluation of Ribose-modified Anilinopyrimidine Derivatives as EGFR Tyrosine Kinase Inhibitors

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Hu, Xiuqin; Wang, Disha; Tong, Yi; Tong, Linjiang; Wang, Xia; Zhu, Lili; Xie, Hua; Li, Shiliang; Yang, You; Xu, Yufang

2017-11-01

The synthesis of a series of ribose-modified anilinopyrimidine derivatives was efficiently achieved by utilizing DBU or tBuOLi-promoted coupling of ribosyl alcohols with 2,4,5-trichloropyrimidine as key step. Preliminary biological evaluation of this type of compounds as new EGFR tyrosine kinase inhibitors for combating EGFR L858R/T790M mutant associated with drug resistance in the treatment of non-small cell lung cancer revealed that 3-N-acryloyl-5-O-anilinopyrimidine ribose derivative 1a possessed potent and specific inhibitory activity against EGFR L858R/T790M over WT EGFR. Based upon molecular docking studies of the binding mode between compound 1a and EGFR, the distance between the Michael receptor and the pyrimidine scaffold is considered as an important factor for the inhibitory potency and future design of selective EGFR tyrosine kinase inhibitors against EGFR L858R/T790M mutants.

Cockayne syndrome group A and B proteins converge on transcription-linked resolution of non-B DNA

DEFF Research Database (Denmark)

Scheibye-Knudsen, Morten; Tseng, Anne; Jensen, Martin Borch

2016-01-01

of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number...... to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans. In conclusion, this work...

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

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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.

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...

A Phase 1 trial of the poly(ADP-ribose) polymerase inhibitor olaparib (AZD2281) in combination with the anti-angiogenic cediranib (AZD2171) in recurrent epithelial ovarian or triple-negative breast cancer.

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Liu, Joyce F; Tolaney, Sara M; Birrer, Michael; Fleming, Gini F; Buss, Mary K; Dahlberg, Suzanne E; Lee, Hang; Whalen, Christin; Tyburski, Karin; Winer, Eric; Ivy, Percy; Matulonis, Ursula A

2013-09-01

Poly(ADP-ribose) polymerase (PARP)-inhibitors and anti-angiogenics have activity in recurrent ovarian and breast cancer; however, the effect of combined therapy against PARP and angiogenesis in this population has not been reported. We investigated the toxicities and recommended phase 2 dosing (RP2D) of the combination of cediranib, a multitargeted inhibitor of vascular endothelial growth factor receptor (VEGFR)-1/2/3 and olaparib, a PARP-inhibitor (NCT01116648). Cediranib tablets once daily and olaparib capsules twice daily were administered orally in a standard 3+3 dose escalation design. Patients with recurrent ovarian or metastatic triple-negative breast cancer were eligible. Patients had measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 or met Gynecologic Cancer InterGroup (GCIG) CA125 criteria. No prior PARP-inhibitors or anti-angiogenics in the recurrent setting were allowed. 28 patients (20 ovarian, 8 breast) enrolled to 4 dose levels. 2 dose limiting toxicities (DLTs) (1 grade 4 neutropenia ≥ 4 days; 1 grade 4 thrombocytopenia) occurred at the highest dose level (cediranib 30 mg daily; olaparib 400 mg twice daily [BID]). The RP2D was cediranib 30 mg daily and olaparib 200 mg BID. Grade 3 or higher toxicities occurred in 75% of patients, and included grade 3 hypertension (25%) and grade 3 fatigue (18%). One grade 3 bowel obstruction occurred. The overall response rate (ORR) in the 18 RECIST-evaluable ovarian cancer patients was 44%, with a clinical benefit rate (ORR plus stable disease (SD) > 24 weeks) of 61%. None of the seven evaluable breast cancer patients achieved clinical response; two patients had stable disease for > 24 weeks. The combination of cediranib and olaparib has haematologic DLTs and anticipated class toxicities, with promising evidence of activity in ovarian cancer patients. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

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Markus Dahl

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

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

International Nuclear Information System (INIS)

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

1998-01-01

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

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

Science.gov (United States)

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

2013-01-01

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

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

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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.

Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

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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

CD38 Structure-Based Inhibitor Design Using the N1-Cyclic Inosine 5'-Diphosphate Ribose Template.

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Christelle Moreau

Full Text Available Few inhibitors exist for CD38, a multifunctional enzyme catalyzing the formation and metabolism of the Ca(2+-mobilizing second messenger cyclic adenosine 5'-diphosphoribose (cADPR. Synthetic, non-hydrolyzable ligands can facilitate structure-based inhibitor design. Molecular docking was used to reproduce the crystallographic binding mode of cyclic inosine 5'-diphosphoribose (N1-cIDPR with CD38, revealing an exploitable pocket and predicting the potential to introduce an extra hydrogen bond interaction with Asp-155. The purine C-8 position of N1-cIDPR (IC50 276 µM was extended with an amino or diaminobutane group and the 8-modified compounds were evaluated against CD38-catalyzed cADPR hydrolysis. Crystallography of an 8-amino N1-cIDPR:CD38 complex confirmed the predicted interaction with Asp-155, together with a second H-bond from a realigned Glu-146, rationalizing the improved inhibition (IC50 56 µM. Crystallography of a complex of cyclic ADP-carbocyclic ribose (cADPcR, IC50 129 µM with CD38 illustrated that Glu-146 hydrogen bonds with the ligand N6-amino group. Both 8-amino N1-cIDPR and cADPcR bind deep in the active site reaching the catalytic residue Glu-226, and mimicking the likely location of cADPR during catalysis. Substantial overlap of the N1-cIDPR "northern" ribose monophosphate and the cADPcR carbocyclic ribose monophosphate regions suggests that this area is crucial for inhibitor design, leading to a new compound series of N1-inosine 5'-monophosphates (N1-IMPs. These small fragments inhibit hydrolysis of cADPR more efficiently than the parent cyclic compounds, with the best in the series demonstrating potent inhibition (IC50 = 7.6 µM. The lower molecular weight and relative simplicity of these compounds compared to cADPR make them attractive as a starting point for further inhibitor design.

Poly (ADP-ribose polymerase plays an important role in intermittent hypoxia-induced cell death in rat cerebellar granule cells

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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.

Poly[ADP-ribose] polymerase-1 expression is related to cold ischemia, acute tubular necrosis, and delayed renal function in kidney transplantation.

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Francisco O'Valle

Full Text Available UNLABELLED: Cold ischemia time especially impacts on outcomes of expanded-criteria donor (ECD transplantation. Ischemia-reperfusion (IR injury produces excessive poly[ADP-Ribose] Polymerase-1 (PARP-1 activation. The present study explored the hypothesis that increased tubular expression of PARP-1 contributes to delayed renal function in suboptimal ECD kidney allografts and in non-ECD allografts that develop posttransplant acute tubular necrosis (ATN. MATERIALS AND METHODS: Nuclear PARP-1 immunohistochemical expression was studied in 326 paraffin-embedded renal allograft biopsies (193 with different degrees of ATN and 133 controls and in murine Parp-1 knockout model of IR injury. RESULTS: PARP-1 expression showed a significant relationship with cold ischemia time (r coefficient = 0.603, time to effective diuresis (r = 0.770, serum creatinine levels at biopsy (r = 0.649, and degree of ATN (r = 0.810 (p = 0.001, Pearson test. In the murine IR model, western blot showed an increase in PARP-1 that was blocked by Parp-1 inhibitor. Immunohistochemical study of PARP-1 in kidney allograft biopsies would allow early detection of possible delayed renal function, and the administration of PARP-1 inhibitors may offer a therapeutic option to reduce damage from IR in donor kidneys by preventing or minimizing ATN. In summary, these results suggest a pivotal role for PARP-1 in the ATN of renal transplantation. We propose the immunohistochemical assessment of PARP-1 in kidney allograft biopsies for early detection of a possible delayed renal function.

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

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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.

Co-targeting Deoxyribonucleic Acid–Dependent Protein Kinase and Poly(Adenosine Diphosphate-Ribose) Polymerase-1 Promotes Accelerated Senescence of Irradiated Cancer Cells

International Nuclear Information System (INIS)

Azad, Arun; Bukczynska, Patricia; Jackson, Susan; Haput, Ygal; Cullinane, Carleen; McArthur, Grant A.; Solomon, Benjamin

2014-01-01

Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination

Co-targeting Deoxyribonucleic Acid–Dependent Protein Kinase and Poly(Adenosine Diphosphate-Ribose) Polymerase-1 Promotes Accelerated Senescence of Irradiated Cancer Cells

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Azad, Arun, E-mail: arun.azad@bccancer.bc.ca [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Department of Pathology, St. Vincent' s Hospital, University of Melbourne, Parkville, Victoria (Australia); Bukczynska, Patricia; Jackson, Susan [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Haput, Ygal; Cullinane, Carleen [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria (Australia); McArthur, Grant A.; Solomon, Benjamin [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Division of Cancer Medicine, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Department of Medicine, St. Vincent' s Hospital, University of Melbourne, Parkville, Victoria (Australia); Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria (Australia)

2014-02-01

Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination.

Protective effect of the poly(ADP-ribose polymerase inhibitor PJ34 on mitochondrial depolarization-mediated cell death in hepatocellular carcinoma cells involves attenuation of c-Jun N-terminal kinase-2 and protein kinase B/Akt activation

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Radnai Balazs

2012-05-01

Full Text Available Abstract Background 2,4-Dimethoxyphenyl-E-4-arylidene-3-isochromanone (IK11 was previously described to induce apoptotic death of A431 tumor cells. In this report, we investigated the molecular action of IK11 in the HepG2 human hepatocellular carcinoma cell line to increase our knowledge of the role of poly (ADP-ribose-polymerase (PARP, protein kinase B/Akt and mitogen activated protein kinase (MAPK activation in the survival and death of tumor cells and to highlight the possible role of PARP-inhibitors in co-treatments with different cytotoxic agents in cancer therapy. Results We found that sublethal concentrations of IK11 prevented proliferation, migration and entry of the cells into their G2 phase. At higher concentrations, IK11 induced reactive oxygen species (ROS production, mitochondrial membrane depolarization, activation of c-Jun N-terminal kinase 2 (JNK2, and substantial loss of HepG2 cells. ROS production appeared marginal in mediating the cytotoxicity of IK11 since N-acetyl cysteine was unable to prevent it. However, the PARP inhibitor PJ34, although not a ROS scavenger, strongly inhibited both IK11-induced ROS production and cell death. JNK2 activation seemed to be a major mediator of the effect of IK11 since inhibition of JNK resulted in a substantial cytoprotection while inhibitors of the other kinases failed to do so. Inhibition of Akt slightly diminished the effect of IK11, while the JNK and Akt inhibitor and ROS scavenger trans-resveratrol completely protected against it. Conclusions These results indicate significant involvement of PARP, a marginal role of ROS and a pro-apoptotic role of Akt in this system, and raise attention to a novel mechanism that should be considered when cancer therapy is augmented with PARP-inhibition, namely the cytoprotection by inhibition of JNK2.

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

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Meng Q

2017-12-01

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

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

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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.

Critical role of poly(ADP-ribose) polymerase-1 in modulating the mode of cell death caused by continuous oxidative stress.

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Son, Young-Ok; Kook, Sung-Ho; Jang, Yong-Suk; Shi, Xianglin; Lee, Jeong-Chae

2009-11-01

Continuously generated hydrogen peroxide (H(2)O(2)) inhibits typical apoptosis and instead initiates a caspase-independent, apoptosis-inducing factor (AIF)-mediated pyknotic cell death. This may be related to H(2)O(2)-mediated DNA damage and subsequent ATP depletion, although the exact mechanisms by which the mode of cell death is decided after H(2)O(2) exposure are still unclear. Accumulated evidence and our previous data led us to hypothesize that continuously generated H(2)O(2), not an H(2)O(2) bolus, induces severe DNA damage, signaling poly(ADP-ribose) polymerase-1 (PARP-1) activation, ATP depletion, and eventually caspase-independent cell death. Results from the present study support that H(2)O(2) generated continuously by glucose oxidase causes excessive DNA damage and PARP-1 activation. Blockage of PARP-1 by a siRNA transfection or by pharmacological inhibitor resulted in the significant inhibition of ATP depletion, loss of mitochondrial membrane potential, nuclear translocation of AIF and endonuclease G, and eventually conversion to caspase-dependent apoptosis. Overall, the current study demonstrates the different roles of PARP-1 inhibition in modulation of cell death according to the method of H(2)O(2) exposure, that is, continuous generation versus a direct addition. (c) 2009 Wiley-Liss, Inc.

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

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Laura Lafon-Hughes

2014-10-01

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

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

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

2016-01-15

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

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

International Nuclear Information System (INIS)

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

2004-01-01

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

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

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

2018-01-18

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

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

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Michèle Rouleau

2011-01-01

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

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

Science.gov (United States)

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

2016-01-15

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

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

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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.

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

International Nuclear Information System (INIS)

Utsumi, Hiroshi; Elkind, M.M.

1994-01-01

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

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

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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.

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

International Nuclear Information System (INIS)

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

2014-01-01

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

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

International Nuclear Information System (INIS)

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

2012-01-01

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

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

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Zhizhi Wang

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

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

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

1982-04-01

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

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

International Nuclear Information System (INIS)

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

1982-01-01

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

Relationship between triterpenoid anticancer drug resistance, autophagy, and caspase-1 in adult T-cell leukemia

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Tsukasa Nakanishi

2016-05-01

Full Text Available We previously reported that the inflammasome inhibitor cucurbitacin D (CuD induces apoptosis in human leukemia cell lines. Here, we investigated the effects of CuD and a B-cell lymphoma extra-large (Bcl-xL inhibitor on autophagy in peripheral blood lymphocytes (PBL isolated from adult T-cell leukemia (ATL patients. CuD induced PBL cell death in patients but not in healthy donors. This effect was not significantly inhibited by treatment with rapamycin or 3-methyladenine (3-MA. The Bcl-xL inhibitor Z36 induced death in primary cells from ATL patients including that induced by CuD treatment, effects that were partly inhibited by 3-MA. Similarly, cell death induced by the steroid prednisolone was enhanced in the presence of Z36. A western blot analysis revealed that Z36 also promoted CuD-induced poly(ADP ribose polymerase cleavage. Interestingly, the effects of CuD and Z36 were attenuated in primary ATL patient cells obtained upon recurrence after umbilical cord blood transplantation, as compared to those obtained before chemotherapy. Furthermore, cells from this patient expressed a high level of caspase-1, and treatment with caspase-1 inhibitor-enhanced CuD-induced cell death. Taken together, these results suggest that rescue from resistance to steroid drugs can enhance chemotherapy, and that caspase-1 is a good marker for drug resistance in ATL patients.

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

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

2016-08-05

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

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

International Nuclear Information System (INIS)

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

2016-01-01

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

Breast Cancer Resistance Protein (BCRP/ABCG2) and P-glycoprotein (P-GP/ABCB1) Restrict Oral Availability and Brain Accumulation of the PARP Inhibitor Rucaparib (AG-014699)

NARCIS (Netherlands)

Durmus, Selvi; Sparidans, Rolf W; van Esch, Anita; Wagenaar, Els; Beijnen, Jos H; Schinkel, Alfred H

BACKGROUND: Rucaparib is a potent, orally available, small-molecule inhibitor of poly ADP-ribose polymerase (PARP) 1 and 2. Ongoing clinical trials are assessing the efficacy of rucaparib alone or in combination with other cytotoxic drugs, mainly in breast and ovarian cancer patients with mutations

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

DEFF Research Database (Denmark)

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

2007-01-01

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

The dual action of poly(ADP-ribose polymerase -1 (PARP-1 inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity

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Slava eRom

2015-08-01

Full Text Available The transcription of HIV-1 (HIV is regulated by complex mechanisms involving various cellular factors and virus-encoded transactivators. Poly(ADP-ribose polymerase 1 (PARP-1 inhibition has emerged recently as a potent anti-inflammatory tool, since PARP-1 is involved in the regulation of some genes through its interaction with various transcription factors. We propose a novel approach to diminish HIV replication via PARP-1 inhibition using human primary monocyte-derived macrophages (MDM as an in vitro model system. PARP-1 inhibitors were able to reduce HIV replication in MDM by 60-80% after 7 days infection. Long Terminal Repeat (LTR acts as a switch in virus replication and can be triggered by several agents such as: Tat, tumor necrosis factor α (TNFα, and phorbol 12-myristate 13-acetate (PMA. Overexpression of Tat in MDM transfected with an LTR reporter plasmid led to a 4.2-fold increase in LTR activation; PARP inhibition resulted in 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85-95%. MDM treated with PARP inhibitors showed 90% reduction in NFκB activity (known to mediate PMA- and TNFα-induced HIV LTR activation. Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These findings suggest that HIV replication in MDM could be suppressed by PARP inhibition via NFκB suppression, diminution of LTR activation and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide a potent approach to treatment of HIV infection.

Cross talk between poly(ADP-ribose) polymerase 1 methylation and oxidative stress involved in the toxic effect of anatase titanium dioxide nanoparticles

Science.gov (United States)

Bai, Wenlin; Chen, Yujiao; Gao, Ai

2015-01-01

Given the tremendous growth in the application of titanium dioxide nanoparticles (TNPs), concerns about the potential health hazards of TNPs to humans have been raised. Poly(ADP-ribose) polymerase 1 (PARP-1), a highly conserved DNA-binding protein, is involved in many molecular and cellular processes. Limited data demonstrated that certain nanomaterials induced the aberrant hypermethylation of PARP-1. However, the mechanism involved in TNP-induced PARP-1 abnormal methylation has not been studied. A549 cells were incubated with anatase TNPs (22.1 nm) for 24 hours pretreatment with or without methyltransferase inhibitor 5-aza-2′-deoxycytidine and the reactive oxygen species (ROS) scavenger α-lipoic acid to assess the possible role of methylation and ROS in the toxic effect of TNPs. After TNPs characterization, a battery of assays was performed to evaluate the toxic effect of TNPs, PARP-1 methylation status, and oxidative damage. Results showed that TNPs decreased the cell viability in a dose-dependent manner, in accordance with the increase of lactate dehydrogenase activity, which indicated membrane damage of cells. Similar to the high level of PARP-1 methylation, the generation of ROS was significantly increased after exposure to TNPs for 24 hours. Furthermore, α-lipoic acid decreased TNP-induced ROS generation and then attenuated TNP-triggered PARP-1 hypermethylation. Meanwhile, 5-aza-2′-deoxycytidine simultaneously decreased the ROS generation induced by TNPs, resulting in the decline of PARP-1 methylation. In summary, TNPs triggered the aberrant hypermethylation of the PARP-1 promoter and there was a cross talk between oxidative stress and PARP-1 methylation in the toxic effect of TNPs. PMID:26366077

Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery.

Science.gov (United States)

Murphy, Anar K; Fitzgerald, Michael; Ro, Teresa; Kim, Jee Hyun; Rabinowitsch, Ariana I; Chowdhury, Dipanjan; Schildkraut, Carl L; Borowiec, James A

2014-08-18

Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress. © 2014 Murphy et al.

Identification of halosalicylamide derivatives as a novel class of allosteric inhibitors of HCV NS5B polymerase.

Science.gov (United States)

Liu, Yaya; Donner, Pamela L; Pratt, John K; Jiang, Wen W; Ng, Teresa; Gracias, Vijaya; Baumeister, Steve; Wiedeman, Paul E; Traphagen, Linda; Warrior, Usha; Maring, Clarence; Kati, Warren M; Djuric, Stevan W; Molla, Akhteruzzaman

2008-06-01

Halosalicylamide derivatives were identified from high-throughput screening as potent inhibitors of HCV NS5B polymerase. The subsequent structure and activity relationship revealed the absolute requirement of the salicylamide moiety for optimum activity. Methylation of either the hydroxyl group or the amide group of the salicylamide moiety abolished the activity while the substitutions on both phenyl rings are acceptable. The halosalicylamide derivatives were shown to be non-competitive with respect to elongation nucleotide and demonstrated broad genotype activity against genotype 1-3 HCV NS5B polymerases. Inhibitor competition studies indicated an additive binding mode to the initiation pocket that is occupied by the thiadiazine class of compounds and an additive binding mode to the elongation pocket that is occupied by diketoacids, but a mutually exclusive binding mode with respect to the allosteric thumb pocket that is occupied by the benzimidazole class of inhibitors. Therefore, halosalicylamides represent a novel class of allosteric inhibitors of HCV NS5B polymerase.

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

Science.gov (United States)

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

2013-05-13

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

Looking for inhibitors of the dengue virus NS5 RNA-dependent RNA-polymerase using a molecular docking approach

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Galiano V

2016-10-01

Full Text Available Vicente Galiano,1 Pablo Garcia-Valtanen,2 Vicente Micol,3,4 José Antonio Encinar3 1Physics and Computer Architecture Department, Miguel Hernández University (UMH, Elche, Spain; 2Experimental Therapeutics Laboratory, Hanson and Sansom Institute for Health Research, School of Pharmacy and Medical Science, University of South Australia, Adelaide, Australia; 3Molecular and Cell Biology Institute, Miguel Hernández University (UMH, Elche, Spain; 4CIBER: CB12/03/30038, Physiopathology of the Obesity and Nutrition, CIBERobn, Instituto de Salud Carlos III, Palma de Mallorca, Spain Abstract: The dengue virus (DENV nonstructural protein 5 (NS5 contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <-10.5 kcal/mol were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo. Keywords: virtual screening, molecular

Dielectric relaxation study of the dynamics of monosaccharides: D-ribose and 2-deoxy-D-ribose

Energy Technology Data Exchange (ETDEWEB)

Kaminski, K; Kaminska, E; Wlodarczyk, P; Paluch, M; Ziolo, J [Institute of Physics, Silesian University, ulica Uniwersytecka 4, 40-007 Katowice (Poland); Ngai, K L [Naval Research Laboratory, Washington, DC 20375-5320 (United States)

2008-08-20

The dielectric loss spectra of two closely related monosaccharides, D-ribose and 2-deoxy-D-ribose, measured at ambient and elevated pressures are presented. 2-deoxy-D-ribose and D-ribose are respectively the building blocks of the backbone chains in the nucleic acids DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Small differences in the structure between D-ribose and 2-deoxy-D-ribose result in changes of the glass transition temperature T{sub g}, as well as the dielectric strength and activation enthalpy of the secondary relaxations. However, the frequency dispersion of the structural {alpha}-relaxation for the same relaxation time remains practically the same. Two secondary relaxations are present in both sugars. The slower secondary relaxation shifts to lower frequencies with increasing applied pressure, but not the faster one. This pressure dependence indicates that the slower secondary relaxation is the important and 'universal' Johari-Goldstein {beta}-relaxation of both sugars according to one of the criteria set up to classify secondary relaxations. Additional confirmation of this conclusion comes from good agreement of the observed relaxation time of the slower secondary relaxation with the primitive relaxation time calculated from the coupling model. All the dynamic properties of D-ribose and 2-deoxy-D-ribose are similar to the other monosaccharides, glucose, fructose, galactose and sorbose, except for the much larger relaxation strength of the {alpha}-relaxation of the former compared to the latter. The difference may distinguish the chemical and biological functions of D-ribose and 2-deoxy-D-ribose from the other monosaccharides.

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

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

International Nuclear Information System (INIS)

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

2012-01-01

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

Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases.

Science.gov (United States)

Sidstedt, Maja; Romsos, Erica L; Hedell, Ronny; Ansell, Ricky; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter; Hedman, Johannes

2017-02-07

Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to 5 U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.

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

International Nuclear Information System (INIS)

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

2007-01-01

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

Ibrutinib synergizes with poly(ADP-ribose) glycohydrolase inhibitors to induce cell death in AML cells via a BTK-independent mechanism.

Science.gov (United States)

Rotin, Lianne E; Gronda, Marcela; MacLean, Neil; Hurren, Rose; Wang, XiaoMing; Lin, Feng-Hsu; Wrana, Jeff; Datti, Alessandro; Barber, Dwayne L; Minden, Mark D; Slassi, Malik; Schimmer, Aaron D

2016-01-19

Targeting Bruton's tyrosine kinase (BTK) with the small molecule BTK inhibitor ibrutinib has significantly improved patient outcomes in several B-cell malignancies, with minimal toxicity. Given the reported expression and constitutive activation of BTK in acute myeloid leukemia (AML) cells, there has been recent interest in investigating the anti-AML activity of ibrutinib. We noted that ibrutinib had limited single-agent toxicity in a panel of AML cell lines and primary AML samples, and therefore sought to identify ibrutinib-sensitizing drugs. Using a high-throughput combination chemical screen, we identified that the poly(ADP-ribose) glycohydrolase (PARG) inhibitor ethacridine lactate synergized with ibrutinib in TEX and OCI-AML2 leukemia cell lines. The combination of ibrutinib and ethacridine induced a synergistic increase in reactive oxygen species that was functionally important to explain the observed cell death. Interestingly, synergistic cytotoxicity of ibrutinib and ethacridine was independent of the inhibitory effect of ibrutinib against BTK, as knockdown of BTK did not sensitize TEX and OCI-AML2 cells to ethacridine treatment. Thus, our findings indicate that ibrutinib may have a BTK-independent role in AML and that PARG inhibitors may have utility as part of a combination therapy for this disease.

Parthanatos, a messenger of death

Science.gov (United States)

David, Karen Kate; Andrabi, Shaida Ahmad; Dawson, Ted Murray; Dawson, Valina Lynn

2015-01-01

Poly-ADP-ribose polymerase-1 (PARP-1)'s multiple roles in the cell span from maintaining life to inducing death. The processes PARP-1 is involved in include, but are not limited to DNA repair, DNA transcription, mitosis, and cell death. Of PARP-1's different cellular functions, its active role in cell death is of particular interest to designing therapies for diseases. Genetic deletion of PARP-1 revealed that PARP-1 over activation underlies cell death in experimental models of stroke, diabetes, inflammation and neurodegeneration. Since interfering with PARP-1 mediated cell death will be clinically beneficial, great effort has been invested into designing PARP-1 inhibitors and understanding mechanisms downstream of PARP-1 over activation. PARP-1 overactivation may kill by depleting cellular energy through nicotinamide adenine dinucleotide (NAD+) consumption, and by releasing the cell death effector apoptosis-inducing factor (AIF). Unexpectedly, recent evidence shows that poly-ADP ribose (PAR) polymer itself, and not the consumption of NAD+ is the source of cytotoxicity. Thus, PAR polymer acts as a cell death effector downstream of PARP-1-mediated cell death signaling. We coined the term parthanatos after Thanatos, the personification of death in Greek mythology, to refer to PAR-mediated cell death. In this review, we will summarize the proposed mechanisms by which PARP-1 overactivation kills. We will present evidence for parthanatos, and the questions raised by these recent findings. It is evident that further understanding of parthanatos opens up new avenues for therapy in ameliorating diseases related to PARP-1 over activation. PMID:19273119

In Silico Screening Hepatitis B Virus DNA Polymerase Inhibitors from Medicinal Plants

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Mokhtar Nosrati

2017-08-01

Full Text Available Abstract Background: Hepatitis B virus infection (HBV is a significant global health problem and is a major cause of morbidity and mortality worldwide. Therefore, currently, introducing novel anti Hepatitis B drugs is taken into consideration. This study was planned to in silico screening novel Hepatitis B virus DNA polymerase inhibitors from two medicinal plants Terminalis chebula and Caesalpinia sappan. Materials and Methods: This is a descriptive-analytic study. In the study, three-dimensional structure of the Hepatitis B virus DNA polymerase was predicted using homology modeling method. A set of phytochemicals from mentioned plants were retrieved from Pubchem database in SDF format. In silico screening was carried out using molecular docking between mentioned phytochemicals and modeled polymerase by iGemdock 2.1 software. Results: Results of the study confirmed that all evaluated ligands have appropriate interactions to the polymerase with least toxicity and without genotoxicity potential. Results also showed that most interactions occur in reverse transcriptase domain which located in 354-694 area in the amino acid sequence of tested polymerase. Analysis of energy and amino acids involved in ligand-polymerase interaction revealed that Terchebin, Chebulinic Acid and Terflavin A have more effective interaction with the polymerase in compared to other ligands. Conclusion: Based on the results it can be concluded that evaluated compounds could be good candidates for in vitro and in vivo research in order to develop novel anti- Hepatitis B drugs.

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.

Histone H2AX is a critical factor for cellular protection against DNA alkylating agents.

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Meador, J A; Zhao, M; Su, Y; Narayan, G; Geard, C R; Balajee, A S

2008-09-25

Histone H2A variant H2AX is a dose-dependent suppressor of oncogenic chromosome translocations. H2AX participates in DNA double-strand break repair, but its role in other DNA repair pathways is not known. In this study, role of H2AX in cellular response to alkylation DNA damage was investigated. Cellular sensitivity to two monofunctional alkylating agents (methyl methane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)) was dependent on H2AX dosage, and H2AX null cells were more sensitive than heterozygous cells. In contrast to wild-type cells, H2AX-deficient cells displayed extensive apoptotic death due to a lack of cell-cycle arrest at G(2)/M phase. Lack of G(2)/M checkpoint in H2AX null cells correlated well with increased mitotic irregularities involving anaphase bridges and gross chromosomal instability. Observation of elevated poly(ADP) ribose polymerase 1 (PARP-1) cleavage suggests that MNNG-induced apoptosis occurs by PARP-1-dependent manner in H2AX-deficient cells. Consistent with this, increased activities of PARP and poly(ADP) ribose (PAR) polymer synthesis were detected in both H2AX heterozygous and null cells. Further, we demonstrate that the increased PAR synthesis and apoptotic death induced by MNNG in H2AX-deficient cells are due to impaired activation of mitogen-activated protein kinase pathway. Collectively, our novel study demonstrates that H2AX, similar to PARP-1, confers cellular protection against alkylation-induced DNA damage. Therefore, targeting either PARP-1 or histone H2AX may provide an effective way of maximizing the chemotherapeutic value of alkylating agents for cancer treatment.

Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization.

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La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca; Caligo, Maria Adelaide; Galli, Alvaro

2015-04-01

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 deubiquitination enzyme gene OTU1, the nuclear pore protein POM152 and the SNT1 that encodes for the Set3C subunit of the histone deacetylase complex. In these strains the PARP-1 level was roughly the same as in the wild type. PARP-1 localized in the nucleus more in the snt1Δ than in the wild type strain; after UV radiation, PARP-1 localized in the nucleus more in hho1 and pom152 deletion strains than in the wild type indicating that these functions may have a role on regulating PARP-1 level and activity in the nucleus. Copyright © 2015 Elsevier B.V. All rights reserved.

Minocycline Blocks Asthma-associated Inflammation in Part by Interfering with the T Cell Receptor-Nuclear Factor κB-GATA-3-IL-4 Axis without a Prominent Effect on Poly(ADP-ribose) Polymerase*

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Naura, Amarjit S.; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C.; Jordan, Joaquin; Catling, Andrew D.; Rezk, Bashir M.; Elmageed, Zakaria Y. Abd; Pyakurel, Kusma; Tarhuni, Abdelmetalab F.; Abughazleh, Mohammad Q.; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C.; Boulares, A. Hamid

2013-01-01

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N′-nitro-N-nitroso-guanidine-treated mice or H2O2-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production. PMID:23184953

Minocycline blocks asthma-associated inflammation in part by interfering with the T cell receptor-nuclear factor κB-GATA-3-IL-4 axis without a prominent effect on poly(ADP-ribose) polymerase.

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Naura, Amarjit S; Kim, Hogyoung; Ju, Jihang; Rodriguez, Paulo C; Jordan, Joaquin; Catling, Andrew D; Rezk, Bashir M; Abd Elmageed, Zakaria Y; Pyakurel, Kusma; Tarhuni, Abdelmetalab F; Abughazleh, Mohammad Q; Errami, Youssef; Zerfaoui, Mourad; Ochoa, Augusto C; Boulares, A Hamid

2013-01-18

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

Novel targeted therapeutics: inhibitors of MDM2, ALK and PARP

Directory of Open Access Journals (Sweden)

Hsueh Chung-Tsen

2011-04-01

Full Text Available Abstract We reviewed preclinical data and clinical development of MDM2 (murine double minute 2, ALK (anaplastic lymphoma kinase and PARP (poly [ADP-ribose] polymerase inhibitors. MDM2 binds to p53, and promotes degradation of p53 through ubiquitin-proteasome degradation. JNJ-26854165 and RO5045337 are 2 small-molecule inhibitors of MDM2 in clinical development. ALK is a transmembrane protein and a member of the insulin receptor tyrosine kinases. EML4-ALK fusion gene is identified in approximately 3-13% of non-small cell lung cancer (NSCLC. Early-phase clinical studies with Crizotinib, an ALK inhibitor, in NSCLC harboring EML4-ALK have demonstrated promising activity with high response rate and prolonged progression-free survival. PARPs are a family of nuclear enzymes that regulates the repair of DNA single-strand breaks through the base excision repair pathway. Randomized phase II study has shown adding PARP-1 inhibitor BSI-201 to cytotoxic chemotherapy improves clinical outcome in patients with triple-negative breast cancer. Olaparib, another oral small-molecule PARP inhibitor, demonstrated encouraging single-agent activity in patients with advanced breast or ovarian cancer. There are 5 other PARP inhibitors currently under active clinical investigation.

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

International Nuclear Information System (INIS)

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

1998-01-01

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

Adipose tissue NAD+-homeostasis, sirtuins and poly(ADP-ribose) polymerases -important players in mitochondrial metabolism and metabolic health.

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Jokinen, Riikka; Pirnes-Karhu, Sini; Pietiläinen, Kirsi H; Pirinen, Eija

2017-08-01

Obesity, a chronic state of energy overload, is characterized by adipose tissue dysfunction that is considered to be the major driver for obesity associated metabolic complications. The reasons for adipose tissue dysfunction are incompletely understood, but one potential contributing factor is adipose tissue mitochondrial dysfunction. Derangements of adipose tissue mitochondrial biogenesis and pathways associate with obesity and metabolic diseases. Mitochondria are central organelles in energy metabolism through their role in energy derivation through catabolic oxidative reactions. The mitochondrial processes are dependent on the proper NAD + /NADH redox balance and NAD + is essential for reactions catalyzed by the key regulators of mitochondrial metabolism, sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs). Notably, obesity is associated with disturbed adipose tissue NAD + homeostasis and the balance of SIRT and PARP activities. In this review we aim to summarize existing literature on the maintenance of intracellular NAD + pools and the function of SIRTs and PARPs in adipose tissue during normal and obese conditions, with the purpose of comprehending their potential role in mitochondrial derangements and obesity associated metabolic complications. Understanding the molecular mechanisms that are the root cause of the adipose tissue mitochondrial derangements is crucial for developing new effective strategies to reverse obesity associated metabolic complications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Quercetin derivatives as non-nucleoside inhibitors for dengue polymerase: molecular docking, molecular dynamics simulation, and binding free energy calculation.

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Anusuya, Shanmugam; Gromiha, M Michael

2017-10-01

Dengue is an important public health problem in tropical and subtropical regions of the world. Neither vaccine nor an antiviral medication is available to treat dengue. This insists the need of drug discovery for dengue. In order to find a potent lead molecule, RNA-dependent RNA polymerase which is essential for dengue viral replication is chosen as a drug target. As Quercetin showed antiviral activity against several viruses, quercetin derivatives developed by combinatorial library synthesis and mined from PubChem databases were screened for a potent anti-dengue viral agent. Our study predicted Quercetin 3-(6″-(E)-p-coumaroylsophoroside)-7-rhamnoside as a dengue polymerase inhibitor. The results were validated by molecular dynamics simulation studies which reveal water bridges and hydrogen bonds as major contributors for the stability of the polymerase-lead complex. Interactions formed by this compound with residues Trp795, Arg792 and Glu351 are found to be essential for the stability of the polymerase-lead complex. Our study demonstrates Quercetin 3-(6″-(E)-p-coumaroylsophoroside)-7-rhamnoside as a potent non-nucleoside inhibitor for dengue polymerase.

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

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Robu, Mihaela; Shah, Rashmi G; Purohit, Nupur K; Zhou, Pengbo; Naegeli, Hanspeter; Shah, Girish M

2017-08-15

Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

Evaluation of candidate biomarkers to predict cancer cell sensitivity or resistance to PARP-1 inhibitor treatment

DEFF Research Database (Denmark)

Oplustilova, L.; Wolanin, K.; Bartkova, J.

2012-01-01

combinations with camptothecin or ionizing radiation. Furthermore, monitoring pARsylation and Rad51 foci formation as surrogate markers for PARP activity and HR, respectively, supported their candidacy for biomarkers of PARP-1i responses. As to resistance mechanisms, we confrmed the role of the multidrug......(ADp-ribose) polymerase-1 (PARP-1), an enzyme critical for repair pathways alternative to HR. While promising, treatment with PARP-1 inhibitors (PARP-1i) faces some hurdles, including (1) acquired resistance, (2) search for other sensitizing, non-BRCA1/2 cancer defects and (3) lack of biomarkers to predict response...

Minocycline attenuates streptomycin-induced cochlear hair cell death by inhibiting protein nitration and poly (ADP-ribose) polymerase activation.

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Wang, Ping; Li, Haonan; Yu, Shuyuan; Jin, Peng; Hassan, Abdurahman; Du, Bo

2017-08-24

This study aimed to elucidate the protective effect of minocycline against streptomycin-induced damage of cochlear hair cells and its mechanism. Cochlear membranes were isolated from newborn Wistar rats and randomly divided into control, 500μmol/L streptomycin, 100μmol/L minocycline, and streptomycin and minocycline treatment groups. Hair cell survival was analyzed by detecting the expression of 3-nitrotyrosine (3-NT) in cochlear hair cells by immunofluorescence and an enzyme-linked immunosorbent assay. Expression of 3-NT and inducible nitric oxide synthase (iNOS), and poly (ADP-Ribose) polymerase (PARP) and caspase-3 activation were evaluated by western blotting. The results demonstrated hair cell loss at 24h after streptomycin treatment. No change was found in supporting cells of the cochleae. Minocycline pretreatment improved hair cell survival and significantly reduced the expression of iNOS and 3-NT in cochlear tissues compared with the streptomycin treatment group. PARP and caspase-3 activation was increased in the streptomycin treatment group compared with the control group, and pretreatment with minocycline decreased cleaved PARP and activated caspase-3 expression. Minocycline protected cochlear hair cells from injury caused by streptomycin in vitro. The mechanism underlying the protective effect may be associated with the inhibition of excessive formation of nitric oxide, reduction of the nitration stress reaction, and inhibition of PARP and caspase-3 activation in cochlear hair cells. Combined minocycline therapy can be applied to patients requiring streptomycin treatment. Copyright © 2017. Published by Elsevier B.V.

Cytological and molecular studies of chromosomal radiosensitivity in Down Syndrome cells

International Nuclear Information System (INIS)

MacLaren, R.A.

1988-01-01

Molecular, cellular and cytogenetic studies were conducted to determine if altered levels of poly(ADP-ribose) polymerase, a DNA repair-related enzyme, is responsible for the reported formation of excess X-ray induced chromosome aberrations in cells derived from Down Syndrome (DS) patients. Nonstimulated lymphocytes from normal and DS subjects were pretreated with 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, for 30 minutes before exposure to X-rays and the levels of induced chromosome aberrations were determined in mitotic cells. DS lymphocytes exhibited significantly higher frequencies of chromosome aberrations in the presence of 3-aminobenzamide that normal lymphocytes. No difference was observed in the absence of 3-aminobenzamide. Additional studies were done using normal and DS lymphoblastoid cell lines which exhibited a similar response at the DNA level as the lymphocytes. Analysis of poly(ADP-ribose) polymerase activity based on incorporation of the substrate, NAD + , into acid insoluble materials, revealed that there was no significant difference in the ability to form poly (ADP-ribose) in the DS or normal cells. 3-aminobenzamide effectively inhibited poly(ADP-ribose) polymerase in both the normal and DS cells

Inhibition of gamma-ray dose-rate effects by D2O and inhibitors of poly(ADP-ribose) synthetase in cultured mammalian L5178Y cells

International Nuclear Information System (INIS)

Ueno, A.M.; Tanaka, O.; Matsudaira, H.

1984-01-01

Effects of deuterium oxide (D 2 O) and 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, on cell proliferation and survival were studied in cultured mammalian L5178Y cells under growing conditions and after acute and low-dose-rate irradiation at about 0.1 to 0.4 Gy/hr of γ rays. Growth of irradiated and unirradiated cells was inhibited by 45% D 2 O but not by 3-aminobenzamide at 10mM, except for treatments longer than 30 hr. The presence of these agents either alone or in combination during irradiation at low dose rates suppressed almost totally the decrease in cell killing due to the decrease in dose rate. Among other inhibitors tested, theobromine and theophylline were found to be effective in eliminating the dose-rate effects of γ rays. Possible mechanisms underlying the inhibition are discussed

NS5B RNA dependent RNA polymerase inhibitors: the promising approach to treat hepatitis C virus infections.

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Deore, R R; Chern, J-W

2010-01-01

Hepatitis C virus (HCV), a causative agent for non-A and non-B hepatitis, has infected approximately 3% of world's population. The current treatment option of ribavirin in combination with pegylated interferon possesses lower sustained virological response rates, and has serious disadvantages. Unfortunately, no prophylactic vaccine has been approved yet. Therefore, there is an unmet clinical need for more effective and safe anti-HCV drugs. HCV NS5B RNA dependent RNA polymerase is currently pursued as the most popular target to develop safe anti-HCV agents, as it is not expressed in uninfected cells. More than 25 pharmaceutical companies and some research groups have developed ≈50 structurally diverse scaffolds to inhibit NS5B. Here we provide comprehensive account of the drug development process of these scaffolds. NS5B polymerase inhibitors have been broadly classified in nucleoside and non nucleoside inhibitors and are sub classified according to their mechanism of action and structural diversities. With some additional considerations about the inhibitor bound NS5B enzyme X-ray crystal structure information and pharmacological aspects of the inhibitors, this review summarizes the lead identification, structure activity relationship (SAR) studies leading to the most potent NS5B inhibitors with subgenomic replicon activity.

Understanding D-Ribose and Mitochondrial Function

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Diane E. Mahoney

2018-02-01

Full Text Available Mitochondria are important organelles referred to as cellular powerhouses for their unique properties of cellular energy production.  With many pathologic conditions and aging, mitochondrial function declines, and there is a reduction in the production of adenosine triphosphate. The energy carrying molecule generated by cellular respiration and by pentose phosphate pathway, an alternative pathway of glucose metabolism. D-ribose is a naturally occurring monosaccharide found in the cells and particularly in the mitochondria is essential in energy production. Without sufficient energy, cells cannot maintain integrity and function. Supplemental D-ribose has been shown to improve cellular processes when there is mitochondrial dysfunction. When individuals take supplemental D-ribose, it can bypass part of the pentose pathway to produce D-ribose-5-phosphate for the production of energy. In this article, we review how energy is produced by cellular respiration, the pentose pathway, and the use of supplemental D-ribose.

Role of glycolysis inhibition and poly(ADP-ribose) polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemic cells.

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Verrax, Julien; Vanbever, Stéphanie; Stockis, Julie; Taper, Henryk; Calderon, Pedro Buc

2007-03-15

Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells. (c) 2006 Wiley-Liss, Inc.

Positive transcriptional regulation of the human micro opioid receptor gene by poly(ADP-ribose) polymerase-1 and increase of its DNA binding affinity based on polymorphism of G-172 -> T.

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Ono, Takeshi; Kaneda, Toshio; Muto, Akihiro; Yoshida, Tadashi

2009-07-24

Micro opioid receptor (MOR) agonists such as morphine are applied widely in clinical practice as pain therapy. The effects of morphine through MOR, such as analgesia and development of tolerance and dependence, are influenced by individual specificity. Recently, we analyzed single nucleotide polymorphisms on the human MOR gene to investigate the factors that contribute to individual specificity. In process of single nucleotide polymorphisms analysis, we found that specific nuclear proteins bound to G(-172) --> T region in exon 1 in MOR gene, and its affinity to DNA was increased by base substitution from G(-172) to T(-172). The isolated protein was identified by mass spectrometry and was confirmed by Western blotting to be poly(ADP-ribose) polymerase-1 (PARP-1). The overexpressed PARP-1 bound to G(-172) --> T and enhanced the transcription of reporter vectors containing G(-172) and T(-172). Furthermore, PARP-1 inhibitor (benzamide) decreased PARP-1 binding to G(-172) --> T without affecting mRNA or protein expression level of PARP-1 and down-regulated the subsequent MOR gene expression in SH-SY5Y cells. Moreover, we found that tumor necrosis factor-alpha enhanced MOR gene expression as well as increased PARP-1 binding to the G(-172) --> T region and G(-172) --> T-dependent transcription in SH-SY5Y cells. These effects were also inhibited by benzamide. In this study, our data suggest that PARP-1 positively regulates MOR gene transcription via G(-172) --> T, which might influence individual specificity in therapeutic opioid effects.

Molecular basis for the regulation of hypoxia-inducible factor-1α levels by 2-deoxy-D-ribose.

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Ikeda, Ryuji; Tabata, Sho; Tajitsu, Yusuke; Nishizawa, Yukihiko; Minami, Kentaro; Furukawa, Tatsuhiko; Yamamoto, Masatatsu; Shinsato, Yoshinari; Akiyama, Shin-Ichi; Yamada, Katsushi; Takeda, Yasuo

2013-09-01

The angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, inhibits the upregulation of hypoxia-inducible factor (HIF) 1α, BNIP3 and caspase-3 induced by hypoxia. In the present study, we investigated the molecular basis for the suppressive effect of 2-deoxy-D-ribose on the upregulation of HIF-1α. 2-Deoxy-D-ribose enhanced the interaction of HIF-1α and the von Hippel-Lindau (VHL) protein under hypoxic conditions. It did not affect the expression of HIF-1α, prolyl hydroxylase (PHD)1/2/3 and VHL mRNA under normoxic or hypoxic conditions, but enhanced the interaction of HIF-1α and PHD2 under hypoxic conditions. 2-Deoxy-D-ribose also increased the amount of hydroxy-HIF-1α in the presence of the proteasome inhibitor MG-132. The expression levels of TP are elevated in many types of malignant solid tumors and, thus, 2-deoxy-D-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis.

Mutagenicity of γ-irradiated oxygenated and deoxygenated solutions of 2-deoxy-D-ribose and D-ribose in Salmonella typhimurium

International Nuclear Information System (INIS)

Wilmer, J.; Leveling, H.; Schubert, J.

1981-01-01

Solutions of 2-deoxy-D-ribose and D-ribose were γ-irradiated under different experimental conditions and tested for mutagenicity, with and without preincubation, in Salmonella typhimurium. The irradiated sugar solutions were mutagenic in the tester strains TA 100 and TA 98. Except for malonaldehyde (MDA), which is not mutagenic in the concentrations produced radiolytically, the relative mutagenicities of the individual radiolytic products are unknown. With irradiated solutions of 2-deoxy-D-ribose, a relationship was found between the level of non-MDA aldehydes and the mutagenicity in TA 100. Heating the irradiated solutions of 2-deoxy-D-ribose resulted in a temperature-dependent reduction fo the mutagenicity. Autoclaved, non-irradiated solutions of 2-deoxy-D-ribose were not mutagenic in the Salmonella test. (orig.)

d-Ribose as a Contributor to Glycated Haemoglobin

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Xixi Chen

2017-11-01

Full Text Available Glycated haemoglobin (HbA1c is the most important marker of hyperglycaemia in diabetes mellitus. We show that d-ribose reacts with haemoglobin, thus yielding HbA1c. Using mass spectrometry, we detected glycation of haemoglobin with d-ribose produces 10 carboxylmethyllysines (CMLs. The first-order rate constant of fructosamine formation for d-ribose was approximately 60 times higher than that for d-glucose at the initial stage. Zucker Diabetic Fatty (ZDF rat, a common model for type 2 diabetes mellitus (T2DM, had high levels of d-ribose and HbA1c, accompanied by a decrease of transketolase (TK in the liver. The administration of benfotiamine, an activator of TK, significantly decreased d-ribose followed by a decline in HbA1c. In clinical investigation, T2DM patients with high HbA1c had a high level of urine d-ribose, though the level of their urine d-glucose was low. That is, d-ribose contributes to HbA1c, which prompts future studies to further explore whether d-ribose plays a role in the pathophysiological mechanism of T2DM.

The nuclear protein Poly(ADP-ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana.

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Rissel, D; Losch, J; Peiter, E

2014-11-01

The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP-ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear-localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3-1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col-0 wild-type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Effects of DNA polymerase inhibitors on replicative and repair DNA synthesis in ultraviolet-irradiated HeLa cells

International Nuclear Information System (INIS)

Morita, T.; Nakamura, H.; Tsutsui, Y.; Nishiyama, Y.; Yoshida, S.

1982-01-01

Aphidicolin specifically inhibits eukaryotic DNA polymerase α, while 2',3'-dideoxythymidine 5'-triphosphate (d 2 TTP) inhibits DNA polymerase ν and ν but not α. 1-ν-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) inhibits both DNA polymerase α and ν although to a different extent. Here we measured the effects of these inhibitors on repair DNA synthesis of U.V.-irradiated HeLa cells by two different methods. Firstly, aphidicolin, 1-ν-D-arabinofuranosylcytosine (araC, a precursor of araCTP) and 2',3'-dideoxythimidine (d 2 Thd, a precursor of d 2 TTP) were added directly to the culture medium. In this case, aphidicolin and araC strongly inhibited replicative DNA synthesis of HeLa cells, and they also inhibited repair synthesis after U.V.-irradiation but to a much lesser extent. In contrast, high concentrations of d 2 Thd inhibited repair DNA synthesis to a higher extent than replicative DNA synthesis. Secondly, the active form of inhibitor, d 2 TTP, was microinjected directly into cytoplasm or nuclei or U.V.-irradiated HeLa cells. Microinjection of d 2 TTP effectively inhibited repair synthesis. The microinjection of d 2 TTP, into either cytoplasm or nucleus, strongly inhibited replicative synthesis. These results might indicate that multiple DNA polymerases are involved in repair synthesis as well as in replicative synthesis

A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor

International Nuclear Information System (INIS)

Yashiroda, Yoko; Okamoto, Reika; Hatsugai, Kaori; Takemoto, Yasushi; Goshima, Naoki; Saito, Tamio; Hamamoto, Makiko; Sugimoto, Yoshikazu; Osada, Hiroyuki; Seimiya, Hiroyuki; Yoshida, Minoru

2010-01-01

The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.

Inhibition of gamma-ray dose-rate effects by D/sup 2/O and inhibitors of poly(ADP-ribose) synthetase in cultured mammalian L5178Y cells

Energy Technology Data Exchange (ETDEWEB)

Ueno, A.M.; Tanaka, O.; Matsudaira, H.

1984-06-01

Effects of deuterium oxide (D/sub 2/O) and 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, on cell proliferation and survival were studied in cultured mammalian L5178Y cells under growing conditions and after acute and low-dose-rate irradiation at about 0.1 to 0.4 Gy/hr of ..gamma.. rays. Growth of irradiated and unirradiated cells was inhibited by 45% D/sub 2/O but not by 3-aminobenzamide at 10mM, except for treatments longer than 30 hr. The presence of these agents either alone or in combination during irradiation at low dose rates suppressed almost totally the decrease in cell killing due to the decrease in dose rate. Among other inhibitors tested, theobromine and theophylline were found to be effective in eliminating the dose-rate effects of ..gamma.. rays. Possible mechanisms underlying the inhibition are discussed.

The dual role of poly(ADP-ribose) polymerase-1 in modulating parthanatos and autophagy under oxidative stress in rat cochlear marginal cells of the stria vascularis.

Science.gov (United States)

Jiang, Hong-Yan; Yang, Yang; Zhang, Yuan-Yuan; Xie, Zhen; Zhao, Xue-Yan; Sun, Yu; Kong, Wei-Jia

2018-04-01

Oxidative stress is reported to regulate several apoptotic and necrotic cell death pathways in auditory tissues. Poly(ADP-ribose) polymerase-1 (PARP-1) can be activated under oxidative stress, which is the hallmark of parthanatos. Autophagy, which serves either a pro-survival or pro-death function, can also be stimulated by oxidative stress, but the role of autophagy and its relationship with parthanatos underlying this activation in the inner ear remains unknown. In this study, we established an oxidative stress model in vitro by glucose oxidase/glucose (GO/G), which could continuously generate low concentrations of H 2 O 2 to mimic continuous exposure to H 2 O 2 in physiological conditions, for investigation of oxidative stress-induced cell death mechanisms and the regulatory role of PARP-1 in this process. We observed that GO/G induced stria marginal cells (MCs) death via upregulation of PARP-1 expression, accumulation of polyADP-ribose (PAR) polymers, decline of mitochondrial membrane potential (MMP) and nuclear translocation of apoptosis-inducing factor (AIF), which all are biochemical features of parthanatos. PARP-1 knockdown rescued GO/G-induced MCs death, as well as abrogated downstream molecular events of PARP-1 activation. In addition, we demonstrated that GO/G stimulated autophagy and PARP-1 knockdown suppressed GO/G-induced autophagy in MCs. Interestingly, autophagy suppression by 3-Methyladenine (3-MA) accelerated GO/G-induced parthanatos, indicating a pro-survival function of autophagy in GO/G-induced MCs death. Taken together, these data suggested that PARP-1 played dual roles by modulating parthanatos and autophagy in oxidative stress-induced MCs death, which may be considered as a promising therapeutic target for ameliorating oxidative stress-related hearing disorders. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

DNA polymerase inhibitors and heat alter fixation of postirradiation sublethal damage in L5178Y-S cells

International Nuclear Information System (INIS)

Kapiszewska, M.; Szumiel, I.; Lange, C.S.

1988-01-01

We have used the inhibitor of DNA polymerase alpha, aphidicolin (apc) (0.5 μg/ml for 1 h), or that of DNA polymerase beta, dideoxythymidine triphosphate (ddTTP) (5 μg/ml), as well as heat (15 min at 43 deg C) to examine fixation of sublethal damage (SLD) induced by X-rays in L5178Y-S (LY-S) cells. This cell line has the unique property of responding to split X-ray doses at 37 deg C by decreased survival. This effect was partly abolished by heating the cells before irradiation with the second dose; the protection was most pronounced when the cells were heated 30-120 min. after the first dose of radiation. Since similar changes in postirradiation survival occurred when ddTTP was applied, we suggest that heat induces a loss of polymerase beta activity. Apc gave a smaller protective effect. We interpreted these results as suggesting that mismatching takes place during DNA semiconservative replication or repair; inhibition of replication results in survival increase, by preventing misrepair. A proper timing of treatment with the inhibitors or heat is essential to obtain the sparing effect, i.e. to prevent SLD fixation. 27 refs., 3 figs., 1 tab. (author)

Niraparib

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... Niraparib is in a class of medications called poly (ADP-ribose) polymerase (PARP) inhibitors. It works by ... sores in the mouth loss of appetite back pain headache dizziness changes in taste difficulty falling asleep ...

Rucaparib

Science.gov (United States)

... Rucaparib is in a class of medications called poly (ADP-ribose) polymerase (PARP) inhibitors. It works by ... not go away: nausea vomiting constipation diarrhea stomach pain loss of appetite bad taste in the mouth ...

Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer

DEFF Research Database (Denmark)

Mirza, Mansoor R; Monk, Bradley J; Herrstedt, Jørn

2016-01-01

Background Niraparib is an oral poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We sought to evaluate the efficacy of niraparib versus placebo as maintenance treatment for patients with platinum-sensitive, ......Background Niraparib is an oral poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We sought to evaluate the efficacy of niraparib versus placebo as maintenance treatment for patients with platinum...... or 4 adverse events that were reported in the niraparib group were thrombocytopenia (in 33.8%), anemia (in 25.3%), and neutropenia (in 19.6%), which were managed with dose modifications. Conclusions Among patients with platinum-sensitive, recurrent ovarian cancer, the median duration of progression...

Taurine prevents arsenic-induced cardiac oxidative stress and apoptotic damage: Role of NF-κB, p38 and JNK MAPK pathway

International Nuclear Information System (INIS)

Ghosh, Jyotirmoy; Das, Joydeep; Manna, Prasenjit; Sil, Parames C.

2009-01-01

Cardiac dysfunction is a major cause of morbidity and mortality worldwide due to its complex pathogenesis. However, little is known about the mechanism of arsenic-induced cardiac abnormalities and the use of antioxidants as the possible protective agents in this pathophysiology. Conditionally essential amino acid, taurine, accounts for 25% to 50% of the amino acid pool in myocardium and possesses antioxidant properties. The present study has, therefore, been carried out to investigate the underlying mechanism of the beneficial role of taurine in arsenic-induced cardiac oxidative damage and cell death. Arsenic reduced cardiomyocyte viability, increased reactive oxygen species (ROS) production and intracellular calcium overload, and induced apoptotic cell death by mitochondrial dependent caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. These changes due to arsenic exposure were found to be associated with increased IKK and NF-κB (p65) phosphorylation. Pre-exposure of myocytes to an IKK inhibitor (PS-1145) prevented As-induced caspase-3 and PARP cleavage. Arsenic also markedly increased the activity of p38 and JNK MAPKs, but not ERK to that extent. Pre-treatment with SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated NF-κB and IKK phosphorylation indicating that p38 and JNK MAPKs are mainly involved in arsenic-induced NF-κB activation. Taurine treatment suppressed these apoptotic actions, suggesting that its protective role in arsenic-induced cardiomyocyte apoptosis is mediated by attenuation of p38 and JNK MAPK signaling pathways. Similarly, arsenic intoxication altered a number of biomarkers related to cardiac oxidative stress and other apoptotic indices in vivo and taurine supplementation could reduce it. Results suggest that taurine prevented arsenic-induced myocardial pathophysiology, attenuated NF-κB activation via IKK, p38 and JNK MAPK signaling pathways and could possibly provide a protection against As

Research progress of PARP-1 inhibitors in antitumor drugs and radionuclide markers

International Nuclear Information System (INIS)

Zhao Lingzhou; Zhang Huabei

2011-01-01

Poly(ADP-ribose)polymerase (PARP) is a new target in the cancer treatment nowadays. PARP not only can repair DNA damage, regulate and control transcription, maintain the stability of intracellular environment and genome, regulate the process of cell survival and death, but also is the main transcription factor in the development of inflammation and the process of cancer. To inhibit PARP activity can reduce the DNA repair function in tumor cells, and increase the sensibility to DNA damage agents, so as to improve the efficacy of radiation therapy and chemotherapy for tumor. A number of studies have suggested that, whether used alone or combination with other chemotherapy drugs, PARP inhibitors show the potential in the anti-tumor therapeutic areas. In this paper, PARP-1 inhibitors were reviewed in antitumor research progress. According to the stage of development , PARP-1 inhibitors are classified. Several representative PARP-1 inhibitors, in clinical trials, with potential clinical value were introduced. Positron emission tomography (PET), uses the main short half-life elementary in human body as tracer, and at the molecular level, achieve the no wound, quantitative and dynamic observation about the different changes of metabolites or drugs in the body. PET is the most advanced contemporary video diagnostic technology, and this paper simply introduce the research progress of PARP-1 inhibitors labeled with radioactive nuclides. (authors)

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

International Nuclear Information System (INIS)

Hammond, R.A.; Miller, M.R.; McClung, J.K.

1990-01-01

The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase α

International Nuclear Information System (INIS)

Maeda, Naoki; Kokai, Yasuo; Ohtani, Seiji; Sahara, Hiroeki; Kuriyama, Isoko; Kamisuki, Shinji; Takahashi, Shunya; Sakaguchi, Kengo; Sugawara, Fumio; Yoshida, Hiromi; Sato, Noriyuki; Mizushina, Yoshiyuki

2007-01-01

In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol α from a fungus (Alternaria tennuis). We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited only mammalian pol α with IC 50 value of 0.5 μM, and did not influence the activities of other replicative pols such as pols δ and ε, but also showed no effect on pol α activity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. The compound also inhibited the proliferation of human cancer cells with LD 50 values of 38.0-44.4 μM. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, dehydroaltenusin was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that increased tumor necrosis and decreased mitotic index were apparently detected by the compound in vivo. Therefore, dehydroaltenusin could be of interest as not only a mammalian pol α-specific inhibitor, but also as a candidate drug for anti-cancer treatment

Synergistic inhibition of Streptococcal biofilm by ribose and xylitol.

Science.gov (United States)

Lee, Heon-Jin; Kim, Se Chul; Kim, Jinkyung; Do, Aejin; Han, Se Yeong; Lee, Bhumgey David; Lee, Hyun Ho; Lee, Min Chan; Lee, So Hui; Oh, Taejun; Park, Sangbin; Hong, Su-Hyung

2015-02-01

Streptococcus mutans and Streptococcus sobrinus are the major causative agents of human dental caries. Therefore, the removal or inhibition of these streptococcal biofilms is essential for dental caries prevention. In the present study, we evaluated the effects of ribose treatment alone or in combination with xylitol on streptococcal biofilm formation for both species. Furthermore, we examined the expression of genes responsible for dextran-dependent aggregation (DDAG). In addition, we investigated whether ribose affects the biofilm formation of xylitol-insensitive streptococci, which results from long-term exposure to xylitol. The viability of streptococci biofilms formed in a 24-well polystyrene plate was quantified by fluorescent staining with the LIVE/DEAD bacterial viability and counting kit, which was followed by fluorescence activated cell sorting analysis. The effects of ribose and/or xylitol on the mRNA expression of DDAG-responsible genes, gbpC and dblB, was evaluated by RT-qPCR. Our data showed that ribose and other pentose molecules significantly inhibited streptococcal biofilm formation and the expression of DDAG-responsible genes. In addition, co-treatment with ribose and xylitol decreased streptococcal biofilm formation to a further extent than ribose or xylitol treatment alone in both streptococcal species. Furthermore, ribose attenuated the increase of xylitol-insensitive streptococcal biofilm, which results in the reduced difference of biofilm formation between S. mutans that are sensitive and insensitive to xylitol. These data suggest that pentose may be used as an additive for teeth-protective materials or in sweets. Furthermore, ribose co-treatment with xylitol might help to increase the anti-cariogenic efficacy of xylitol. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cediranib, a pan-VEGFR inhibitor, and olaparib, a PARP inhibitor, in combination therapy for high grade serous ovarian cancer.

Science.gov (United States)

Ivy, S Percy; Liu, Joyce F; Lee, Jung-Min; Matulonis, Ursula A; Kohn, Elise C

2016-01-01

An estimated 22,000 women are diagnosed annually with ovarian cancer in the United States. Initially chemo-sensitive, recurrent disease ultimately becomes chemoresistant and may kill ~14,000 women annually. Molecularly targeted therapy with cediranib (AZD2171), a vascular endothelial growth factor receptor (VEGFR)-1, 2, and 3 signaling blocker, and olaparib (AZD2281), a poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitor, administered orally in combination has shown anti-tumor activity in the treatment of high grade serous ovarian cancer (HGSOC). This combination has the potential to change the treatment of HGSOC. Preclinical and clinical studies of single agent cediranib and olaparib or their combination are reviewed. Data are presented from peer-reviewed published manuscripts, completed and ongoing early phase clinical trials registered in ClinicalTrials.gov, National Cancer Institute-sponsored clinical trials, and related recent abstracts. Advances in the treatment of HGSOC that improve progression-free and overall survival have proven elusive despite examination of molecularly targeted therapy. HGSOC patients with deleterious germline or somatic mutations in BRCA1 or BRCA2 (BRCAm) are most responsive to PARP inhibitors (PARPi). PARPi combined with angiogenesis inhibition improved anti-cancer response and duration in both BRCAm and BRCA wild type HGSOC patients, compared to olaparib single agent treatment, demonstrating therapeutic chemical and contextual synthetic lethality.

Functional genomic analysis of drug sensitivity pathways to guide adjuvant strategies in breast cancer

DEFF Research Database (Denmark)

Swanton, Charles; Szallasi, Zoltan Imre; Brenton, James D.

2008-01-01

The widespread introduction of high throughput RNA interference screening technology has revealed tumour drug sensitivity pathways to common cytotoxics such as paclitaxel, doxorubicin and 5-fluorouracil, targeted agents such as trastuzumab and inhibitors of AKT and Poly(ADP-ribose) polymerase (PARP...

Diabetic Neuropathy and Oxidative Stress: Therapeutic Perspectives

Directory of Open Access Journals (Sweden)

Asieh Hosseini

2013-01-01

Full Text Available Diabetic neuropathy (DN is a widespread disabling disorder comprising peripheral nerves' damage. DN develops on a background of hyperglycemia and an entangled metabolic imbalance, mainly oxidative stress. The majority of related pathways like polyol, advanced glycation end products, poly-ADP-ribose polymerase, hexosamine, and protein kinase c all originated from initial oxidative stress. To date, no absolute cure for DN has been defined; although some drugs are conventionally used, much more can be found if all pathophysiological links with oxidative stress would be taken into account. In this paper, although current therapies for DN have been reviewed, we have mainly focused on the links between DN and oxidative stress and therapies on the horizon, such as inhibitors of protein kinase C, aldose reductase, and advanced glycation. With reference to oxidative stress and the related pathways, the following new drugs are under study such as taurine, acetyl-L-carnitine, alpha lipoic acid, protein kinase C inhibitor (ruboxistaurin, aldose reductase inhibitors (fidarestat, epalrestat, ranirestat, advanced glycation end product inhibitors (benfotiamine, aspirin, aminoguanidine, the hexosamine pathway inhibitor (benfotiamine, inhibitor of poly ADP-ribose polymerase (nicotinamide, and angiotensin-converting enzyme inhibitor (trandolapril. The development of modern drugs to treat DN is a real challenge and needs intensive long-term comparative trials.

Diabetic Neuropathy and Oxidative Stress: Therapeutic Perspectives

Science.gov (United States)

Hosseini, Asieh; Abdollahi, Mohammad

2013-01-01

Diabetic neuropathy (DN) is a widespread disabling disorder comprising peripheral nerves' damage. DN develops on a background of hyperglycemia and an entangled metabolic imbalance, mainly oxidative stress. The majority of related pathways like polyol, advanced glycation end products, poly-ADP-ribose polymerase, hexosamine, and protein kinase c all originated from initial oxidative stress. To date, no absolute cure for DN has been defined; although some drugs are conventionally used, much more can be found if all pathophysiological links with oxidative stress would be taken into account. In this paper, although current therapies for DN have been reviewed, we have mainly focused on the links between DN and oxidative stress and therapies on the horizon, such as inhibitors of protein kinase C, aldose reductase, and advanced glycation. With reference to oxidative stress and the related pathways, the following new drugs are under study such as taurine, acetyl-L-carnitine, alpha lipoic acid, protein kinase C inhibitor (ruboxistaurin), aldose reductase inhibitors (fidarestat, epalrestat, ranirestat), advanced glycation end product inhibitors (benfotiamine, aspirin, aminoguanidine), the hexosamine pathway inhibitor (benfotiamine), inhibitor of poly ADP-ribose polymerase (nicotinamide), and angiotensin-converting enzyme inhibitor (trandolapril). The development of modern drugs to treat DN is a real challenge and needs intensive long-term comparative trials. PMID:23738033

Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer

NARCIS (Netherlands)

Mirza, M. R.; Monk, B. J.; Herrstedt, J.; Oza, A. M.; Mahner, S.; Redondo, A.; Fabbro, M.; Ledermann, J. A.; Lorusso, D.; Vergote, I.; Ben-Baruch, N. E.; Marth, C.; Madry, R.; Christensen, R. D.; Berek, J. S.; Dorum, A.; Tinker, A. V.; du Bois, A.; Gonzalez-Martin, A.; Follana, P.; Benigno, B.; Rosenberg, P.; Gilbert, L.; Rimel, B. J.; Buscema, J.; Balser, J. P.; Agarwal, S.; Matulonis, U. A.; van der Zee, A.G.J.

2016-01-01

BACKGROUND Niraparib is an oral poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We sought to evaluate the efficacy of niraparib versus placebo as maintenance treatment for patients with platinum-sensitive,

Nucleolar integrity is required for the maintenance of long-term synaptic plasticity.

Directory of Open Access Journals (Sweden)

Kim D Allen

Full Text Available Long-term memory (LTM formation requires new protein synthesis and new gene expression. Based on our work in Aplysia, we hypothesized that the rRNA genes, stimulation-dependent targets of the enzyme Poly(ADP-ribose polymerase-1 (PARP-1, are primary effectors of the activity-dependent changes in synaptic function that maintain synaptic plasticity and memory. Using electrophysiology, immunohistochemistry, pharmacology and molecular biology techniques, we show here, for the first time, that the maintenance of forskolin-induced late-phase long-term potentiation (L-LTP in mouse hippocampal slices requires nucleolar integrity and the expression of new rRNAs. The activity-dependent upregulation of rRNA, as well as L-LTP expression, are poly(ADP-ribosylation (PAR dependent and accompanied by an increase in nuclear PARP-1 and Poly(ADP ribose molecules (pADPr after forskolin stimulation. The upregulation of PARP-1 and pADPr is regulated by Protein kinase A (PKA and extracellular signal-regulated kinase (ERK--two kinases strongly associated with long-term plasticity and learning and memory. Selective inhibition of RNA Polymerase I (Pol I, responsible for the synthesis of precursor rRNA, results in the segmentation of nucleoli, the exclusion of PARP-1 from functional nucleolar compartments and disrupted L-LTP maintenance. Taken as a whole, these results suggest that new rRNAs (28S, 18S, and 5.8S ribosomal components--hence, new ribosomes and nucleoli integrity--are required for the maintenance of long-term synaptic plasticity. This provides a mechanistic link between stimulation-dependent gene expression and the new protein synthesis known to be required for memory consolidation.

Cockayne syndrome group A and B proteins converge on transcription-linked resolution of non-B DNA.

Science.gov (United States)

Scheibye-Knudsen, Morten; Tseng, Anne; Borch Jensen, Martin; Scheibye-Alsing, Karsten; Fang, Evandro Fei; Iyama, Teruaki; Bharti, Sanjay Kumar; Marosi, Krisztina; Froetscher, Lynn; Kassahun, Henok; Eckley, David Mark; Maul, Robert W; Bastian, Paul; De, Supriyo; Ghosh, Soumita; Nilsen, Hilde; Goldberg, Ilya G; Mattson, Mark P; Wilson, David M; Brosh, Robert M; Gorospe, Myriam; Bohr, Vilhelm A

2016-11-01

Cockayne syndrome is a neurodegenerative accelerated aging disorder caused by mutations in the CSA or CSB genes. Although the pathogenesis of Cockayne syndrome has remained elusive, recent work implicates mitochondrial dysfunction in the disease progression. Here, we present evidence that loss of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number of cell lines. Furthermore, machine-learning algorithms predict that diseases with defects in ribosomal DNA (rDNA) transcription have mitochondrial dysfunction, and, accordingly, this is found when factors involved in rDNA transcription are knocked down. Mechanistically, loss of CSA or CSB leads to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans In conclusion, this work supports a role for impaired ribosomal DNA transcription in Cockayne syndrome and suggests that transcription-coupled resolution of secondary structures may be a mechanism to repress spurious activation of a DNA damage response.

Activity of a potent hepatitis C virus polymerase inhibitor in the chimpanzee model.

Science.gov (United States)

Chen, Chih-Ming; He, Yupeng; Lu, Liangjun; Lim, Hock Ben; Tripathi, Rakesh L; Middleton, Tim; Hernandez, Lisa E; Beno, David W A; Long, Michelle A; Kati, Warren M; Bosse, Todd D; Larson, Daniel P; Wagner, Rolf; Lanford, Robert E; Kohlbrenner, William E; Kempf, Dale J; Pilot-Matias, Tami J; Molla, Akhteruzzaman

2007-12-01

A-837093 is a potent and specific nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase. It possesses nanomolar potencies in both enzymatic and replicon-based cell culture assays. In rats and dogs this compound demonstrated an oral plasma half-life of greater than 7 h, and its bioavailability was >60%. In monkeys it had a half-life of 1.9 h and 15% bioavailability. Its antiviral efficacy was evaluated in two chimpanzees infected with HCV in a proof-of-concept study. The design included oral dosing of 30 mg per kg of body weight twice a day for 14 days, followed by a 14-day posttreatment observation. Maximum viral load reductions of 1.4 and 2.5 log(10) copies RNA/ml for genotype 1a- and 1b-infected chimpanzees, respectively, were observed within 2 days after the initiation of treatment. After this initial drop in the viral load, a rebound of plasma HCV RNA was observed in the genotype 1b-infected chimpanzee, while the genotype 1a-infected chimpanzee experienced a partial rebound that lasted throughout the treatment period. Clonal analysis of NS5B gene sequences derived from the plasma of A-837093-treated chimpanzees revealed the presence of several mutations associated with resistance to A-837093, including Y448H, G554D, and D559G in the genotype 1a-infected chimpanzee and C316Y and G554D in the genotype 1b-infected chimpanzee. The identification of resistance-associated mutations in both chimpanzees is consistent with the findings of in vitro selection studies, in which many of the same mutations were selected. These findings validate the antiviral efficacy and resistance development of benzothiadiazine HCV polymerase inhibitors in vivo.

Hyperthermal (1-100 eV) nitrogen ion scattering damage to D-ribose and 2-deoxy-D-ribose films.

Science.gov (United States)

Deng, Zongwu; Bald, Ilko; Illenberger, Eugen; Huels, Michael A

2007-10-14

Highly charged heavy ion traversal of a biological medium can produce energetic secondary fragment ions. These fragment ions can in turn cause collisional and reactive scattering damage to DNA. Here we report hyperthermal (1-100 eV) scattering of one such fragment ion (N(+)) from biologically relevant sugar molecules D-ribose and 2-deoxy-D-ribose condensed on polycrystalline Pt substrate. The results indicate that N(+) ion scattering at kinetic energies down to 10 eV induces effective decomposition of both sugar molecules and leads to the desorption of abundant cation and anion fragments. Use of isotope-labeled molecules (5-(13)C D-ribose and 1-D D-ribose) partly reveals some site specificity of the fragment origin. Several scattering reactions are also observed. Both ionic and neutral nitrogen atoms abstract carbon from the molecules to form CN(-) anion at energies down to approximately 5 eV. N(+) ions also abstract hydrogen from hydroxyl groups of the molecules to form NH(-) and NH(2) (-) anions. A fraction of OO(-) fragments abstract hydrogen to form OH(-). The formation of H(3)O(+) ions also involves hydrogen abstraction as well as intramolecular proton transfer. These findings suggest a variety of severe damaging pathways to DNA molecules which occur on the picosecond time scale following heavy ion irradiation of a cell, and prior to the late diffusion-limited homogeneous chemical processes.

Assessment of Hematological and Biochemical parameters with extended D-Ribose ingestion

Directory of Open Access Journals (Sweden)

Frelich Angela

2008-09-01

Full Text Available Abstract D-ribose, a naturally occurring pentose carbohydrate, has been shown to replenish high- energy phosphates following myocardial ischemia and high intensity, repetitive exercise. Human studies have mainly involved short-term assessment, including potential toxicity. Reports describing adverse effects of D-ribose with prolonged ingestion have been lacking. Therefore, this study assessed the toxicity of extended consumption of D-ribose in healthy adults. Nineteen subjects ingested 20 grams/Day (10 grams, twice a Day of ribose with serial measurements of biochemical and hematological parameters at Days 0, 7, and 14. No significant toxic changes over the 14-day assessment period occurred in complete blood count, albumin, alkaline phosphatase, gamma glutamyltransferase, alanine amiotransferase, and aspartate aminotransferase. However, D-ribose did produce an asymptomatic, mild hypoglycemia of short duration. Uric acid levels increased at Day 7, but decreased to baseline values by Day 14. D-ribose consumption for 14 days appears not to produce significant toxic changes in both hematological and biochemical parameters in healthy human volunteers.

Effect of veliparib (ABT-888) on cardiac repolarization in patients with advanced solid tumors : a randomized, placebo-controlled crossover study

NARCIS (Netherlands)

Munasinghe, Wijith; Stodtmann, Sven; Tolcher, Anthony; Calvo, Emiliano; Gordon, Michael; Jalving, Mathilde; de Vos-Geelen, Judith; Medina, Diane; Bergau, Dennis; Nuthalapati, Silpa; Hoffman, David; Shepherd, Stacie; Xiong, Hao

2016-01-01

Veliparib (ABT-888) is an orally bioavailable potent inhibitor of poly(ADP-ribose) polymerase (PARP)-1 and PARP-2. This phase 1 study evaluated the effect of veliparib on corrected QT interval using Fridericia's formula (QTcF). Eligible patients with advanced solid tumors received single-dose oral

Combinations of PARP Inhibitors with Temozolomide Drive PARP1 Trapping and Apoptosis in Ewing's Sarcoma.

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Gill, Sonja J; Travers, Jon; Pshenichnaya, Irina; Kogera, Fiona A; Barthorpe, Syd; Mironenko, Tatiana; Richardson, Laura; Benes, Cyril H; Stratton, Michael R; McDermott, Ultan; Jackson, Stephen P; Garnett, Mathew J

2015-01-01

Ewing's sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing's sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing's sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing's sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing's sarcoma patients with PARP inhibitors.

Spirulina maxima Extract Prevents Neurotoxicity via Promoting Activation of BDNF/CREB Signaling Pathways in Neuronal Cells and Mice.

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Koh, Eun-Jeong; Seo, Young-Jin; Choi, Jia; Lee, Hyeon Yong; Kang, Do-Hyung; Kim, Kui-Jin; Lee, Boo-Yong

2017-08-17

Spirulina maxima is a microalgae which contains flavonoids and other polyphenols. Although Spirulina maxima 70% ethanol extract (SM70EE) has diverse beneficial effects, its effects on neurotoxicity have not been fully understood. In this study, we investigated the neuroprotective effects of SM70EE against trimethyltin (TMT)-induced neurotoxicity in HT-22 cells. SM70EE inhibited the cleavage of poly-ADP ribose polymerase (PARP). Besides, ROS production was decreased by down-regulating oxidative stress-associated enzymes. SM70EE increased the factors of brain-derived neurotrophic factor (BDNF)/cyclic AMPresponsive elementbinding protein (CREB) signalling pathways. Additionally, acetylcholinesterase (AChE) was suppressed by SM70EE. Furthermore, we investigated whether SM70EE prevents cognitive deficits against scopolamine-induced neurotoxicity in mice by applying behavioral tests. SM70EE increased step-through latency time and decreased the escape latency time. Therefore, our data suggest that SM70EE may prevent TMT neurotoxicity through promoting activation of BDNF/CREB neuroprotective signaling pathways in neuronal cells. In vivo study, SM70EE would prevent cognitive deficits against scopolamine-induced neurotoxicity in mice.

MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption.

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Posavec Marjanović, Melanija; Hurtado-Bagès, Sarah; Lassi, Maximilian; Valero, Vanesa; Malinverni, Roberto; Delage, Hélène; Navarro, Miriam; Corujo, David; Guberovic, Iva; Douet, Julien; Gama-Perez, Pau; Garcia-Roves, Pablo M; Ahel, Ivan; Ladurner, Andreas G; Yanes, Oscar; Bouvet, Philippe; Suelves, Mònica; Teperino, Raffaele; Pospisilik, J Andrew; Buschbeck, Marcus

2017-11-01

Histone variants are structural components of eukaryotic chromatin that can replace replication-coupled histones in the nucleosome. The histone variant macroH2A1.1 contains a macrodomain capable of binding NAD + -derived metabolites. Here we report that macroH2A1.1 is rapidly induced during myogenic differentiation through a switch in alternative splicing, and that myotubes that lack macroH2A1.1 have a defect in mitochondrial respiratory capacity. We found that the metabolite-binding macrodomain was essential for sustained optimal mitochondrial function but dispensable for gene regulation. Through direct binding, macroH2A1.1 inhibits basal poly-ADP ribose polymerase 1 (PARP-1) activity and thus reduces nuclear NAD + consumption. The resultant accumulation of the NAD + precursor NMN allows for maintenance of mitochondrial NAD + pools that are critical for respiration. Our data indicate that macroH2A1.1-containing chromatin regulates mitochondrial respiration by limiting nuclear NAD + consumption and establishing a buffer of NAD + precursors in differentiated cells.

Cardioprotective effect of vitamin D2 on isoproterenol-induced myocardial infarction in diabetic rats.

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El Agaty, Sahar M

2018-03-08

To assess the effect of vitamin D 2 and to elucidate the underlying mechanisms on acute myocardial injury induced by isoproterenol (ISO) in diabetic rats. Rats were divided into control rats, diabetic rats (DM), diabetic rats received ISO (DM-ISO), and diabetic rats pretreated with vitamin D 2 and received ISO (DM-D 2 -ISO). Vitamin D 2 pretreatment significantly decreased fasting glucose and myocardial malondialdehyde, associated with increased insulin, myocardial glutathione and superoxide dismutase in DM-D 2 -ISO versus DM-ISO. The serum triglycerides, total cholesterol, and LDL were significantly decreased, along with increased HDL and adiponectin. Poly-ADP ribose polymerase, cyclooxygenase-2, tumour necrosis factor alpha, interleukin-6, caspase-3, BAX, and p53 were significantly downregulated in myocardium of DM-D 2 -ISO versus DM-ISO. Histological studies showed diminished inflammatory cells infiltration in myocardium of DM-D 2 -ISO versus DM-ISO. Vitamin D 2 ameliorates hyperglycaemia, dyslipidaemia, redox imbalance, inflammatory and apoptotic processes, protecting the myocardium of diabetic rats against acute myocardial infarction.

The cyclin-dependent kinase inhibitor flavopiridol disrupts sodium butyrate-induced p21WAF1/CIP1 expression and maturation while reciprocally potentiating apoptosis in human leukemia cells.

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Rosato, Roberto R; Almenara, Jorge A; Cartee, Leanne; Betts, Vicki; Chellappan, Srikumar P; Grant, Steven

2002-02-01

Interactions between the cyclin-dependent kinase inhibitor flavopiridol (FP) and the histone deacetylase inhibitor sodium butyrate (SB) have been examined in human leukemia cells (U937) in relation to differentiation and apoptosis. Whereas 1 mM of SB or 100 nM of FP minimally induced apoptosis (4% and 10%, respectively) at 24 h, simultaneous exposure of U937 cells to these agents dramatically increased cell death (e.g., approximately 60%), reflected by both morphological and Annexin/propidium iodide-staining features, procaspase 3 activation, and poly(ADP-ribose) polymerase cleavage. Similar interactions were observed in human promyelocytic (HL-60), B-lymphoblastic (Raji), and T-lymphoblastic (Jurkat) leukemia cells. Coadministration of FP opposed SB-mediated accumulation of cells in G0G1 and differentiation, reflected by reduced CD11b expression, but instead dramatically increased procaspase-3, procaspase-8, Bid, and poly(ADP-ribose) polymerase cleavage, as well as mitochondrial damage (e.g., loss of mitochondrial membrane potential and cytochrome c release). FP also blocked SB-related p21WAF1-CIP1 induction through a caspase-independent mechanism and triggered the caspase-mediated cleavage of p27KIP1 and retinoblastoma protein. The latter event was accompanied by a marked reduction in retinoblastoma protein/E2F1 complex formation. However, FP did not modify the extent of SB-associated acetylation of histones H3 and H4. Treatment of cells with FP/SB also resulted in the caspase-mediated cleavage of Bcl-2 and caspase-independent down-regulation of Mcl-1. Levels of cyclins A, D1, and E, and X-linked inhibitor of apoptosis also declined in SB/FP-treated cells. Finally, FP/SB coexposure potently induced apoptosis in two primary acute myelogenous leukemia samples. Together, these findings demonstrate that FP, when combined with SB, induces multiple perturbations in cell cycle and apoptosis regulatory proteins, which oppose leukemic cell differentiation but instead

Structural exploration for the refinement of anticancer matrix metalloproteinase-2 inhibitor designing approaches through robust validated multi-QSARs

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Adhikari, Nilanjan; Amin, Sk. Abdul; Saha, Achintya; Jha, Tarun

2018-03-01

Matrix metalloproteinase-2 (MMP-2) is a promising pharmacological target for designing potential anticancer drugs. MMP-2 plays critical functions in apoptosis by cleaving the DNA repair enzyme namely poly (ADP-ribose) polymerase (PARP). Moreover, MMP-2 expression triggers the vascular endothelial growth factor (VEGF) having a positive influence on tumor size, invasion, and angiogenesis. Therefore, it is an urgent need to develop potential MMP-2 inhibitors without any toxicity but better pharmacokinetic property. In this article, robust validated multi-quantitative structure-activity relationship (QSAR) modeling approaches were attempted on a dataset of 222 MMP-2 inhibitors to explore the important structural and pharmacophoric requirements for higher MMP-2 inhibition. Different validated regression and classification-based QSARs, pharmacophore mapping and 3D-QSAR techniques were performed. These results were challenged and subjected to further validation to explain 24 in house MMP-2 inhibitors to judge the reliability of these models further. All these models were individually validated internally as well as externally and were supported and validated by each other. These results were further justified by molecular docking analysis. Modeling techniques adopted here not only helps to explore the necessary structural and pharmacophoric requirements but also for the overall validation and refinement techniques for designing potential MMP-2 inhibitors.

Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxia-inducible factor-1alpha expression via poly(ADP-ribose) polymerase-1.

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Pan, Rong; Chen, Chen; Liu, Wen-Lan; Liu, Ke-Jian

2013-07-01

Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study tests the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-h hypoxic treatment. Although 3-h hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc-concentration-dependent manner. Exposure of astrocytes to hypoxia for 3 h remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably, HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. © 2013 John Wiley & Sons Ltd.

Molecular docking and 3D-QSAR studies on inhibitors of DNA damage signaling enzyme human PARP-1.

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Fatima, Sabiha; Bathini, Raju; Sivan, Sree Kanth; Manga, Vijjulatha

2012-08-01

Poly (ADP-ribose) polymerase-1 (PARP-1) operates in a DNA damage signaling network. Molecular docking and three dimensional-quantitative structure activity relationship (3D-QSAR) studies were performed on human PARP-1 inhibitors. Docked conformation obtained for each molecule was used as such for 3D-QSAR analysis. Molecules were divided into a training set and a test set randomly in four different ways, partial least square analysis was performed to obtain QSAR models using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Derived models showed good statistical reliability that is evident from their r², q²(loo) and r²(pred) values. To obtain a consensus for predictive ability from all the models, average regression coefficient r²(avg) was calculated. CoMFA and CoMSIA models showed a value of 0.930 and 0.936, respectively. Information obtained from the best 3D-QSAR model was applied for optimization of lead molecule and design of novel potential inhibitors.

SOX4 expression is associated with treatment failure and chemoradioresistance in oral squamous cell carcinoma

International Nuclear Information System (INIS)

Yoon, Tae Mi; Kim, Sun-Ae; Cho, Wan Seok; Lee, Dong Hoon; Lee, Joon Kyoo; Park, Young-Lan; Lee, Kyung-Hwa; Lee, Jae Hyuk; Kweon, Sun-Seog; Chung, Ik-Joo; Lim, Sang Chul; Joo, Young-Eun

2015-01-01

In humans, sex-determining region-Y (SRY) related high-mobility-group box 4 (SOX4) is linked to development and tumorigenesis. SOX4 is over-expressed in several cancers and has prognostic significance. This study evaluated whether SOX4 affects oncogenic behavior and chemoradiotherapy response in head and neck squamous cell carcinoma (HNSCC) cells, and documented the relationship between its expression and prognosis in oral squamous cell carcinoma (OSCC). We used small interfering RNA in HNSCC cells to evaluate the effect of SOX4 on cell proliferation, apoptosis, chemoradiation-induced apoptosis, invasion, and migration. SOX4 expression in OSCC tissues was investigated by immunohistochemistry. SOX4 knockdown (KO) decreased cell proliferation and induced apoptosis by activating caspases-3 and −7, and poly-ADP ribose polymerase and suppressing X-linked inhibitor of apoptosis protein in HNSCC cells; it also enhanced radiation/cisplatin-induced apoptosis; and suppressed tumor cell invasion and migration. Immunostaining showed SOX4 protein was significantly increased in OSCC tissues compared with adjacent normal mucosa. SOX4 expression was observed in 51.8 % of 85 OSCC tissues, and was significantly correlated with treatment failure (P = 0.032) and shorter overall survival (P = 0.036) in patients with OSCC. SOX4 may contribute to oncogenic phenotypes of HNSCC cells by promoting cell survival and causing chemoradioresistance. It could be a potential prognostic marker for OSCC. The online version of this article (doi:10.1186/s12885-015-1875-8) contains supplementary material, which is available to authorized users

The Juxtaposition of Ribose Hydroxyl Groups: The Root of Biological Catalysis and the RNA World?

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Bernhardt, Harold S.

2015-06-01

We normally think of enzymes as being proteins; however, the RNA world hypothesis suggests that the earliest biological catalysts may have been composed of RNA. One of the oldest surviving RNA enzymes we are aware of is the peptidyl transferase centre (PTC) of the large ribosomal RNA, which joins amino acids together to form proteins. Recent evidence indicates that the enzymatic activity of the PTC is principally due to ribose 2 '-OHs. Many other reactions catalyzed by RNA and/or in which RNA is a substrate similarly utilize ribose 2 '-OHs, including phosphoryl transfer reactions that involve the cleavage and/or ligation of the ribose-phosphate backbone. It has recently been proposed by Yakhnin (2013) that phosphoryl transfer reactions were important in the prebiotic chemical evolution of RNA, by enabling macromolecules composed of polyols joined by phosphodiester linkages to undergo recombination reactions, with the reaction energy supplied by the phosphodiester bond itself. The almost unique juxtaposition of the ribose 2'-hydroxyl and 3'-oxygen in ribose-containing polymers such as RNA, which gives ribose the ability to catalyze such reactions, may have been an important factor in the selection of ribose as a component of the first biopolymer. In addition, the juxtaposition of hydroxyl groups in free ribose: (i) allows coordination of borate ions, which could have provided significant and preferential stabilization of ribose in a prebiotic environment; and (ii) enhances the rate of permeation by ribose into a variety of lipid membrane systems, possibly favouring its incorporation into early metabolic pathways and an ancestral ribose-phosphate polymer. Somewhat more speculatively, hydrogen bonds formed by juxtaposed ribose hydroxyl groups may have stabilized an ancestral ribose-phosphate polymer against degradation (Bernhardt and Sandwick 2014). I propose that the almost unique juxtaposition of ribose hydroxyl groups constitutes the root of both biological

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

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

2009-12-15

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

Inhibitors of the Hepatitis C Virus Polymerase; Mode of Action and Resistance.

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Eltahla, Auda A; Luciani, Fabio; White, Peter A; Lloyd, Andrew R; Bull, Rowena A

2015-09-29

The hepatitis C virus (HCV) is a pandemic human pathogen posing a substantial health and economic burden in both developing and developed countries. Controlling the spread of HCV through behavioural prevention strategies has met with limited success and vaccine development remains slow. The development of antiviral therapeutic agents has also been challenging, primarily due to the lack of efficient cell culture and animal models for all HCV genotypes, as well as the large genetic diversity between HCV strains. On the other hand, the use of interferon-α-based treatments in combination with the guanosine analogue, ribavirin, achieved limited success, and widespread use of these therapies has been hampered by prevalent side effects. For more than a decade, the HCV RNA-dependent RNA polymerase (RdRp) has been targeted for antiviral development, and direct-acting antivirals (DAA) have been identified which bind to one of at least six RdRp inhibitor-binding sites, and are now becoming a mainstay of highly effective and well tolerated antiviral treatment for HCV infection. Here we review the different classes of RdRp inhibitors and their mode of action against HCV. Furthermore, the mechanism of antiviral resistance to each class is described, including naturally occurring resistance-associated variants (RAVs) in different viral strains and genotypes. Finally, we review the impact of these RAVs on treatment outcomes with the newly developed regimens.

Potent host-directed small-molecule inhibitors of myxovirus RNA-dependent RNA-polymerases.

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Stefanie A Krumm

Full Text Available Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid

Formation of nicotinamide ribose diphosphate ribose, a new metabolite of the NAD pathway, by growing mycelium of Aspergillus niger

International Nuclear Information System (INIS)

Kuwahara, Masaaki

1976-01-01

A new step of NAD metabolism was shown in Aspergillus niger. Radioactive nicotinic acid and nicotinamide were incorporated into nicotinamide ribose diphosphate ribose (NAm-RDPR), which had been isolated from the culture filtrate. Its content in the culture medium increased with an increase of culture time, and this compound was proved to be a terminal metabolite in the NAD pathway. The experimental results also showed that the Preiss-Handler pathway and the NAD cycling system function in the NAD biosynthesis in A. niger. A part of the radioactive precursors was also incorporated into an unknown compound. (auth.)

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

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Hwan-Young Kim

2014-01-01

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

PARP inhibitors protect against sex- and AAG-dependent alkylation-induced neural degeneration.

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Allocca, Mariacarmela; Corrigan, Joshua J; Fake, Kimberly R; Calvo, Jennifer A; Samson, Leona D

2017-09-15

Alkylating agents are commonly used to treat cancer. Although base excision repair (BER) is a major pathway for repairing DNA alkylation damage, under certain conditions, the initiation of BER produces toxic repair intermediates that damage healthy tissues. The initiation of BER by the alkyladenine DNA glycosylase (AAG, a.k.a. MPG) can mediate alkylation-induced cytotoxicity in specific cells in the retina and cerebellum of male mice. Cytotoxicity in both wild-type and Aag -transgenic ( AagTg ) mice is abrogated in the absence of Poly(ADP-ribose) polymerase-1 (PARP1). Here, we tested whether PARP inhibitors can also prevent alkylation-induced retinal and cerebellar degeneration in male and female WT and AagTg mice. Importantly, we found that WT mice display sex-dependent alkylation-induced retinal damage (but not cerebellar damage), with WT males being more sensitive than females. Accordingly, estradiol treatment protects males against alkylation-induced retinal degeneration. In AagTg male and female mice, the alkylation-induced tissue damage in both the retina and cerebellum is exacerbated and the sex difference in the retina is abolished. PARP inhibitors, much like Parp1 gene deletion, protect against alkylation-induced AAG-dependent neuronal degeneration in WT and AagTg mice, regardless of the gender, but their efficacy in preventing alkylation-induced neuronal degeneration depends on PARP inhibitor characteristics and doses. The recent surge in the use of PARP inhibitors in combination with cancer chemotherapeutic alkylating agents might represent a powerful tool for obtaining increased therapeutic efficacy while avoiding the collateral effects of alkylating agents in healthy tissues.

Inhibitor candidates's identification of HCV's RNA polymerase NS5B using virtual screening against iPPI-library

Science.gov (United States)

Sulistyawati, Indah; Sulistyo Dwi K., P.; Ichsan, Mochammad

2016-03-01

Hepatitis C is one of the major causes of chronic liver failure that caused by Hepatitis C Virus (HCV). Preventing the progression of HCV's replication through the inhibition of The RNA polymerase NS5B of Hepatitis C virus (NS5B) can be achieved via 4 binding regions: Site I (Thumb I), Site II (Thumb II), Site III (Palm I), and Site IV (Palm II). The aim of this research is to identify a candidate of NS5B inhibitor as an alternative for Hepatitis C treatment. An NS5B's 3D structure (PDB ID = 3D5M) used in this study has met some criteria of a good model to be used in virtual screening againts iPPI-lib using MTiOpenScreen webserver. The top two natural compounds resulted here then docked using Pyrix 0.8 and discovered trans-6-Benzamido-2-methyldecahydroisoquinoline (-9,1kcal/mol) and 2,4-dichloro-5-[4-(2 methoxyphenyl) piperazine-1-carbonyl]-N-[3-(trifluoromethyl)phenyl] benzenesulfonamide (9,4 kcal/mol) can bind to Tyr448 similar with all three established inhibitors, such as setrobuvir (-11,4 kcal/mol; site 3 inhibitor), CHEMBL379677 (-9,1 kcal/mol; site 1 inhibitor), and nesbuvir (-7,7 kcal/mol; site 4 inhibitor). The results of this study are relatively still needs to be tested, both in vitro and in vivo, in order to obtain more comprehensive knowledges as a follow-up of this predictive study.

Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.

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Siew Pheng Lim

2016-08-01

Full Text Available Flaviviruses comprise major emerging pathogens such as dengue virus (DENV or Zika virus (ZIKV. The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp domain of non-structural protein 5 (NS5. This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a "de novo" initiation mechanism. Crystal structures of the flavivirus RdRp revealed a "closed" conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the "GDD" active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed "N pocket". Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.

Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo

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Masaaki Yasukawa

2016-02-01

Full Text Available Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy. Poly(ADP-ribosylation is reported to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties, poly(ADP-ribose polymerase (PARP inhibitors are used for treatment of cancers, such as BRCA1/2 mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs. However, the effects on oral cancer have not been fully evaluated. In this study, we examined the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib showed synergetic effects with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281 significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have the potential to improve therapeutic strategies for oral cancer.

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

International Nuclear Information System (INIS)

Doucet-Chabeaud, G.

2000-01-01

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

Targeting EGFR induced oxidative stress by PARP1 inhibition in glioblastoma therapy.

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Nitta, Masayuki; Kozono, David; Kennedy, Richard; Stommel, Jayne; Ng, Kimberly; Zinn, Pascal O; Kushwaha, Deepa; Kesari, Santosh; Inda, Maria-del-Mar; Wykosky, Jill; Furnari, Frank; Hoadley, Katherine A; Chin, Lynda; DePinho, Ronald A; Cavenee, Webster K; D'Andrea, Alan; Chen, Clark C

2010-05-24

Despite the critical role of Epidermal Growth Factor Receptor (EGFR) in glioblastoma pathogenesis, EGFR targeted therapies have achieved limited clinical efficacy. Here we propose an alternate therapeutic strategy based on the conceptual framework of non-oncogene addiction. A directed RNAi screen revealed that glioblastoma cells over-expressing EGFRvIII, an oncogenic variant of EGFR, become hyper-dependent on a variety of DNA repair genes. Among these, there was an enrichment of Base Excision Repair (BER) genes required for the repair of Reactive Oxygen Species (ROS)-induced DNA damage, including poly-ADP ribose polymerase 1 (PARP1). Subsequent studies revealed that EGFRvIII over-expression in glioblastoma cells caused increased levels of ROS, DNA strand break accumulation, and genome instability. In a panel of primary glioblastoma lines, sensitivity to PARP1 inhibition correlated with the levels of EGFR activation and oxidative stress. Gene expression analysis indicated that reduced expression of BER genes in glioblastomas with high EGFR expression correlated with improved patient survival. These observations suggest that oxidative stress secondary to EGFR hyper-activation necessitates increased cellular reliance on PARP1 mediated BER, and offer critical insights into clinical trial design.

Targeting EGFR induced oxidative stress by PARP1 inhibition in glioblastoma therapy.

Directory of Open Access Journals (Sweden)

Masayuki Nitta

Full Text Available Despite the critical role of Epidermal Growth Factor Receptor (EGFR in glioblastoma pathogenesis, EGFR targeted therapies have achieved limited clinical efficacy. Here we propose an alternate therapeutic strategy based on the conceptual framework of non-oncogene addiction. A directed RNAi screen revealed that glioblastoma cells over-expressing EGFRvIII, an oncogenic variant of EGFR, become hyper-dependent on a variety of DNA repair genes. Among these, there was an enrichment of Base Excision Repair (BER genes required for the repair of Reactive Oxygen Species (ROS-induced DNA damage, including poly-ADP ribose polymerase 1 (PARP1. Subsequent studies revealed that EGFRvIII over-expression in glioblastoma cells caused increased levels of ROS, DNA strand break accumulation, and genome instability. In a panel of primary glioblastoma lines, sensitivity to PARP1 inhibition correlated with the levels of EGFR activation and oxidative stress. Gene expression analysis indicated that reduced expression of BER genes in glioblastomas with high EGFR expression correlated with improved patient survival. These observations suggest that oxidative stress secondary to EGFR hyper-activation necessitates increased cellular reliance on PARP1 mediated BER, and offer critical insights into clinical trial design.

Mechanical stiffness of TMJ condylar cartilage increases after artificial aging by ribose.

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Mirahmadi, Fereshteh; Koolstra, Jan Harm; Lobbezoo, Frank; van Lenthe, G Harry; Ghazanfari, Samaneh; Snabel, Jessica; Stoop, Reinout; Everts, Vincent

2018-03-01

Aging is accompanied by a series of changes in mature tissues that influence their properties and functions. Collagen, as one of the main extracellular components of cartilage, becomes highly crosslinked during aging. In this study, the aim was to examine whether a correlation exists between collagen crosslinking induced by artificial aging and mechanical properties of the temporomandibular joint (TMJ) condyle. To evaluate this hypothesis, collagen crosslinks were induced using ribose incubation. Porcine TMJ condyles were incubated for 7 days with different concentrations of ribose. The compressive modulus and stiffness ratio (incubated versus control) was determined after loading. Glycosaminoglycan and collagen content, and the number of crosslinks were analyzed. Tissue structure was visualized by microscopy using different staining methods. Concomitant with an increasing concentration of ribose, an increase of collagen crosslinks was found. The number of crosslinks increased almost 50 fold after incubation with the highest concentration of ribose. Simultaneously, the stiffness ratio of the samples showed a significant increase after incubation with the ribose. Pearson correlation analyses showed a significant positive correlation between the overall stiffness ratio and the crosslink level; the higher the number of crosslinks the higher the stiffness. The present model, in which ribose was used to mimic certain aspects of age-related changes, can be employed as an in vitro model to study age-related mechanical changes in the TMJ condyle. Copyright © 2017 Elsevier Ltd. All rights reserved.

c-MYC G-quadruplex binding by the RNA polymerase I inhibitor BMH-21 and analogues revealed by a combined NMR and biochemical Approach.

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Musso, Loana; Mazzini, Stefania; Rossini, Anna; Castagnoli, Lorenzo; Scaglioni, Leonardo; Artali, Roberto; Di Nicola, Massimo; Zunino, Franco; Dallavalle, Sabrina

2018-03-01

Pyridoquinazolinecarboxamides have been reported as RNA polymerase I inhibitors and represent a novel class of potential antitumor agents. BMH-21, was reported to intercalate with GC-rich rDNA, resulting in nucleolar stress as a primary mechanism of cytotoxicity. The interaction of BMH-21 and analogues with DNA G-quadruplex structures was studied by NMR and molecular modelling. The cellular response was investigated in a panel of human tumor cell lines and protein expression was examined by Western Blot analysis. We explored the ability of BMH-21 and its analogue 2 to bind to G-quadruplex present in the c-MYC promoter, by NMR and molecular modelling studies. We provide evidence that both compounds are not typical DNA intercalators but are effective binders of the tested G-quadruplex. The interaction with c-MYC G-quadruplex was reflected in down-regulation of c-Myc expression in human tumor cells. The inhibitory effect was almost complete in lymphoma cells SUDHL4 characterized by overexpression of c-Myc protein. This downregulation reflected an early and persistent modulation of cMyc mRNA. Given the relevance of c-MYC in regulation of ribosome biogenesis, it is conceivable that the inhibition of c-MYC contributes to the perturbation of nuclear functions and RNA polymerase I activity. Similar experiments with CX-5461, another RNA polymerase I transcription inhibitor, indicate the same behaviour in G-quadruplex stabilization. Our results support the hypothesis that BMH-21 and analogue compounds share the same mechanism, i.e. G-quadruplex binding as a primary event of a cascade leading to inhibition of RNA polymerase I and apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

International Nuclear Information System (INIS)

Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

2006-01-01

RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

Ribose facilitates thallium-201 redistribution in patients with coronary artery disease

International Nuclear Information System (INIS)

Perlmutter, N.S.; Wilson, R.A.; Angello, D.A.; Palac, R.T.; Lin, J.; Brown, B.G.

1991-01-01

To investigate whether i.v. infusion of ribose, an adenine nucleotide precursor, postischemia facilitates thallium-201 (201Tl) redistribution and improves identification of ischemic myocardium in patients with coronary artery disease (CAD), 17 patients underwent two exercise 201Tl stress tests, performed 1-2 wk apart. After immediate postexercise planar imaging, patients received either i.v. ribose (3.3 mg/kg/min x 30 min) or saline as a control. Additional imaging was performed 1 and 4 hr postexercise. Reversible defects were identified by count-profile analysis. Significantly more (nearly twice as many) reversible 201Tl defects were identified on the post-ribose images compared to the post-saline (control) images at both 1 and 4 hr postexercise (p less than 0.001). Quantitative analyses of the coronary arteriogram was available in 13 patients and confirmed that the additional reversible defects were in myocardial regions supplied by stenosed arteries. We conclude that ribose appears to facilitate 201Tl redistribution in patients with CAD and enhances identification of ischemic myocardium

Enantioselective synthesis of tetrafluorinated ribose and fructose.

Science.gov (United States)

Linclau, Bruno; Boydell, A James; Timofte, Roxana S; Brown, Kylie J; Vinader, Victoria; Weymouth-Wilson, Alexander C

2009-02-21

A perfluoroalkylidene lithium mediated cyclisation approach for the enantioselective synthesis of a tetrafluorinated aldose (ribose) and of a tetrafluorinated ketose (fructose), both in the furanose and in the pyranose form, is described.

Metabolic Enhancer Piracetam Attenuates the Translocation of Mitochondrion-Specific Proteins of Caspase-Independent Pathway, Poly [ADP-Ribose] Polymerase 1 Up-regulation and Oxidative DNA Fragmentation.

Science.gov (United States)

Verma, Dinesh Kumar; Gupta, Sonam; Biswas, Joyshree; Joshi, Neeraj; Sivarama Raju, K; Wahajuddin, Mu; Singh, Sarika

2018-03-12

Piracetam, a nootropic drug, has been clinically used for decades; however, its mechanism of action still remains enigmatic. The present study was undertaken to evaluate the role of mitochondrion-specific factors of caspase-independent pathway like apoptotic-inducing factor (AIF) and endonuclease-G (endo-G) in piracetam-induced neuroprotection. N2A cells treated with lipopolysaccharide (LPS) exhibited significant cytotoxicity, impaired mitochondrial activity, and reactive oxygen species generation which was significantly attenuated with piracetam co-treatment. Cells co-treated with LPS and piracetam exhibited significant uptake of piracetam in comparison to only piracetam-treated cells as estimated by liquid chromatography-mass spectrometry (LC-MSMS). LPS treatment caused significant translocation of AIF and endonuclease-G in neuronal N2A cells which were significantly attenuated with piracetam co-treatment. Significant over-expression of proinflammatory cytokines was also observed after treatment of LPS to cells which was inhibited with piracetam co-treatment demonstrating its anti-inflammatory property. LPS-treated cells exhibited significant oxidative DNA fragmentation and poly [ADP-ribose] polymerase-1 (PARP-1) up-regulation in nucleus, both of which were attenuated with piracetam treatment. Antioxidant melatonin but not z-VAD offered the inhibited LPS-induced DNA fragmentation indicating the involvement of oxidative DNA fragmentation. Further, we did not observe the altered caspase-3 level after LPS treatment initially while at a later time point, significantly augmented level of caspase-3 was observed which was not inhibited with piracetam treatment. In total, our findings indicate the interference of piracetam in mitochondrion-mediated caspase-independent pathway, as well as its anti-inflammatory and antioxidative properties. Graphical Abstract Graphical abstract indicating the novel interference of metabolic enhancer piracetam (P) in neuronal death

PARP1 inhibitors: contemporary attempts at their use in anticancer therapy and future perspective

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Ewelina Wiśnik

2016-04-01

Full Text Available Current cancer therapies are based mainly on the use of compounds that cause DNA damage. Unfortunately, even the combination therapies do not give rewarding effects, due to the high efficiency of DNA damage repair mechanisms in tumor cells. Therefore, the present studies should be focused on proteins that are involved in DNA repair systems. Poly(ADP-ribose polymerase-1 is an example of a protein commonly known as an enzyme that plays a role in the detection of DNA damage and repair. Activation of PARP1 in response to DNA damage leads to poly-ADP-ribosylation of proteins contributing to DNA repair systems, therefore facilitating the maintenance of genome stability. On the other hand, inhibition of PARP1 enzyme results in the accumulation of DNA damage, which in turn contributes to cell death. Studies on inhibitors of PARP1 are still ongoing, and some of them are currently in the third phase of clinical trials. To date, only one representative of the PARP1 inhibitors, called olaparib, has been approved for anti-cancer therapy in the EU and the USA. Moreover, a growing body of evidence indicates a role of this protein in various intracellular processes such as bioenergetics, proliferation, regulation of gene expression, cell death as well as immunoregulation. A number of different intracellular processes regulated by PARP1 give rise to potential wider use of PARP1 inhibitors in treatment of other diseases, including immune or autoimmune disorders.

Allosteric inhibitors of Coxsackie virus A24 RNA polymerase.

Science.gov (United States)

Schein, Catherine H; Rowold, Diane; Choi, Kyung H

2016-02-15

Coxsackie virus A24 (CVA24), a causative agent of acute hemorrhagic conjunctivitis, is a prototype of enterovirus (EV) species C. The RNA polymerase (3D(pol)) of CVA24 can uridylylate the viral peptide linked to the genome (VPg) from distantly related EV and is thus, a good model for studying this reaction. Once UMP is bound, VPgpU primes RNA elongation. Structural and mutation data have identified a conserved binding surface for VPg on the RNA polymerase (3D(pol)), located about 20Å from the active site. Here, computational docking of over 60,000 small compounds was used to select those with the lowest (best) specific binding energies (BE) for this allosteric site. Compounds with varying structures and low BE were assayed for their effect on formation of VPgU by CVA24-3D(pol). Two compounds with the lowest specific BE for the site inhibited both uridylylation and formation of VPgpolyU at 10-20μM. These small molecules can be used to probe the role of this allosteric site in polymerase function, and may be the basis for novel antiviral compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

D-ribose--an additive with caffeine.

Science.gov (United States)

Herrick, Jim; Shecterle, L M; St Cyr, J A

2009-05-01

Caffeine acts as a stimulant, in which approximately 90% of people in the United States consume daily. Besides its beneficial effects, many individuals have experienced unpleasant reactions following the consumption of caffeine: such as insomnia, an increase in heart rate, feelings of nervousness, headaches, occasional lightheadedness, a state of "jitters," and a potential "crash" state following its metabolism. Researchers have proposed mechanisms responsible for caffeine's interactions, which include its blocking capacity of adenosine receptors, its role with the pituitary gland, increasing levels of dopamine, and its role with the intracellular release of calcium from the sarcoplasmic reticulum, which is dependent on intracellular adenosine triphosphate levels. Specific substrates have been investigated to lessen the undesirable effects of caffeine and still preserve its stimulatory benefits. The results of these investigations have produced no positive consensus. However, D-ribose, an important pentose carbohydrate in the energy molecule of adenosine triphosphate, as well as our genetic code and other cellular processes, could offer such a solution to this problem. D-ribose could potentially aid in maintaining or potentially lowering extra-cellular adenosine concentrations, aid in the flux of intracellular calcium, aid in intracellular energy production, and potentially lessen the perceived "crash" state felt by many. Every cell requires adequate levels of energy to maintain its integrity and function. Caffeine has the potential to task this energy equilibrium. D-ribose with caffeine may be the substrate to aid in the potential intracellular energy demand, aid in lessening the perceived unpleasant side effects of caffeine, and still preserving the desired benefits of this stimulant consumed by all of us daily.

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

International Nuclear Information System (INIS)

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

1989-01-01

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

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

International Nuclear Information System (INIS)

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

1984-01-01

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

Effect of MRE11 loss on PARP-inhibitor sensitivity in endometrial cancer in vitro.

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Romana Koppensteiner

Full Text Available To evaluate the frequency of MRE11/RAD50/NBS1 (MRN-complex loss of protein expression in endometrial cancers (EC and to determine whether loss of MRE11 renders the cancer cells sensitive to Poly(ADP-ribose polymerase (PARP-inhibitory treatment.MRN expression was examined in 521 samples of endometrial carcinomas and in 10 cancer cell lines. A putative mutation hotspot in the form of an intronic poly(T allele in MRE11 was sequenced in selected cases (n = 26. Sensitivity to the PARP-inhibitor, BMN673 was tested in colony formation assays before and after MRE11 silencing using siRNA. Homologous recombination (HR DNA repair was evaluated by RAD51-foci formation assay upon irradiation and drug treatment.Loss of MRE11 protein was found in 30.7% of EC tumours and significantly associated with loss of RAD50, NBS1 and mismatch repair protein expression. One endometrial cell line showed a markedly reduced MRE11 expression due to a homozygous poly(T mutation of MRE11, thereby exhibiting an increased sensitivity to BMN673. MRE11 depletion sensitizes MRE11 expressing EC cell lines to the treatment with BMN673. The increased sensitivity to PARP-inhibition correlates with reduced RAD51 foci formation upon ionizing radiation in MRE11-depleted cells.Loss of the MRE11 protein predicts sensitivity to PARP-inhibitor sensitivity in vitro, defining it as an additional synthetic lethal gene with PARP. The high incidence of MRE11 loss in ECs can be potentially exploited for PARP-inhibitor therapy. Furthermore, MRE11 protein expression using immunohistochemistry could be investigated as a predictive biomarker for PARP-inhibitor treatment.

Involvement of the ribose operon repressor RbsR in regulation of purine nucleotide synthesis in Escherichia coli.

Science.gov (United States)

Shimada, Tomohiro; Kori, Ayako; Ishihama, Akira

2013-07-01

Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides and its salvage pathway. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

PARP inhibitor rucaparib induces changes in NAD levels in cells and liver tissues as assessed by MRS.

Science.gov (United States)

Almeida, Gilberto S; Bawn, Carlo M; Galler, Martin; Wilson, Ian; Thomas, Huw D; Kyle, Suzanne; Curtin, Nicola J; Newell, David R; Maxwell, Ross J

2017-09-01

Poly(adenosine diphosphate ribose) polymerases (PARPs) are multifunctional proteins which play a role in many cellular processes. Namely, PARP1 and PARP2 have been shown to be involved in DNA repair, and therefore are valid targets in cancer treatment with PARP inhibitors, such as rucaparib, currently in clinical trials. Proton magnetic resonance spectroscopy ( 1 H-MRS) was used to study the impact of rucaparib in vitro and ex vivo in liver tissue from mice, via quantitative analysis of nicotinamide adenosine diphosphate (NAD + ) spectra, to assess the potential of MRS as a biomarker of the PARP inhibitor response. SW620 (colorectal) and A2780 (ovarian) cancer cell lines, and PARP1 wild-type (WT) and PARP1 knock-out (KO) mice, were treated with rucaparib, temozolomide (methylating agent) or a combination of both drugs. 1 H-MRS spectra were obtained from perchloric acid extracts of tumour cells and mouse liver. Both cell lines showed an increase in NAD + levels following PARP inhibitor treatment in comparison with temozolomide treatment. Liver extracts from PARP1 WT mice showed a significant increase in NAD + levels after rucaparib treatment compared with untreated mouse liver, and a significant decrease in NAD + levels in the temozolomide-treated group. The combination of rucaparib and temozolomide did not prevent the NAD + depletion caused by temozolomide treatment. The 1 H-MRS results show that NAD + levels can be used as a biomarker of PARP inhibitor and methylating agent treatments, and suggest that in vivo measurement of NAD + would be valuable. Copyright © 2017 John Wiley & Sons, Ltd.

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

Science.gov (United States)

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

1999-02-01

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

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.

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

International Nuclear Information System (INIS)

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

2013-01-01

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

Glycyrrhetinic acid and its derivatives as inhibitors of poly(ADP-ribosepolymerases 1 and 2, apurinic/apyrimidinic endonuclease 1 and DNA polymerase β

Directory of Open Access Journals (Sweden)

Salakhutdinov N. F.

2012-06-01

Full Text Available Aim. For strengthening the efficiency of monofunctional alkylating antineoplastic drugs it is important to lower the capacity of base excision repair (BER system which corrects the majority of DNA damages caused by these reagents. The objective was to create inhibitors of the key BER enzymes (PARP1, PARP2, DNA polymerase β, and APE1 by the directed modification of glycyrrhetinic acid (GA. Methods. Amides of GA were produced from the GA acetate by formation of the corresponding acyl chloride, amidation with the appropriate amine and subsequent deacylation. Small library of 2-cyano substituted derivatives of GA methyl esters was obtained by the structural modification of GA framework and carboxylic acid group. The inhibitory capacity of the compounds was estimated by comparison of the enzyme activities in specific tests in the presence of compounds versus their absence. Results. None of tested compounds inhibits PARP1 significantly. Unmodified GA and its morpholinic derivative were shown to be weak inhibitors of PARP2. The derivatives of GA containing keto-group in 11 triterpene framework were shown to be moderate inhibitors of pol β. Compound 3, containing 12-oxo-9(11-en moiety in the ring C, was shown to be a single inhibitor of APE1 among all compounds studied. Conclusions. The class of GA derivatives, selective pol β inhibitors, was found out. The selective inhibitor of APE1 and weak selective inhibitor of PARP2 were also revealed.

Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

Energy Technology Data Exchange (ETDEWEB)

Abdelali, Ala [Department of Anatomy, Faculty of Medicine, Kuwait University (Kuwait); Al-Bader, Maie [Department of Physiology, Faculty of Medicine, Kuwait University (Kuwait); Kilarkaje, Narayana, E-mail: knarayana@hsc.edu.kw [Department of Anatomy, Faculty of Medicine, Kuwait University (Kuwait)

2016-11-15

Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis. - Highlights: • Resveratrol inhibits diabetes-induced abnormal sperm morphogenesis • Resveratrol recovers

Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

International Nuclear Information System (INIS)

Abdelali, Ala; Al-Bader, Maie; Kilarkaje, Narayana

2016-01-01

Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis. - Highlights: • Resveratrol inhibits diabetes-induced abnormal sperm morphogenesis • Resveratrol recovers

Neer Award 2016: reduced muscle degeneration and decreased fatty infiltration after rotator cuff tear in a poly(ADP-ribose) polymerase 1 (PARP-1) knock-out mouse model.

Science.gov (United States)

Kuenzler, Michael B; Nuss, Katja; Karol, Agnieszka; Schär, Michael O; Hottiger, Michael; Raniga, Sumit; Kenkel, David; von Rechenberg, Brigitte; Zumstein, Matthias A

2017-05-01

Disturbed muscular architecture, atrophy, and fatty infiltration remain irreversible in chronic rotator cuff tears even after repair. Poly (adenosine 5'-diphosphate-ribose) polymerase 1 (PARP-1) is a key regulator of inflammation, apoptosis, muscle atrophy, muscle regeneration, and adipocyte development. We hypothesized that the absence of PARP-1 would lead to a reduction in damage to the muscle subsequent to combined tenotomy and neurectomy in a PARP-1 knockout (KO) mouse model. PARP-1 KO and wild-type C57BL/6 (WT group) mice were analyzed at 1, 6, and 12 weeks (total n = 84). In all mice, the supraspinatus and infraspinatus muscles of the left shoulder were detached and denervated. Macroscopic analysis, magnetic resonance imaging, gene expression analysis, immunohistochemistry, and histology were used to assess the differences in PARP-1 KO and WT mice. The muscles in the PARP-1 KO group had significantly less retraction, atrophy, and fatty infiltration after 12 weeks than in the WT group. Gene expression of inflammatory, apoptotic, adipogenic, and muscular atrophy genes was significantly decreased in PARP-1 KO mice in the first 6 weeks. Absence of PARP-1 leads to a reduction in muscular architectural damage, early inflammation, apoptosis, atrophy, and fatty infiltration after combined tenotomy and neurectomy of the rotator cuff muscle. Although the macroscopic reaction to injury is similar in the first 6 weeks, the ability of the muscles to regenerate was much greater in the PARP-1 KO group, leading to a near-normalization of the muscle after 12 weeks. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

Efficient biosynthesis of d-ribose using a novel co-feeding strategy in Bacillus subtilis without acid formation.

Science.gov (United States)

Cheng, J; Zhuang, W; Li, N N; Tang, C L; Ying, H J

2017-01-01

Normally, low d-ribose production was identified as responsible for plenty of acid formation by Bacillus subtilis due to its carbon overflow. An approach of co-feeding glucose and sodium citrate is developed here and had been proved to be useful in d-ribose production. This strategy is critical because it affects the cell concentration, the productivity of d-ribose and, especially, the formation of by-products such as acetoin, lactate and acetate. d-ribose production was increased by 59·6% from 71·06 to 113·41 g l -1 without acid formation by co-feeding 2·22 g l -1  h -1 glucose and 0·036 g l -1  h -1 sodium citrate to a 60 g l -1 glucose reaction system. Actually, the cell density was also enhanced from 11·51 to 13·84 g l -1 . These parameters revealed the importance of optimization and modelling of the d-ribose production process. Not only could zero acid formation was achieved over a wide range of co-feeding rate by reducing glycolytic flux drastically but also the cell density and d-ribose yield were elevated by increasing the hexose monophosphate pathway flux. Bacillus subtilis usually produce d-ribose accompanied by plenty of organic acids when glucose is used as a carbon source, which is considered to be a consequence of mismatched glycolytic and tricarboxylic acid cycle capacities. This is the first study to provide high-efficiency biosynthesis of d-ribose without organic acid formation in B. subtilis, which would be lower than the cost of separation and purification. The strain transketolase-deficient B. subtilis CGMCC 3720 can be potentially applied to the production of d-ribose in industry. © 2016 The Society for Applied Microbiology.

An all-atom, active site exploration of antiviral drugs that target Flaviviridae polymerases

Czech Academy of Sciences Publication Activity Database

Valdés, James J.; Gil, V.A.; Butterill, Philip T.; Růžek, Daniel

2016-01-01

Roč. 97, OCT (2016), s. 2552-2565 ISSN 0022-1317 R&D Projects: GA MŠk(CZ) EE2.3.30.0032; GA ČR GB14-36098G; GA MZd(CZ) NV16-34238A EU Projects: European Commission(XE) 316304 - MODBIOLIN Institutional support: RVO:60077344 Keywords : dependent RNA-polymerase * c virus polymerase * de-novo initiation * hepatitis C * allosteric inhibitors * nucleoside inhibitors * molecular dynamics * encephalitis virus * protein-structure * cluster-analysis Subject RIV: EE - Microbiology, Virology Impact factor: 2.838, year: 2016

Differential transactivation by orphan nuclear receptor NOR1 and its fusion gene product EWS/NOR1: possible involvement of poly(ADP-ribose) polymerase I, PARP-1.

Science.gov (United States)

Ohkura, Naganari; Nagamura, Yuko; Tsukada, Toshihiko

2008-10-15

In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins. (c) 2008 Wiley-Liss, Inc.

Common changes in global gene expression induced by RNA polymerase inhibitors in Shigella flexneri.

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Hua Fu

Full Text Available Characterization of expression profile of organisms in response to antimicrobials provides important information on the potential mechanism of action of the drugs. The special expression signature can be used to predict whether other drugs act on the same target. Here, the common response of Shigella flexneri to two inhibitors of RNA polymerase was examined using gene expression profiling. Consistent with similar effects of the two drugs, the gene expression profiles indicated that responses of the bacteria to these drugs were roughly the same, with 225 genes affected commonly. Of them, 88 were induced and 137 were repressed. Real-time PCR was performed for selected genes to verify the microarray results. Analysis of the expression data revealed that more than 30% of the plasmid-encoded genes on the array were up-regulated by the antibiotics including virF regulon, other virulence-related genes, and genes responsible for plasmid replication, maintenance, and transfer. In addition, some chromosome-encoded genes involved in virulence and genes acquired from horizontal transfer were also significantly up-regulated. However, the expression of genes encoding the beta-subunit of RNA polymerase was increased moderately. The repressed genes include those that code for products associated with the ribosome, citrate cycle, glycolysis, thiamine biosynthesis, purine metabolism, fructose metabolism, mannose metabolism, and cold shock proteins. This study demonstrates that the two antibiotics induce rapid cessation of RNA synthesis resulting in inhibition of translation components. It also indicates that the production of virulence factors involved in intercellular dissemination, tissue invasion and inflammatory destruction may be enhanced through derepressing horizontal transfer genes by the drugs.

Combination Treatment with PPARγ Ligand and Its Specific Inhibitor GW9662 Downregulates BIS and 14-3-3 Gamma, Inhibiting Stem-Like Properties in Glioblastoma Cells.

Science.gov (United States)

Im, Chang-Nim

2017-01-01

PPAR γ is a nuclear receptor that regulates differentiation and proliferation and is highly expressed in many cancer cells. Its synthetic ligands, such as rosiglitazone and ciglitazone, and its inhibitor GW9662, were shown to induce cellular differentiation, inhibit proliferation, and lead to apoptosis. Glioblastoma is a common brain tumor with poor survival prospects. Recently, glioblastoma stem cells (GSCs) have been examined as a potential target for anticancer therapy; however, little is known about the combined effect of various agents on GSCs. In this study, we found that cotreatment with PPAR γ ligands and GW9662 inhibited stem-like properties in GSC-like spheres, which significantly express SOX2. In addition, this treatment decreased the activation of STAT3 and AKT and decreased the amounts of 14-3-3 gamma and BIS proteins. Moreover, combined administration of small-interfering RNA (siRNA) transfection with PPAR γ ligands induced downregulation of SOX2 and MMP2 activity together with inhibition of sphere-forming activity regardless of poly(ADP-ribose) polymerase (PARP) cleavage. Taken together, our findings suggest that a combination therapy using PPAR γ ligands and its inhibitor could be a potential therapeutic strategy targeting GSCs.

Combination Treatment with PPARγ Ligand and Its Specific Inhibitor GW9662 Downregulates BIS and 14-3-3 Gamma, Inhibiting Stem-Like Properties in Glioblastoma Cells

Directory of Open Access Journals (Sweden)

Chang-Nim Im

2017-01-01

Full Text Available PPARγ is a nuclear receptor that regulates differentiation and proliferation and is highly expressed in many cancer cells. Its synthetic ligands, such as rosiglitazone and ciglitazone, and its inhibitor GW9662, were shown to induce cellular differentiation, inhibit proliferation, and lead to apoptosis. Glioblastoma is a common brain tumor with poor survival prospects. Recently, glioblastoma stem cells (GSCs have been examined as a potential target for anticancer therapy; however, little is known about the combined effect of various agents on GSCs. In this study, we found that cotreatment with PPARγ ligands and GW9662 inhibited stem-like properties in GSC-like spheres, which significantly express SOX2. In addition, this treatment decreased the activation of STAT3 and AKT and decreased the amounts of 14-3-3 gamma and BIS proteins. Moreover, combined administration of small-interfering RNA (siRNA transfection with PPARγ ligands induced downregulation of SOX2 and MMP2 activity together with inhibition of sphere-forming activity regardless of poly(ADP-ribose polymerase (PARP cleavage. Taken together, our findings suggest that a combination therapy using PPARγ ligands and its inhibitor could be a potential therapeutic strategy targeting GSCs.

Occurrence and stability of lone pair–π stacking interactions between ribose and nucleobases in functional RNAs

KAUST Repository

Chawla, Mohit; Chermak, Edrisse; Zhang, Qingyun; Bujnicki, Janusz M.; Oliva, Romina; Cavallo, Luigi

2017-01-01

The specific folding pattern and function of RNA molecules lies in various weak interactions, in addition to the strong base-base pairing and stacking. One of these relatively weak interactions, characterized by the stacking of the O4′ atom of a ribose on top of the heterocycle ring of a nucleobase, has been known to occur but has largely been ignored in the description of RNA structures. We identified 2015 ribose–base stacking interactions in a high-resolution set of non-redundant RNA crystal structures. They are widespread in structured RNA molecules and are located in structural motifs other than regular stems. Over 50% of them involve an adenine, as we found ribose-adenine contacts to be recurring elements in A-minor motifs. Fewer than 50% of the interactions involve a ribose and a base of neighboring residues, while approximately 30% of them involve a ribose and a nucleobase at least four residues apart. Some of them establish inter-domain or inter-molecular contacts and often implicate functionally relevant nucleotides. In vacuo ribose-nucleobase stacking interaction energies were calculated by quantum mechanics methods. Finally, we found that lone pair–π stacking interactions also occur between ribose and aromatic amino acids in RNA–protein complexes.

Occurrence and stability of lone pair–π stacking interactions between ribose and nucleobases in functional RNAs

KAUST Repository

Chawla, Mohit

2017-08-18

The specific folding pattern and function of RNA molecules lies in various weak interactions, in addition to the strong base-base pairing and stacking. One of these relatively weak interactions, characterized by the stacking of the O4′ atom of a ribose on top of the heterocycle ring of a nucleobase, has been known to occur but has largely been ignored in the description of RNA structures. We identified 2015 ribose–base stacking interactions in a high-resolution set of non-redundant RNA crystal structures. They are widespread in structured RNA molecules and are located in structural motifs other than regular stems. Over 50% of them involve an adenine, as we found ribose-adenine contacts to be recurring elements in A-minor motifs. Fewer than 50% of the interactions involve a ribose and a base of neighboring residues, while approximately 30% of them involve a ribose and a nucleobase at least four residues apart. Some of them establish inter-domain or inter-molecular contacts and often implicate functionally relevant nucleotides. In vacuo ribose-nucleobase stacking interaction energies were calculated by quantum mechanics methods. Finally, we found that lone pair–π stacking interactions also occur between ribose and aromatic amino acids in RNA–protein complexes.

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118

International Nuclear Information System (INIS)

Lobley, Carina M. C.; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E.; Nettleship, Joanne E.; Brandao-Neto, Jose; Owens, Raymond J.; O’Toole, Paul W.; Walsh, Martin A.

2012-01-01

The crystal structure of ribose 5-phosphate isomerase has been determined to 1.72 Å resolution and is presented with a brief comparison to other known ribose 5-phosphate isomerase A structures. The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β d-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography

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

Science.gov (United States)

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

2013-08-30

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

The Multiple Functions of the Nucleolus in Plant Development, Disease and Stress Responses

Science.gov (United States)

Kalinina, Natalia O.; Makarova, Svetlana; Makhotenko, Antonida; Love, Andrew J.; Taliansky, Michael

2018-01-01

The nucleolus is the most conspicuous domain in the eukaryotic cell nucleus, whose main function is ribosomal RNA (rRNA) synthesis and ribosome biogenesis. However, there is growing evidence that the nucleolus is also implicated in many other aspects of cell biology, such as regulation of cell cycle, growth and development, senescence, telomerase activity, gene silencing, responses to biotic and abiotic stresses. In the first part of the review, we briefly assess the traditional roles of the plant nucleolus in rRNA synthesis and ribosome biogenesis as well as possible functions in other RNA regulatory pathways such as splicing, nonsense-mediated mRNA decay and RNA silencing. In the second part of the review we summarize recent progress and discuss already known and new hypothetical roles of the nucleolus in plant growth and development. In addition, this part will highlight studies showing new nucleolar functions involved in responses to pathogen attack and abiotic stress. Cross-talk between the nucleolus and Cajal bodies is also discussed in the context of their association with poly(ADP ribose)polymerase (PARP), which is known to play a crucial role in various physiological processes including growth, development and responses to biotic and abiotic stresses. PMID:29479362

The heat shock protein 90 inhibitor 17-AAG suppresses growth and induces apoptosis in human cholangiocarcinoma cells.

Science.gov (United States)

Zhang, Jianjun; Zheng, Zhichao; Zhao, Yan; Zhang, Tao; Gu, Xiaohu; Yang, Wei

2013-11-01

The aim of this study was to investigate the effects of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor, on the proliferation, cell cycle, and apoptosis of human cholangiocarcinoma (CCA) cells. Cell proliferation and cell cycle distribution were measured by the MTT assay and flow cytometry analysis, respectively. Induction of apoptosis was determined by flow cytometry and Hoechst staining. The expressions of cleaved poly ADP-ribose polymerase (PARP), Bcl-2, Survivin, and Cyclin B1 were detected by Western blot analysis. The activity of caspase-3 was also examined. We found that 17-AAG inhibited cell growth and induced G2/M cell cycle arrest and apoptosis in CCA cells together with the down-regulation of Bcl-2, Survivin and Cyclin B1, and the up-regulation of cleaved PARP. Moreover, increased caspase-3 activity was also observed in CCA cells treated with 17-AAG. In conclusion, our data suggest that the inhibition of HSP90 function by 17-AAG may provide a promising therapeutic strategy for the treatment of human CCA.

Ribose mediated crosslinking of collagen-hydroxyapatite hybrid scaffolds for bone tissue regeneration using biomimetic strategies.

Science.gov (United States)

Krishnakumar, Gopal Shankar; Gostynska, Natalia; Campodoni, Elisabetta; Dapporto, Massimiliano; Montesi, Monica; Panseri, Silvia; Tampieri, Anna; Kon, Elizaveta; Marcacci, Maurilio; Sprio, Simone; Sandri, Monica

2017-08-01

This study explores for the first time the application of ribose as a highly biocompatible agent for the crosslinking of hybrid mineralized constructs, obtained by bio-inspired mineralization of self-assembling Type I collagen matrix with magnesium-doped-hydroxyapatite nanophase, towards a biomimetic mineralized 3D scaffolds (MgHA/Coll) with excellent compositional and structural mimicry of bone tissue. To this aim, two different crosslinking mechanisms in terms of pre-ribose glycation (before freeze drying) and post-ribose glycation (after freeze drying) were investigated. The obtained results explicate that with controlled freeze-drying, highly anisotropic porous structures with opportune macro-micro porosity are obtained. The physical-chemical features of the scaffolds characterized by XRD, FTIR, ICP and TGA demonstrated structural mimicry analogous to the native bone. The influence of ribose greatly assisted in decreasing solubility and increased enzymatic resistivity of the scaffolds. In addition, enhanced mechanical behaviour in response to compressive forces was achieved. Preliminary cell culture experiments reported good cytocompatibility with extensive cell adhesion, proliferation and colonization. Overall, scaffolds developed by pre-ribose glycation process are preferred, as the related crosslinking technique is more facile and robust to obtain functional scaffolds. As a proof of concept, we have demonstrated that ribose crosslinking is cost-effective, safe and functionally effective. This study also offers new insights and opportunities in developing promising scaffolds for bone tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

New approaches of PARP-1 inhibitors in human lung cancer cells and cancer stem-like cells by some selected anthraquinone-derived small molecules.

Directory of Open Access Journals (Sweden)

Yu-Ru Lee

Full Text Available Poly (ADP-ribose polymerase-1 (PARP-1 and telomerase, as well as DNA damage response pathways are targets for anticancer drug development, and specific inhibitors are currently under clinical investigation. The purpose of this work is to evaluate anticancer activities of anthraquinone-derived tricyclic and tetracyclic small molecules and their structure-activity relationships with PARP-1 inhibition in non-small cell lung cancer (NSCLC and NSCLC-overexpressing Oct4 and Nanog clone, which show high-expression of PARP-1 and more resistance to anticancer drug. We applied our library selected compounds to NCI's 60 human cancer cell-lines (NCI-60 in order to generate systematic profiling data. Based on our analysis, it is hypothesized that these drugs might be, directly and indirectly, target components to induce mitochondrial permeability transition and the release of pro-apoptotic factors as potential anti-NSCLC or PARP inhibitor candidates. Altogether, the most active NSC747854 showed its cytotoxicity and dose-dependent PARP inhibitory manner, thus it emerges as a promising structure for anti-cancer therapy with no significant negative influence on normal cells. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and telomerase inhibitors, in particular. Together, the data presented here expand our insight into the PARP inhibitors and support the resource-demanding lead optimization of structurally related small molecules for human cancer therapy.

New Approaches of PARP-1 Inhibitors in Human Lung Cancer Cells and Cancer Stem-Like Cells by Some Selected Anthraquinone-Derived Small Molecules

Science.gov (United States)

Yu, Dah-Shyong; Huang, Kuo-Feng; Chou, Shih-Jie; Chen, Tsung-Chih; Lee, Chia-Chung; Chen, Chun-Liang; Chiou, Shih-Hwa; Huang, Hsu-Shan

2013-01-01

Poly (ADP-ribose) polymerase-1 (PARP-1) and telomerase, as well as DNA damage response pathways are targets for anticancer drug development, and specific inhibitors are currently under clinical investigation. The purpose of this work is to evaluate anticancer activities of anthraquinone-derived tricyclic and tetracyclic small molecules and their structure-activity relationships with PARP-1 inhibition in non-small cell lung cancer (NSCLC) and NSCLC-overexpressing Oct4 and Nanog clone, which show high-expression of PARP-1 and more resistance to anticancer drug. We applied our library selected compounds to NCI's 60 human cancer cell-lines (NCI-60) in order to generate systematic profiling data. Based on our analysis, it is hypothesized that these drugs might be, directly and indirectly, target components to induce mitochondrial permeability transition and the release of pro-apoptotic factors as potential anti-NSCLC or PARP inhibitor candidates. Altogether, the most active NSC747854 showed its cytotoxicity and dose-dependent PARP inhibitory manner, thus it emerges as a promising structure for anti-cancer therapy with no significant negative influence on normal cells. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and telomerase inhibitors, in particular. Together, the data presented here expand our insight into the PARP inhibitors and support the resource-demanding lead optimization of structurally related small molecules for human cancer therapy. PMID:23451039

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

Science.gov (United States)

Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

2014-01-01

DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

Directory of Open Access Journals (Sweden)

Tom eKillelea

2014-05-01

Full Text Available DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR, cDNA cloning, genome sequencing and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3’ primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

Effect of 3-aminobenzamide, PARP inhibitor, on matrix metalloproteinase-9 level in plasma and brain of ischemic stroke model

International Nuclear Information System (INIS)

Koh, Seong-Ho; Chang, Dae-Il; Kim, Hee-Tae; Kim, Juhan; Kim, Myung-Ho; Kim, Kyung Suk; Bae, Inhee; Kim, Haekwon; Kim, Dong Won; Kim, Seung Hyun

2005-01-01

We investigated the effect of poly(ADP-ribose) polymerase (PARP) inhibitor on the levels of plasma and brain matrix metalloproteinase-9 (MMP-9) and the expression of nuclear factor kappa B (NF-κB) during experimental focal cerebral ischemia. The 3-aminobenzamide (3-AB), a PARP inhibitor, and saline were administered to 80 Sprague-Dawley rats [3-AB group; 5 rats for plasma sampling, 35 for brain sampling, and 40 for TTC staining] and to 85 rats (10, 35, and 40, respectively), respectively, 10 min before the occlusion of the left middle cerebral artery (MCAo) for 2 h. Infarct volume was measured by TTC staining, the serial levels of plasma and brain MMP-9 were measured by zymography just before and 2, 4, 8, 24, 48, and 72 h after MCAo, brain NF-κB activity was determined by Western blotting, and neutrophil infiltration was evaluated by assessing myeloperoxidase activity. Compared with control group, the levels of plasma and brain MMP-9, brain NF-κB, and MPO activities were significantly reduced in 3-AB group at each time point (p < 0.05). Plasma MMP-9 increased maximally at 4 h and then decreased rapidly, brain MMP-9 increased maximally at 24 h and persisted until 72 h, and NF-κB increased maximally at 24 h and then decreased slowly in both groups. Therefore, the PARP inhibitor reduces the expression of MMP-9 and NF-κB and the infiltration of neutrophils in ischemic stroke

Sigma-2 ligands and PARP inhibitors synergistically trigger cell death in breast cancer cells

International Nuclear Information System (INIS)

McDonald, Elizabeth S.; Mankoff, Julia; Makvandi, Mehran; Chu, Wenhua; Chu, Yunxiang; Mach, Robert H.; Zeng, Chenbo

2017-01-01

The sigma-2 receptor is overexpressed in proliferating cells compared to quiescent cells and has been used as a target for imaging solid tumors by positron emission tomography. Recent work has suggested that the sigma-2 receptor may also be an effective therapeutic target for cancer therapy. Poly (ADP-ribose) polymerase (PARP) is a family of enzymes involved in DNA damage response. In this study, we looked for potential synergy of cytotoxicity between PARP inhibitors and sigma-2 receptor ligands in breast cancer cell lines. We showed that the PARP inhibitor, YUN3-6, sensitized mouse breast cancer cell line, EMT6, to sigma-2 receptor ligand (SV119, WC-26, and RHM-138) induced cell death determined by cell viability assay and colony forming assay. The PARP inhibitor, olaparib, sensitized tumor cells to a different sigma-2 receptor ligand SW43-induced apoptosis and cell death in human triple negative cell line, MDA-MB-231. Olaparib inhibited PARP activity and cell proliferation, and arrested cells in G2/M phase of the cell cycle in MDA-MB-231 cells. Subsequently cells became sensitized to SW43 induced cell death. In conclusion, the combination of sigma-2 receptor ligands and PARP inhibitors appears to hold promise for synergistically triggering cell death in certain types of breast cancer cells and merits further investigation. - Highlights: • PARPi, YUN3-6 and olaparib, and σ2 ligands, SV119 and SW43, were evaluated. • Mouse and human breast cancer cells, EMT6 and MDA-MB-231 respectively, were used. • YUN3-6 and SV119 synergistically triggered cell death in EMT6 cells. • Olaparib and SW43 additively triggered cell death in MDA-MB-231 cells. • Olaparib arrested cells in G2/M in MDA-MB-231 cells.

Rucaparib: a novel PARP inhibitor for BRCA advanced ovarian cancer

Directory of Open Access Journals (Sweden)

Colombo I

2018-03-01

Full Text Available Ilaria Colombo, Stephanie Lheureux, Amit Manulal Oza Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada Abstract: Rucaparib is a potent small-molecule inhibitor of poly (ADP-ribose polymerase (PARP proteins (PARP-1, PARP-2 and PARP-3 that play an important role in repairing DNA damage and maintaining genomic stability. Tumors with mutations in BRCA1/2 or other homologous recombination deficiency (HRD genes are particularly sensitive to PARP inhibitors because of “synthetic lethality”, whereby a therapeutic agent can take advantage of an intrinsic weakness in DNA repair. Rucaparib has been investigated in several preclinical and clinical studies showing promising activity in BRCA-mutant and BRCA–wild-type epithelial ovarian cancers (EOCs. Dose-escalation Phase I studies have established the recommended Phase II dose to be 600 mg twice a day for oral rucaparib. Phase II and III studies have defined its role as treatment for BRCA-mutant recurrent high-grade EOC and as maintenance treatment for platinum-sensitive relapsed EOC following response to platinum-based chemotherapy. Genomic loss of heterozygosity has also been investigated as a potential signature of HRD and as a potential predictive biomarker of response. Treatment-induced adverse events (AEs have been observed in almost all patients treated with rucaparib, but mainly lower grade; with the most common being nausea, vomiting, asthenia/fatigue, anemia and transient transaminitis. The majority of AEs occurred early in treatment, were transient and have been easily managed with supportive treatment, dose interruption or discontinuation. This review will analyze the results of clinical trials investigating efficacy and safety of rucaparib in patients with ovarian cancer. Keywords: rucaparib, ovarian cancer, BRCA mutations, homologous recombination deficiency, maintenance treatment, PARP inhibitor

Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells

OpenAIRE

Kyoung-jin Min; Ju-Ock Nam; Taeg Kyu Kwon

2017-01-01

Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki) cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (...

Suppression of Homologous Recombination by insulin-like growth factor-1 inhibition sensitizes cancer cells to PARP inhibitors

International Nuclear Information System (INIS)

Amin, Oreekha; Beauchamp, Marie-Claude; Nader, Paul Abou; Laskov, Ido; Iqbal, Sanaa; Philip, Charles-André; Yasmeen, Amber; Gotlieb, Walter H.

2015-01-01

Impairment of homologous recombination (HR) is found in close to 50 % of ovarian and breast cancer. Tumors with BRCA1 mutations show increased expression of the Insulin-like growth factor type 1 receptor (IGF-1R). We previously have shown that inhibition of IGF-1R results in growth inhibition and apoptosis of ovarian tumor cells. In the current study, we aimed to investigate the correlation between HR and sensitivity to IGF-1R inhibition. Further, we hypothesized that IGF-1R inhibition might sensitize HR proficient cancers to Poly ADP ribose polymerase (PARP) inhibitors. Using ovarian and breast cancer cellular models with known BRCA1 status, we evaluated their HR functionality by RAD51 foci formation assay. The 50 % lethal concentration (LC50) of Insulin-like growth factor type 1 receptor kinase inhibitor (IGF-1Rki) in these cells was assessed, and western immunoblotting was performed to determine the expression of proteins involved in the IGF-1R pathway. Moreover, IGF-1R inhibitors were added on HR proficient cell lines to assess mRNA and protein expression of RAD51 by qPCR and western blot. Also, we explored the interaction between RAD51 and Insulin receptor substance 1 (IRS-1) by immunoprecipitation. Next, combination effect of IGF-1R and PARP inhibitors was evaluated by clonogenic assay. Cells with mutated/methylated BRCA1 showed an impaired HR function, and had an overactivation of the IGF-1R pathway. These cells were more sensitive to IGF-1R inhibition compared to HR proficient cells. In addition, the IGF-IR inhibitor reduced RAD51 expression at mRNA and protein levels in HR proficient cells, and sensitized these cells to PARP inhibitor. Targeting IGF-1R might lead to improved personalized therapeutic approaches in cancer patients with HR deficiency. Targeting both PARP and IGF-1R might increase the clinical efficacy in HR deficient patients and increase the population of patients who may benefit from PARP inhibitors

Synthesis, biological evaluation and molecular modeling of 2-Hydroxyisoquinoline-1,3-dione analogues as inhibitors of HIV reverse transcriptase associated ribonuclease H and polymerase.

Science.gov (United States)

Tang, Jing; Vernekar, Sanjeev Kumar V; Chen, Yue-Lei; Miller, Lena; Huber, Andrew D; Myshakina, Nataliya; Sarafianos, Stefan G; Parniak, Michael A; Wang, Zhengqiang

2017-06-16

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not clinically validated as an antiviral target. 2-Hydroxyisoquinoline-1,3-dione (HID) is known to confer active site directed inhibition of divalent metal-dependent enzymatic functions, such as HIV RNase H, integrase (IN) and hepatitis C virus (HCV) NS5B polymerase. We report herein the synthesis and biochemical evaluation of a few C-5, C-6 or C-7 substituted HID subtypes as HIV RNase H inhibitors. Our data indicate that while some of these subtypes inhibited both the RNase H and polymerase (pol) functions of RT, potent and selective RNase H inhibition was achieved with subtypes 8-9 as exemplified with compounds 8c and 9c. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Growth and gas production of a novel obligatory heterofermentative Cheddar cheese nonstarter lactobacilli species on ribose and galactose.

Science.gov (United States)

Ortakci, Fatih; Broadbent, Jeffery R; Oberg, Craig J; McMahon, Donald J

2015-06-01

An obligatory heterofermentative lactic acid bacterium, Lactobacillus wasatchii sp. nov., isolated from gassy Cheddar cheese was studied for growth, gas formation, salt tolerance, and survival against pasteurization treatments at 63°C and 72°C. Initially, Lb. wasatchii was thought to use only ribose as a sugar source and we were interested in whether it could also utilize galactose. We conducted experiments to determine the rate and extent of growth and gas production in carbohydrate-restricted (CR) de Man, Rogosa, and Sharpe (MRS) medium under anaerobic conditions with various combinations of ribose and galactose at 12, 23, and 37°C, with 23°C being the optimum growth temperature of Lb. wasatchii among the 3 temperatures studied. When Lb. wasatchii was grown on ribose (0.1, 0.5, and 1%), maximum specific growth rates (µmax) within each temperature were similar. When galactose was the only sugar, compared with ribose, µmax was 2 to 4 times lower. At all temperatures, the highest final cell densities (optical density at 640 nm) of Lb. wasatchii were achieved in CR-MRS plus 1% ribose, 0.5% ribose and 0.5% galactose, or 1% ribose and 1% galactose. Similar µmax values and final cell densities were achieved when 50% of the ribose in CR-MRS was substituted with galactose. Such enhanced utilization of galactose in the presence of ribose to support bacterial growth has not previously been reported. It appears that Lb. wasatchii co-metabolizes ribose and galactose, utilizing ribose for energy and galactose for other functions such as cell wall biosynthesis. Co-utilization of both sugars could be an adaptation mechanism of Lb. wasatchii to the cheese environment to efficiently ferment available sugars for maximizing metabolism and growth. As expected, gas formation by the heterofermenter was observed only when galactose was present in the medium. Growth experiments with MRS plus 1.5% ribose at pH 5.2 or 6.5 with 0, 1, 2, 3, 4, or 5% NaCl revealed that Lb. wasatchii is

Combination strategy of PARP inhibitor with antioxidant prevent bioenergetic deficits and inflammatory changes in CCI-induced neuropathy.

Science.gov (United States)

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

2017-02-01

Neuropathic pain, a debilitating pain condition and the underlying pathogenic mechanisms are complex and interwoven amongst each other and still there is scant information available regarding therapies which promise to treat the condition. Evidence indicate that oxidative/nitrosative stress induced poly (ADP-ribose) polymerase (PARP) overactivation initiate neuroinflammation and bioenergetic crisis culminating into neurodegenerative changes following nerve injury. Hence, we investigated the therapeutic effect of combining an antioxidant, quercetin and a PARP inhibitor, 4-amino 1, 8-naphthalimide (4-ANI) on the hallmark deficits induced by chronic constriction injury (CCI) of sciatic nerve in rats. Quercetin (25 mg/kg, p.o.) and 4-ANI (3 mg/kg, p.o.) were administered either alone or in combination for 14 days to examine sciatic functional index, allodynia and hyperalgesia using walking track analysis, Von Frey, acetone spray and hot plate tests respectively. Malondialdehyde, nitrite and glutathione levels were estimated to detect oxidative/nitrosative stress; mitochondrial membrane potential and cytochrome c oxidase activity to assess mitochondrial function; NAD & ATP levels to examine the bioenergetic status and levels of inflammatory markers were evaluated in ipsilateral sciatic nerve. Quercetin and 4-ANI alone improved the pain behaviour and biochemical alterations but the combination therapy demonstrated an appreciable reversal of CCI-induced changes. Nitrotyrosine and Poly ADP-Ribose (PAR) immunopositivity was decreased and nuclear factor erythroid 2-related factor (Nrf-2) levels were increased significantly in micro-sections of the sciatic nerve and dorsal root ganglion (DRG) of treatment group. These results suggest that simultaneous inhibition of oxidative stress-PARP activation cascade may potentially be useful strategies for management of trauma induced neuropathic pain. Copyright © 2016 Elsevier Ltd. All rights reserved.

Combining the ABL1 kinase inhibitor ponatinib and the histone deacetylase inhibitor vorinostat: a potential treatment for BCR-ABL-positive leukemia.

Science.gov (United States)

Okabe, Seiichi; Tauchi, Tetsuzo; Kimura, Shinya; Maekawa, Taira; Kitahara, Toshihiko; Tanaka, Yoko; Ohyashiki, Kazuma

2014-01-01

Resistance to imatinib (Gleevec®) in cancer cells is frequently because of acquired point mutations in the kinase domain of BCR-ABL. Ponatinib, also known as AP24534, is an oral multi-targeted tyrosine kinase inhibitor (TKI), and it has been investigated in a pivotal phase 2 clinical trial. The histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid) has been evaluated for its significant clinical activity in hematological malignancies. Thus, treatments combining ABL TKIs with additional drugs may be a promising strategy in the treatment of leukemia. In the current study, we analyzed the efficacy of ponatinib and vorinostat treatment by using BCR-ABL-positive cell lines. Treatment with ponatinib for 72 h inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner. We found that ponatinib potently inhibited the growth of Ba/F3 cells ectopically expressing BCR-ABL T315I mutation. Upon BCR-ABL phosphorylation, Crk-L was decreased, and poly (ADP-ribose) polymerase (PARP) was activated in a dose-dependent manner. Combined treatment of Ba/F3 T315I mutant cells with vorinostat and ponatinib resulted in significantly increased cytotoxicity. Additionally, the intracellular signaling of ponatinib and vorinostat was examined. Caspase 3 and PARP activation increased after combination treatment with ponatinib and vorinostat. Moreover, an increase in the phosphorylation levels of γH2A.X was observed. Previously established ponatinib-resistant Ba/F3 cells were also resistant to imatinib, nilotinib, and dasatinib. We investigated the difference in the efficacy of ponatinib and vorinostat by using ponatinib-resistant Ba/F3 cells. Combined treatment of ponatinib-resistant cells with ponatinib and vorinostat caused a significant increase in cytotoxicity. Thus, combined administration of ponatinib and vorinostat may be a powerful strategy against BCR-ABL mutant cells and could enhance the cytotoxic effects of ponatinib in those BCR

The Effects of Ribose on Mechanical and Physicochemical Properties of Cold Water Fish Gelatin Films

Directory of Open Access Journals (Sweden)

Neda Javadian

2014-06-01

Full Text Available Native fish gelatin has some disadvantages such as high hydrophilic, and solubility in cold water. Mixing with other biopolymers and crosslinking by sugars may improve functional properties of fish gelatin. So in this research, the effects of ribose were investigated on moisture sorption isotherm, solubility in water, and mechanical properties of cold water fish gelatin (CWFG films. Ribose sugar was incorporated into CWFG solutions at different concentrations (e.g. 0, 2, 4, and 6% w/w dried gelatin. Physicochemical properties such as water solubility, moisture sorption isotherm and mechanical properties of the films were measured according to ASTM standards. Results showed that incorporation of ribose sugar significantly improved functional properties of CWFG films. Solubility, moisture content and monolayer water content of the matrixes were decreased by increasing the ribose contents. Mechanical properties of biocomposites were improved more than 20% and moisture sorption isotherm curve significantly shifted to lower moisture contents. The results of this study could be explored for commercial use, depending on industrial needs for either production of edible films or for packaging purposes.

Escherichia coli rpiA gene encoding ribose phosphate isomerase A

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne; Maigaard, Marianne

1993-01-01

The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment was seque......The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment...

COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

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Masashi Miura

2013-12-01

Full Text Available SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

The ribose and glycine Maillard reaction in the interstellar medium ...

Indian Academy of Sciences (India)

WINTEC

mechanics are briefly addressed in this work. Keywords. Density functional computational study; ribose; glycine; Maillard reaction; gaseous phase .... following the total mass balance of the reaction. Thus, ..... Jalbout A F Origin Life Evol. Biosph ...

Inhibition of polymerases-alpha and -beta completely blocks DNA repair induced by UV irradiation in cultured mouse neuronal cells

International Nuclear Information System (INIS)

Licastro, F.; Sarafian, T.; Verity, A.M.; Walford, R.L.

1985-01-01

The effects of hydroxyurea, aphidicolin and dideoxythymidine on UV-induced DNA repair of mouse neuronal granular cells were studied. Aphidicolin, which is considered a specific inhibitor of polymerase-alpha, decreased spontaneous DNA synthesis by 93% and totally suppressed DNA repair. Dideoxythymidine, an inhibitor of polymerase-beta, was more potent in decreasing scheduled DNA synthesis than aphidicolin, and also completely blocked the UV-induced DNA repair. Hydroxyurea, a specific inhibitor of ribonucleotide reductase, inhibited scheduled DNA synthesis, but unscheduled DNA synthesis after UV irradiation was always well detectable. Our data suggest that in neuronal cells from 5 to 10 days old mice both polymerases-alpha and -beta are required for both DNA synthesis and repair. These two enzymes may act jointly in filling up the gaps along the DNA molecule and elongating the DNA chain

DNA repair synthesis in human fibroblasts requires DNA polymerase delta

International Nuclear Information System (INIS)

Nishida, C.; Reinhard, P.; Linn, S.

1988-01-01

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

A Wheat SIMILAR TO RCD-ONE Gene Enhances Seedling Growth and Abiotic Stress Resistance by Modulating Redox Homeostasis and Maintaining Genomic Integrity[C][W

Science.gov (United States)

Liu, Shuantao; Liu, Shuwei; Wang, Mei; Wei, Tiandi; Meng, Chen; Wang, Meng; Xia, Guangmin

2014-01-01

Plant growth inhibition is a common response to salinity. Under saline conditions, Shanrong No. 3 (SR3), a bread wheat (Triticum aestivum) introgression line, performs better than its parent wheat variety Jinan 177 (JN177) with respect to both seedling growth and abiotic stress tolerance. Furthermore, the endogenous reactive oxygen species (ROS) was also elevated in SR3 relative to JN177. The SR3 allele of sro1, a gene encoding a poly(ADP ribose) polymerase (PARP) domain protein, was identified to be crucial for both aspects of its superior performance. Unlike RADICAL-INDUCED CELL DEATH1 and other Arabidopsis thaliana SIMILAR TO RCD-ONE (SRO) proteins, sro1 has PARP activity. Both the overexpression of Ta-sro1 in wheat and its heterologous expression in Arabidopsis promote the accumulation of ROS, mainly by enhancing the activity of NADPH oxidase and the expression of NAD(P)H dehydrogenase, in conjunction with the suppression of alternative oxidase expression. Moreover, it promotes the activity of ascorbate-GSH cycle enzymes and GSH peroxidase cycle enzymes, which regulate ROS content and cellular redox homeostasis. sro1 is also found to be involved in the maintenance of genomic integrity. We show here that the wheat SRO has PARP activity; such activity could be manipulated to improve the growth of seedlings exposed to salinity stress by modulating redox homeostasis and maintaining genomic stability. PMID:24443520

Stability of the resistance to the thiosemicarbazone derived from 5,6-Dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus

OpenAIRE

Castro, Eliana Florencia; Campos, Rodolfo Hector; Cavallaro, Lucía Vicenta

2017-01-01

Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1–5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of...

Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

International Nuclear Information System (INIS)

Chen Haibing; Jia Weiping; Xu Xun; Fan Ying; Zhu Dongqing; Wu Haixiang; Xie Zhenggao; Zheng Zhi

2008-01-01

Poly(ADP-ribose)polymerase (PARP) inhibitors decrease angiogenesis through reducing vascular endothelium growth factor (VEGF) induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). In contrast to VEGF, pigment epithelium-derived factor (PEDF) has been demonstrated to act as a strong endogenous inhibitor of angiogenesis. Here, we show that PARP inhibition with a specific inhibitor PJ-34 or specific PARP antisense oligonucleotide upregulates hyperglycemia-induced PEDF expression in HUVECs in a dose-dependent manner. This results in the retard of activation of p38 MAP kinase and the concomitant decrease in cell apoptosis. These results give the first direct demonstration that PEDF might represent a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent

Ribose Supplementation Alone or with Elevated Creatine Does Not Preserve High Energy Nucleotides or Cardiac Function in the Failing Mouse Heart.

Directory of Open Access Journals (Sweden)

Kiterie M E Faller

Full Text Available Reduced levels of creatine and total adenine nucleotides (sum of ATP, ADP and AMP are hallmarks of chronic heart failure and restoring these pools is predicted to be beneficial by maintaining the diseased heart in a more favourable energy state. Ribose supplementation is thought to support both salvage and re-synthesis of adenine nucleotides by bypassing the rate-limiting step. We therefore tested whether ribose would be beneficial in chronic heart failure in control mice and in mice with elevated myocardial creatine due to overexpression of the creatine transporter (CrT-OE.FOUR GROUPS WERE STUDIED: sham; myocardial infarction (MI; MI+ribose; MI+CrT-OE+ribose. In a pilot study, ribose given in drinking water was bioavailable, resulting in a two-fold increase in myocardial ribose-5-phosphate levels. However, 8 weeks post-surgery, total adenine nucleotide (TAN pool was decreased to a similar amount (8-14% in all infarcted groups irrespective of the treatment received. All infarcted groups also presented with a similar and substantial degree of left ventricular (LV dysfunction (3-fold reduction in ejection fraction and LV hypertrophy (32-47% increased mass. Ejection fraction closely correlated with infarct size independently of treatment (r(2 = 0.63, p<0.0001, but did not correlate with myocardial creatine or TAN levels.Elevating myocardial ribose and creatine levels failed to maintain TAN pool or improve post-infarction LV remodeling and function. This suggests that ribose is not rate-limiting for purine nucleotide biosynthesis in the chronically failing mouse heart and that alternative strategies to preserve TAN pool should be investigated.

Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

Science.gov (United States)

Kempf, Brian J.; Peersen, Olve B.

2016-01-01

ABSTRACT RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a

Improvement of D-Ribose Production from Corn Starch Hydrolysate by a Transketolase-Deficient Strain Bacillus subtilis UJS0717

Science.gov (United States)

Wei, Zhuan; Zhou, Jue; Sun, WenJing; Cui, FengJie; Xu, QinHua; Liu, ChangFeng

2015-01-01

D-Ribose is a five-carbon sugar and generally used as an energy source to improve athletic performance and the ability. The culture conditions for maximum D-ribose production performance from cheap raw material corn starch hydrolysate were improved by using one-factor-at-a-time experiments and a three-level Box-Behnken factorial design. The optimal fermentation parameters were obtained as 36°C culture temperature, 10% inoculum volume, and 7.0 initial pH. The mathematical model was then developed to show the effect of each medium composition and their interactions on the production of D-ribose and estimated that the optimized D-ribose production performance with the concentration of 62.13 g/L, yield of 0.40 g/g, and volumetric productivity of 0.86 g/L·h could be obtained when the medium compositions were set as 157 g/L glucose, 21 g/L corn steep liquor, 3.2 g/L (NH4)2SO4, 1 g/L yeast extract, 0.05 g/L MnSO4·H2O, and 20 g/L CaCO3. These findings indicated the D-ribose production performance was significantly improved compared to that under original conditions. PMID:26759810

Identification of Critical Residues for the Tight Binding of Both Correct and Incorrect Nucleotides to Human DNA Polymerase λ

Science.gov (United States)

Brown, Jessica A.; Pack, Lindsey R.; Sherrer, Shanen M.; Kshetry, Ajay K.; Newmister, Sean A.; Fowler, Jason D.; Taylor, John-Stephen; Suo, Zucai

2010-01-01

DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β (Pol β). Pre-steady state kinetic studies have shown that the Pol λ•DNA complex binds both correct and incorrect nucleotides 130-fold tighter on average than the Pol β•DNA complex, although, the base substitution fidelity of both polymerases is 10−4 to 10−5. To better understand Pol λ’s tight nucleotide binding affinity, we created single- and double-substitution mutants of Pol λ to disrupt interactions between active site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding with each of the common structural moieties in the following order: triphosphate ≫ base > ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and template base led to a moderate increase in the Kd. The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template. PMID:20851705

Antioxidant activity and inhibitory effects of 2-hydroxy-3-methylcyclopent-2-enone isolated from ribose-histidine Maillard reaction products on aldose reductase and tyrosinase.

Science.gov (United States)

Hwang, Seung Hwan; Wang, Zhiqiang; Suh, Hong-Won; Lim, Soon Sung

2018-03-01

This study aimed to better understand the functional properties of ribose and 20 amino acid Maillard reaction products (MRPs). The ABTS + radical scavenging ability of the ribose-20 amino acid MRPs was evaluated. Among the MRPs, ribose-histidine MRPs (RH-MRPs) showed the highest inhibitory activities on the ABTS + radical scavenging ability, aldose reductase (AR), and tyrosinase compared to other MRPs. Functional compounds with antioxidant and AR inhibitory activities have been recognized as an important strategy in the prevention and treatment of diabetic complications, and the search for tyrosinase inhibitors is important for the treatment of hyperpigmentation, development of skin-whitening agents, and use as preservatives in the food industry. On this basis, we sought to isolate and identify compounds with inhibitory activities against AR and tyrosinase. RH-MRPs were heated at 120 °C for 2 h and fractionated using four solvents: methylene chloride (MC), ethyl acetate, n-butanol, and water. The highest inhibitions were found in the MC fraction. The two compounds from this fraction were purified by silica gel column and preparative thin layer chromatography, and identified as 2-hydroxy-3-methylcyclopent-2-enone and furan-3-carboxylic acid. AR inhibition, tyrosinase inhibition, and ABTS + scavenging (IC 50 ) of 2-hydroxy-3-methylcyclopent-2-enone were 4.47, 721.91 and 9.81 μg mL -1 , respectively. In this study, inhibitory effects of 2-hydroxy-3-methylcyclopent-2-enone isolated from RH-MRP were demonstrated on AR, tyrosinase, and its antioxidant activity for the first time. RH-MRP and its constituents can be developed as beneficial functional food sources and cosmetic materials and should be investigated further as potential functional food sources.

Prebiotic synthesis of 2-deoxy-d-ribose from interstellar building blocks promoted by amino esters or amino nitriles.

Science.gov (United States)

Steer, Andrew M; Bia, Nicolas; Smith, David K; Clarke, Paul A

2017-09-25

Understanding the prebiotic genesis of 2-deoxy-d-ribose, which forms the backbone of DNA, is of crucial importance to unravelling the origins of life, yet remains open to debate. Here we demonstrate that 20 mol% of proteinogenic amino esters promote the selective formation of 2-deoxy-d-ribose over 2-deoxy-d-threopentose in combined yields of ≥4%. We also demonstrate the first aldol reaction promoted by prebiotically-relevant proteinogenic amino nitriles (20 mol%) for the enantioselective synthesis of d-glyceraldehyde with 6% ee, and its subsequent conversion into 2-deoxy-d-ribose in yields of ≥ 5%. Finally, we explore the combination of these two steps in a one-pot process using 20 mol% of an amino ester or amino nitrile promoter. It is hence demonstrated that three interstellar starting materials, when mixed together with an appropriate promoter, can directly lead to the formation of a mixture of higher carbohydrates, including 2-deoxy-d-ribose.

Mechanism of chromatin degradation in thymocytes of irradiated rats

International Nuclear Information System (INIS)

Zotova, R.N.; Umanskij, S.R.; Tokarskaya, V.I.

1983-01-01

A biphase change in poly (ADP-ribose) polymerase activity of the thymocyte chromatin was observed after 10 Gy irradiation of rats: during the first minutes the incorporation of 14 C-NAD increased by 40% then started decreasing to make 110, 60 and 35% after 1, 2 and 3 h, respectively. Irradiation of rat thymus chromatin in vitro sharply decreased poly (ADP-ribose) polymerase activity. The possible role of changes in the poly (ADP-ribose) synthesis in the activation of nuclear Ca/Mg-dependent endonuclease and in the postirradiation degradation of the thymocyte chromatin is discussed

Chemoproteomics-Aided Medicinal Chemistry for the Discovery of EPHA2 Inhibitors.

Science.gov (United States)

Heinzlmeir, Stephanie; Lohse, Jonas; Treiber, Tobias; Kudlinzki, Denis; Linhard, Verena; Gande, Santosh Lakshmi; Sreeramulu, Sridhar; Saxena, Krishna; Liu, Xiaofeng; Wilhelm, Mathias; Schwalbe, Harald; Kuster, Bernhard; Médard, Guillaume

2017-06-21

The receptor tyrosine kinase EPHA2 has gained attention as a therapeutic drug target for cancer and infectious diseases. However, EPHA2 research and EPHA2-based therapies have been hampered by the lack of selective small-molecule inhibitors. Herein we report the synthesis and evaluation of dedicated EPHA2 inhibitors based on the clinical BCR-ABL/SRC inhibitor dasatinib as a lead structure. We designed hybrid structures of dasatinib and the previously known EPHA2 binders CHEMBL249097, PD-173955, and a known EPHB4 inhibitor in order to exploit both the ATP pocket entrance as well as the ribose pocket as binding epitopes in the kinase EPHA2. Medicinal chemistry and inhibitor design were guided by a chemical proteomics approach, allowing early selectivity profiling of the newly synthesized inhibitor candidates. Concomitant protein crystallography of 17 inhibitor co-crystals delivered detailed insight into the atomic interactions that underlie the structure-affinity relationship. Finally, the anti-proliferative effect of the inhibitor candidates was confirmed in the glioblastoma cell line SF-268. In this work, we thus discovered a novel EPHA2 inhibitor candidate that features an improved selectivity profile while maintaining potency against EPHA2 and anticancer activity in SF-268 cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Glycosidation of Methanol with Ribose: An Interdisciplinary Undergraduate Laboratory Experiment

Science.gov (United States)

Simon, Erin; Cook, Katie; Pritchard, Meredith R.; Stripe, Wayne; Bruch, Martha; Bendinskas, Kestutis

2010-01-01

This exercise provides students hands-on experience with the topics of glycosidation, hemiacetal and acetal formation, proton nuclear magnetic resonance ([superscript 1]H NMR) spectroscopy, and kinetic and thermodynamic product formation. In this laboratory experiment, the methyl acetal of ribose is synthesized, and the kinetic and thermodynamic…

Targeting the unmet medical need: the Abbott Laboratories oncology approach.

Science.gov (United States)

Carlson, Dawn M; Steinberg, Joyce L; Gordon, Gary

2005-09-01

While significant advances in the treatment of cancer occured during the last half of the twentieth century, parallel decreases in overall cancer death rates were not observed. Cancer therapy remains an area of significant unmet medical need. Abbott's oncology research programs are focused on pioneering trageted, less toxic therapies, aimed at different aspects of tumor growth and development. Oncology drugs in development at Abbott target several mechanisms of cancer progression by interfering with multiple processes necessary for tumor growth: recruitment of a blood supply, cell proliferation, and the development of metastases. They include a selective endothelin A-receptor antagonist (atrasentan/Xinlay), 3 angiogenesis inhibitors (ABT 510, a thrombospondin mimetic: ABT-869, a multitargeted receptor tyrosine kinase inhibitor; and ABT 828, recombinant human plasminogen kringle 5), a cell proliferation inhibitor (ABT-751, an antimitotic agent), an apoptosis inducer (ABT 737, a Bcl-2 family inhibitor), and a poly(ADP-ribose)polymerase inhibitor.

Heat shock protein 90 inhibitor enhances apoptosis by inhibiting the AKT pathway in thermal-stimulated SK-MEL-2 human melanoma cell line.

Science.gov (United States)

Shin, Min Kyung; Jeong, Ki-Heon; Choi, Hyeongwon; Ahn, Hye-Jin; Lee, Mu-Hyoung

2018-02-08

Heat shock proteins (Hsps) are chaperone proteins, which are upregulated after various stresses. Hsp90 inhibitors have been investigated as adjuvant therapies for the treatment of melanoma. Thermal ablation could be a treatment option for surgically unresectable melanoma or congenital nevomelanocytic nevi, however, there is a limitation such as the possibility of recurrence. We evaluated apoptosis in a melanoma cell line treated with the Hsp90 inhibitor 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), in hyperthermic conditions. SK-MEL-2 cells were stimulated at 43 °C for 1 h and treated with 0, 0.1 and 1 μM 17-DMAG. We evaluated the cell viability using MTT and apoptosis with HSP 90 inhibitor. We studied the protein expression of AKT, phospho-AKT, ERK, phospho-ERK, MAPK, and phospho-MAPK, caspase 3,7,9, and anti-poly (ADP-ribose) polymerase. 17-DMAG significantly inhibited the proliferation of the SK-MEL-2 cells at 37 °C (0.1 μM: 44.47% and 1 μM: 61.23%) and 43 °C (0.1 μM: 49.21% and 1 μM: 63.60%), suggesting synergism between thermal stimulation and 17-DMAG. 17-DMAG treatment increased the frequency of apoptotic cell populations to 2.17% (0.1 μM) and 3.05% (1 μM) in 37 °C controls, and 4.40% (0.1 μM) and 4.97% (1 μM) in the group stimulated at 43 °C. AKT phosphorylation were activated by thermal stimulation and inhibited by 17-DMAG. Hsp90 inhibitor treatment may be clinically applicable to enhance the apoptosis of melanoma cells in hyperthermic condition. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Overcoming Resistance of Cancer Cells to PARP-1 Inhibitors with Three Different Drug Combinations.

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Michal Yalon

Full Text Available Inhibitors of poly[ADP-ribose] polymerase 1 (PARPis show promise for treatment of cancers which lack capacity for homologous recombination repair (HRR. However, new therapeutic strategies are required in order to overcome innate and acquired resistance to these drugs and thus expand the array of cancers that could benefit from them. We show that human cancer cell lines which respond poorly to ABT-888 (a PARPi, become sensitive to it when co-treated with vorinostat (a histone deacetylase inhibitor (HDACi. Vorinostat also sensitized PARPis insensitive cancer cell lines to 6-thioguanine (6-TG-a drug that targets PARPis sensitive cells. The sensitizing effect of vorinostat was associated with increased phosphorylation of eukaryotic initiation factor (eIF 2α which in and of itself increases the sensitivity of cancer cells to ABT-888. Importantly, these drug combinations did not affect survival of normal fibroblasts and breast cells, and significantly increased the inhibition of xenograft tumor growth relative to each drug alone, without affecting the mice weight or their liver and kidney function. Our results show that combination of vorinostat and ABT-888 could potentially prove useful for treatment of cancer with innate resistance to PARPis due to active HRR machinery, while the combination of vorinostat and 6-TG could potentially overcome innate or acquired resistance to PARPis due to secondary or reversal BRCA mutations, to decreased PARP-1 level or to increased expression of multiple drug resistant proteins. Importantly, drugs which increase phosphorylation of eIF2α may mimic the sensitizing effect of vorinostat on cellular response to PARPis or to 6-TG, without activating all of its downstream effectors.

Functional role of zinc in poly(A) synthesis catalyzed by nuclear poly(A) polymerase

Energy Technology Data Exchange (ETDEWEB)

Rose, K M; Allen, M S; Crawford, I L; Jacob, S T [Pennsylvania State Univ., Hershey (USA). Dept. of Pharmacology; Pennsylvania State Univ., Hershey (USA). Specialized Cancer Research Center; Texas Univ., Dallas (USA). Dept. of Neurology; Texas Univ., Dallas (USA). Dept. of Pharmacology)

1978-07-01

The functional role of transition metals in poly(A) synthesis was elucidated by investigating the effect of the metal chelator o-Phenanthroline on purified nuclear poly(A) polymerase. This chelator inhibited the enzyme activity in a manner competitive with respect to the polynucleotide primer concentration. o-phenanthroline was a non-competitive inhibitor with regard to ATP concentration and an 'uncompetitive' inhibitor with regard to dithiothreitor levels. The metal content of the purified enzyme preparations from rat liver and Morris hepatoma 3924A was determined using atomic absorption spectrometry. Of the transition metals measured, only zinc was present in detectable quantities at levels less than 1 mol/mol of enzyme. Hepatoma enzyme contained 2-3 times as much zinc as the corresponding liver enzyme. Hepatoma poly(A) polymerase was also radioactively labelled in vivo by injection of tumor-bearing animals with /sup 65/Zn. Dialysis experiments with highly purified radiolabelled poly(A) polymerase showed that the enzyme-zinc complex was labile and that a reduction in /sup 65/Zn content correlated with a loss in enzyme activity.

Functional role of zinc in poly(A) synthesis catalyzed by nuclear poly(A) polymerase

International Nuclear Information System (INIS)

Rose, K.M.; Allen, M.S.; Crawford, I.L.; Jacob, S.T.; Pennsylvania State Univ., Hershey; Texas Univ., Dallas; Texas Univ., Dallas

1978-01-01

The functional role of transition metals in poly(A) synthesis was elucidated by investigating the effect of the metal chelator o-phenanthroline on purified nuclear poly(A) polymerase. This chelator inhibited the enzyme activity in a manner competitive with respect to the polynucleotide primer concentration. o-phenanthroline was a non-competitive inhibitor with regard to ATP concentration and an 'uncompetitive' inhibitor with regard to dithiothreitor levels. The metal content of the purified enzyme preparations from rat liver and Morris hepatoma 3924A was determined using atomic absorption spectrometry. Of the transition metals measured, only zinc was present in detectable quantities, at levels less than 1 mol/mol of enzyme. Hepatoma enzyme contained 2-3 times as much zinc as the corresponding liver enzyme. Hepatoma poly(A) polymerase was also radioactively labelled in vivo by injection of tumor-bearing animals with 65 Zn. Dialysis experiments with highly purified radiolabelled poly(A) polymerase showed that the enzyme-zinc complex was labile and that a reduction in 65 Zn content correlated with a loss in enzyme activity. (orig./AJ) [de

Molecularly imprinted nanoparticles for inhibiting ribonuclease in reverse transcriptase polymerase chain reaction

DEFF Research Database (Denmark)

Feng, Xiaotong; Ashley, Jon; Zhou, Tongchang

2018-01-01

Molecularly imprinted nanoparticles (nanoMIPs) are synthesized via a solid-phase approach using RNase as the template. The feasibility of employing the nanoMIPs as RNase inhibitor is successfully demonstrated in reverse transcriptase polymerase chain reaction (RT-PCR) assays, suggesting the tailor...

A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

Science.gov (United States)

Patel, Manisha J; Akhani, Rekha C; Patel, Arti T; Dedania, Samir R; Patel, Darshan H

2017-02-01

l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency. Copyright © 2016 Elsevier Inc. All rights reserved.

EBV DNA polymerase inhibition of tannins from Eugenia uniflora.

Science.gov (United States)

Lee, M H; Chiou, J F; Yen, K Y; Yang, L L

2000-06-30

Nasopharyngeal carcinoma (NPC) is one of the high population malignant tumors among Chinese in southern China and southeast Asia. Epstein-Barr virus (EBV) is a human B lymphotropic herpes virus which is known to be closely associated with NPC. EBV DNA polymerase is a key enzyme during EBV replication and is measured by its radioactivity. The addition of phorbol 12-myristate 13-acetate to Raji cell cultures led to a large increase in EBV DNA polymerase, which was purified by sequential DEAE-cellulose, phosphocellulose and DNA-cellulose column chromatography. Four tannins were isolated from the active fractions of Eugenia uniflora L., which were tested for the inhibition of EBV DNA polymerase. The results showed the 50% inhibitory concentration (IC(50)) values of gallocatechin, oenothein B, eugeniflorins D(1) and D(2) were 26.5 62.3, 3.0 and 3.5 microM, respectively. Furthermore, when compared with the positive control (phosphonoacetic acid), an inhibitor of EBV replication, the IC(50) value was 16.4 microM. In view of the results, eugeniflorins D(1) and D(2) are the potency principles in the inhibition of EBV DNA polymerase from E. uniflora.

Cloning and characterization of a thermostable 2- deoxy-D-ribose-5 ...

African Journals Online (AJOL)

Analysis of the presumptive 2-deoxy-D-ribose 5-phosphate aldolase gene from Aciduliprofundum boonei revealed an open reading frame (ORF) encoding 222 amino acids, which was subcloned and then expressed in Escherichia coli. The recombinant DERA protein was purified to apparent homogeneity. The enzyme ...

Daily supplementation of D-ribose shows no therapeutic benefits in the MHC-I transgenic mouse model of inflammatory myositis.

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William Coley

Full Text Available BACKGROUND: Current treatments for idiopathic inflammatory myopathies (collectively called myositis focus on the suppression of an autoimmune inflammatory response within the skeletal muscle. However, it has been observed that there is a poor correlation between the successful suppression of muscle inflammation and an improvement in muscle function. Some evidence in the literature suggests that metabolic abnormalities in the skeletal muscle underlie the weakness that continues despite successful immunosuppression. We have previously shown that decreased expression of a purine nucleotide cycle enzyme, adenosine monophosphate deaminase (AMPD1, leads to muscle weakness in a mouse model of myositis and may provide a mechanistic basis for muscle weakness. One of the downstream metabolites of this pathway, D-ribose, has been reported to alleviate symptoms of myalgia in patients with a congenital loss of AMPD1. Therefore, we hypothesized that supplementing exogenous D-ribose would improve muscle function in the mouse model of myositis. We treated normal and myositis mice with daily doses of D-ribose (4 mg/kg over a 6-week time period and assessed its effects using a battery of behavioral, functional, histological and molecular measures. RESULTS: Treatment with D-ribose was found to have no statistically significant effects on body weight, grip strength, open field behavioral activity, maximal and specific forces of EDL, soleus muscles, or histological features. Histological and gene expression analysis indicated that muscle tissues remained inflamed despite treatment. Gene expression analysis also suggested that low levels of the ribokinase enzyme in the skeletal muscle might prevent skeletal muscle tissue from effectively utilizing D-ribose. CONCLUSIONS: Treatment with daily oral doses of D-ribose showed no significant effect on either disease progression or muscle function in the mouse model of myositis.

Daily Supplementation of D-ribose Shows No Therapeutic Benefits in the MHC-I Transgenic Mouse Model of Inflammatory Myositis

Science.gov (United States)

Coley, William; Rayavarapu, Sree; van der Meulen, Jack H.; Duba, Ayyappa S.; Nagaraju, Kanneboyina

2013-01-01

Background Current treatments for idiopathic inflammatory myopathies (collectively called myositis) focus on the suppression of an autoimmune inflammatory response within the skeletal muscle. However, it has been observed that there is a poor correlation between the successful suppression of muscle inflammation and an improvement in muscle function. Some evidence in the literature suggests that metabolic abnormalities in the skeletal muscle underlie the weakness that continues despite successful immunosuppression. We have previously shown that decreased expression of a purine nucleotide cycle enzyme, adenosine monophosphate deaminase (AMPD1), leads to muscle weakness in a mouse model of myositis and may provide a mechanistic basis for muscle weakness. One of the downstream metabolites of this pathway, D-ribose, has been reported to alleviate symptoms of myalgia in patients with a congenital loss of AMPD1. Therefore, we hypothesized that supplementing exogenous D-ribose would improve muscle function in the mouse model of myositis. We treated normal and myositis mice with daily doses of D-ribose (4 mg/kg) over a 6-week time period and assessed its effects using a battery of behavioral, functional, histological and molecular measures. Results Treatment with D-ribose was found to have no statistically significant effects on body weight, grip strength, open field behavioral activity, maximal and specific forces of EDL, soleus muscles, or histological features. Histological and gene expression analysis indicated that muscle tissues remained inflamed despite treatment. Gene expression analysis also suggested that low levels of the ribokinase enzyme in the skeletal muscle might prevent skeletal muscle tissue from effectively utilizing D-ribose. Conclusions Treatment with daily oral doses of D-ribose showed no significant effect on either disease progression or muscle function in the mouse model of myositis. PMID:23785461

Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself.

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Cian M McCrudden

Full Text Available Therapeutic inhibition of poly(ADP-ribose polymerase (PARP, as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699, induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

QSAR study of HCV NS5B polymerase inhibitors using the genetic algorithm-multiple linear regression (GA-MLR).

Science.gov (United States)

Rafiei, Hamid; Khanzadeh, Marziyeh; Mozaffari, Shahla; Bostanifar, Mohammad Hassan; Avval, Zhila Mohajeri; Aalizadeh, Reza; Pourbasheer, Eslam

2016-01-01

Quantitative structure-activity relationship (QSAR) study has been employed for predicting the inhibitory activities of the Hepatitis C virus (HCV) NS5B polymerase inhibitors . A data set consisted of 72 compounds was selected, and then different types of molecular descriptors were calculated. The whole data set was split into a training set (80 % of the dataset) and a test set (20 % of the dataset) using principle component analysis. The stepwise (SW) and the genetic algorithm (GA) techniques were used as variable selection tools. Multiple linear regression method was then used to linearly correlate the selected descriptors with inhibitory activities. Several validation technique including leave-one-out and leave-group-out cross-validation, Y-randomization method were used to evaluate the internal capability of the derived models. The external prediction ability of the derived models was further analyzed using modified r(2), concordance correlation coefficient values and Golbraikh and Tropsha acceptable model criteria's. Based on the derived results (GA-MLR), some new insights toward molecular structural requirements for obtaining better inhibitory activity were obtained.

Probes of eukaryotic DNA-dependent RNA polymerase II-I. Binding of 9-beta-D-arabinofuranosyl-6-mercaptopurine to the elongation subsite.

Science.gov (United States)

Cho, J M; Kimball, A P

1982-08-15

9-beta-D-Arabinofuranosyl-6-mercaptopurine (ara-6-MP) was used to affinity-label wheat germ DNA-dependent RNA polymerase II (or B) (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). This nucleoside analogue was found to be a competitive inhibitor with respect to [3H]UMP incorporation. Natural substrates protected the enzyme from inactivation by ara-6-MP when the enzyme was preincubated with excess concentrations of substrates, suggesting that the inhibitor binds at the elongation subsite. The inhibitor bound the catalytic center of the enzyme with a stoichiometry of 0.6:1. The sulfhydryl reagent, dithiothreitol, reversed the inhibition by ara-6-MP, suggesting that the 6-thiol group of the inhibitor was interacting closely with an essential cysteine residue in the catalytic center of the enzyme. Chromatographic analysis of the pronase-digestion products of the RNA polymerase II-ara-6-MP complex also showed that ara-6-MP had bound a cysteine residue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured [6-35S]ara-6-MP-labeled RNA polymerase II revealed that over 80% of the radioactivity was associated with the IIb subunit of the enzyme.

Isorhynchophylline, a Potent Plant Alkaloid, Induces Apoptotic and Anti-Metastatic Effects in Human Hepatocellular Carcinoma Cells through the Modulation of Diverse Cell Signaling Cascades.

Science.gov (United States)

Lee, Hanwool; Baek, Seung Ho; Lee, Jong Hyun; Kim, Chulwon; Ko, Jeong-Hyeon; Lee, Seok-Geun; Chinnathambi, Arunachalam; Alharbi, Sulaiman Ali; Yang, Woong Mo; Um, Jae-Young; Sethi, Gautam; Ahn, Kwang Seok

2017-05-19

Isorhynchophylline (Rhy) is an active pharmacological component of Uncaria rhynchophylla that has been reported previously to exert significant antihypertensive and neuroprotective effects. However, very little is known about its potential anti-cancer activities. This study was carried out to evaluate the anticancer effects of Rhy against various human carcinoma cell lines. We found that Rhy exhibited substantial cytotoxic effect against human hepatocellular carcinoma HepG2 cells when compared with other human carcinoma cell lines including those of lung, pancreas, prostate, head and neck, breast, multiple myeloma, brain and renal cell carcinoma. Rhy induced apoptosis as characterized by accumulation of cells in sub G1 phase; positive Annexin V binding; activation of caspase-8, -9, and -3; and cleavage of PARP (poly-ADP ribose polymerase). This effect of Rhy correlated with the down-regulation of various proteins that mediated cell proliferation, cell survival, metastasis, and angiogenesis. Moreover, cell proliferation, migration, and constitutive CXCR4 (C-X-C chemokine receptor type 4), MMP-9 (Matrix metallopeptidase-9), and MMP-2 expression were inhibited upon Rhy treatment. We further investigated the effect of Rhy on the oncogenic cell signaling cascades through phospho-kinase array profiling assay. Rhy was found to abrogate phospho-p38, ERK, JNK, CREB, c-Jun, Akt, and STAT3 signals, but interestingly enhanced phospho-p53 signal. Overall, our results indicate, for the first time, that Rhy could exert anticancer and anti-metastatic effects through regulation of multiple signaling cascades in hepatocellular carcinoma cells.

Pyridine nucleotide cycling and control of intracellular redox state in relation to poly (ADP-ribose) polymerase activity and nuclear localization of glutathione during exponential growth of Arabidopsis cells in culture.

Science.gov (United States)

Pellny, Till K; Locato, Vittoria; Vivancos, Pedro Diaz; Markovic, Jelena; De Gara, Laura; Pallardó, Federico V; Foyer, Christine H

2009-05-01

Pyridine nucleotides, ascorbate and glutathione are major redox metabolites in plant cells, with specific roles in cellular redox homeostasis and the regulation of the cell cycle. However, the regulation of these metabolite pools during exponential growth and their precise functions in the cell cycle remain to be characterized. The present analysis of the abundance of ascorbate, glutathione, and pyridine nucleotides during exponential growth of Arabidopsis cells in culture provides evidence for the differential regulation of each of these redox pools. Ascorbate was most abundant early in the growth cycle, but glutathione was low at this point. The cellular ascorbate to dehydroascorbate and reduced glutathione (GSH) to glutathione disulphide ratios were high and constant but the pyridine nucleotide pools were largely oxidized over the period of exponential growth and only became more reduced once growth had ceased. The glutathione pool increased in parallel with poly (ADP-ribose) polymerase (PARP) activities and with increases in the abundance of PARP1 and PARP2 mRNAs at a time of high cell cycle activity as indicated by transcriptome information. Marked changes in the intracellular partitioning of GSH between the cytoplasm and nucleus were observed. Extension of the exponential growth phase by dilution or changing the media led to increases in the glutathione and nicotinamide adenine dinucleotide, oxidized form (NAD)-plus-nicotinamide adenine dinucleotide, reduced form (NADH) pools and to higher NAD/NADH ratios but the nicotinamide adenine dinucleotide phosphate, oxidized form (NADP)-plus-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) pool sizes, and NAPD/NADPH ratios were much less affected. The ascorbate, glutathione, and pyridine nucleotide pools and PARP activity decreased before the exponential growth phase ended. We conclude that there are marked changes in intracellular redox state during the growth cycle but that redox homeostasis is

Natural Polymorphisms Conferring Resistance to HCV Protease and Polymerase Inhibitors in Treatment-Naïve HIV/HCV Co-Infected Patients in China.

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Kali Zhou

Full Text Available The advent of direct-acting agents (DAAs has improved treatment of HCV in HIV co-infection, but may be limited by primary drug resistance. This study reports the prevalence of natural polymorphisms conferring resistance to NS3/4A protease inhibitors and NS5B polymerase inhibitors in treatment-naïve HIV/HCV co-infected individuals in China.Population based NS3/4A sequencing was completed for 778 treatment-naïve HIV/HCV co-infected patients from twelve provinces. NS3 sequences were amplified by nested PCR using in-house primers for genotypes 1-6. NS5B sequencing was completed for genotyping in 350 sequences. Resistance-associated variants (RAVs were identified in positions associated with HCV resistance.Overall, 72.8% (566/778 of all HCV sequences had at least one RAV associated with HCV NS3/4A protease inhibitor resistance. Variants were found in 3.6% (7/193 of genotype 1, 100% (23/23 of genotype 2, 100% (237/237 of genotype 3 and 92% (299/325 of genotype 6 sequences. The Q80K variant was present in 98.4% of genotype 6a sequences. High-level RAVs were rare, occurring in only 0.8% of patients. 93% (64/69 patients with genotype 1b also carried the C316N variant associated with NS5B low-level resistance.The low frequency of high-level RAVs associated with primary HCV DAA resistance among all genotypes in HIV/HCV co-infected patients is encouraging. Further phenotypic studies and clinical research are needed.

A uv-sensitive Chinese hamster lung fibroblast cell line (V79/UC) with a possible defect in DNA polymerase activity is deficient in DNA repair

International Nuclear Information System (INIS)

Creissen, D.M.; Hill, C.K.

1991-01-01

Studies of repair enzyme activities in a uv-sensitive cell line (V79/UC) derived from Chinese hamster V79 cells have revealed levels of total DNA polymerase that are about 50% of the levels in the parental cell line. There are a number of DNA polymerase inhibitors available which allow us to distinguish between the major forms of DNA polymerase (alpha, beta, gamma, and delta) identified in mammalian cells. Enzyme assays with these inhibitors indicate that the aphidicolin-sensitive DNA polymerase is defective in the V79/UC cell line. This could be either polymerase alpha or delta, or both. The V79/UC cells do not express resistance to aphidicolin in standard toxicity studies. However, when aphidicolin is added postirradiation in survival assays designed to measure the extent of inhibitable repair, V79/UC cells do not respond with the further decrease in survival seen in the parental line. Further evidence of a polymerase-dependent repair defect is evident from alkaline elution data. In this case the V79/UC cells show the appearance of single-strand breaks following uv irradiation in the absence of any added inhibitor. Cells of the V79/M12G parental line, on the other hand, show the appearance of single-strand breaks only when aphidicolin is present

Generation and comprehensive analysis of an influenza virus polymerase cellular interaction network.

Science.gov (United States)

Tafforeau, Lionel; Chantier, Thibault; Pradezynski, Fabrine; Pellet, Johann; Mangeot, Philippe E; Vidalain, Pierre-Olivier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent

2011-12-01

The influenza virus transcribes and replicates its genome inside the nucleus of infected cells. Both activities are performed by the viral RNA-dependent RNA polymerase that is composed of the three subunits PA, PB1, and PB2, and recent studies have shown that it requires host cell factors to transcribe and replicate the viral genome. To identify these cellular partners, we generated a comprehensive physical interaction map between each polymerase subunit and the host cellular proteome. A total of 109 human interactors were identified by yeast two-hybrid screens, whereas 90 were retrieved by literature mining. We built the FluPol interactome network composed of the influenza virus polymerase (PA, PB1, and PB2) and the nucleoprotein NP and 234 human proteins that are connected through 279 viral-cellular protein interactions. Analysis of this interactome map revealed enriched cellular functions associated with the influenza virus polymerase, including host factors involved in RNA polymerase II-dependent transcription and mRNA processing. We confirmed that eight influenza virus polymerase-interacting proteins are required for virus replication and transcriptional activity of the viral polymerase. These are involved in cellular transcription (C14orf166, COPS5, MNAT1, NMI, and POLR2A), translation (EIF3S6IP), nuclear transport (NUP54), and DNA repair (FANCG). Conversely, we identified PRKRA, which acts as an inhibitor of the viral polymerase transcriptional activity and thus is required for the cellular antiviral response.

Mangiferin, a novel nuclear factor kappa B-inducing kinase inhibitor, suppresses metastasis and tumor growth in a mouse metastatic melanoma model

Energy Technology Data Exchange (ETDEWEB)

Takeda, Tomoya; Tsubaki, Masanobu; Sakamoto, Kotaro; Ichimura, Eri; Enomoto, Aya; Suzuki, Yuri [Division of Pharmacotherapy, Kinki University School of Pharmacy, Kowakae, Higashi-, Osaka (Japan); Itoh, Tatsuki [Department of Food Science and Nutrition, Kinki University School of Agriculture, Nara, Nara (Japan); Imano, Motohiro [Department of Surgery, Kinki University School of Medicine, Osakasayama, Osaka (Japan); Tanabe, Genzoh; Muraoka, Osamu [Laboratory of Pharmaceutical Organic Chemistry, School of Pharmacy, Kinki University, Kowakae, Higashi-, Osaka (Japan); Matsuda, Hideaki [Department of Natural Drugs Resources, Kinki University School of Pharmacy, Kowakae, Higashi-, Osaka (Japan); Satou, Takao [Department of Pathology, Kinki University School of Medicine, Osakasayama, Osaka (Japan); Nishida, Shozo, E-mail: nishida@phar.kindai.ac.jp [Division of Pharmacotherapy, Kinki University School of Pharmacy, Kowakae, Higashi-, Osaka (Japan)

2016-09-01

Advanced metastatic melanoma, one of the most aggressive malignancies, is currently without reliable therapy. Therefore, new therapies are urgently needed. Mangiferin is a naturally occurring glucosylxanthone and exerts many beneficial biological activities. However, the effect of mangiferin on metastasis and tumor growth of metastatic melanoma remains unclear. In this study, we evaluated the effect of mangiferin on metastasis and tumor growth in a mouse metastatic melanoma model. We found that mangiferin inhibited spontaneous metastasis and tumor growth. Furthermore, mangiferin suppressed the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated NF-κB-inducing kinase (NIK), inhibitor of kappa B kinase (IKK), and inhibitor of kappa B (IκB) and increases the expression of IκB protein in vivo. In addition, we found that mangiferin inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs) in vivo. Mangiferin treatment also increased the expression of cleaved caspase-3, cleaved Poly ADP ribose polymerase-1 (PARP-1), p53 upregulated modulator of apoptosis (PUMA), p53, and phosphorylated p53 proteins, and decreased the expression of Survivin and Bcl-associated X (Bcl-xL) proteins in vivo. These results indicate that mangiferin selectivity suppresses the NF-κB pathway via inhibition of NIK activation, thereby inhibiting metastasis and tumor growth. Importantly, the number of reported NIK selective inhibitors is limited. Taken together, our data suggest that mangiferin may be a potential therapeutic agent with a new mechanism of targeting NIK for the treatment of metastatic melanoma. - Highlights: • Mangiferin prolongs survival in mice by inhibiting metastasis and tumor growth • Mangiferin selectivity suppresses the NF-κB pathway via inhibition of NIK activation • Mangiferin regulates the expression of MMPs, VLAs, and apoptosis regulatory proteins.

Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

Energy Technology Data Exchange (ETDEWEB)

Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Tajima, Shigeru [Department of Virology I, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640 (Japan); Hikono, Hirokazu; Saito, Takehiko [Influenza and Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856 (Japan); Aida, Yoko, E-mail: aida@riken.jp [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

2014-07-18

Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified a novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC{sub 50} values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets.

Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

International Nuclear Information System (INIS)

Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn; Tajima, Shigeru; Hikono, Hirokazu; Saito, Takehiko; Aida, Yoko

2014-01-01

Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified a novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC 50 values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets

Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans-Expression and characterization.

Directory of Open Access Journals (Sweden)

Marcin Olszewski

Full Text Available DNA polymerases are present in all organisms and are important enzymes that synthesise DNA molecules. They are used in various fields of science, predominantly as essential components for in vitro DNA syntheses, known as PCR. Modern diagnostics, molecular biology and genetic engineering need DNA polymerases which demonstrate improved performance. This study was aimed at obtaining a new NeqSSB-TaqS fusion DNA polymerase from the Taq DNA Stoffel domain and a single-stranded DNA binding-like protein of Nanoarchaeum equitans in order to significantly improve the properties of DNA polymerase. The DNA coding sequence of Taq Stoffel DNA polymerase and the nonspecific DNA-binding protein of Nanoarchaeum equitans (NeqSSB-like protein were fused. A novel recombinant gene was obtained which was cloned into the pET-30 Ek/LIC vector and introduced into E. coli for expression. The recombinant enzyme was purified and its enzymatic properties including DNA polymerase activity, PCR amplification rate, thermostability, processivity and resistance to inhibitors, were tested. The yield of the target protein reached approximately 18 mg/l after 24 h of the IPTG induction. The specific activity of the polymerase was 2200 U/mg. The recombinant NeqSSB-TaqS exhibited a much higher extension rate (1000 bp template in 20 s, processivity (19 nt, thermostability (half-life 35 min at 95°C and higher tolerance to PCR inhibitors (0.3-1.25% of whole blood, 0.84-13.5 μg of lactoferrin and 4.7-150 ng of heparin than Taq Stoffel DNA polymerase. Furthermore, our studies show that NeqSSB-TaqS DNA polymerase has a high level of flexibility in relation to Mg2+ ions (from 1 to 5 mM and KCl or (NH42SO4 salts (more than 60 mM and 40 mM, respectively. Using NeqSSB-TaqS DNA polymerase instead of the Taq DNA polymerase could be a better choice in many PCR applications.

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

Science.gov (United States)

Lobley, Carina M C; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E; Nettleship, Joanne E; Brandao-Neto, Jose; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

2012-12-01

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

CRISPR/Cas9-Mediated Correction of the FANCD1 Gene in Primary Patient Cells

Directory of Open Access Journals (Sweden)

Karolina Skvarova Kramarzova

2017-06-01

Full Text Available Fanconi anemia (FA is an inherited condition characterized by impaired DNA repair, physical anomalies, bone marrow failure, and increased incidence of malignancy. Gene editing holds great potential to precisely correct the underlying genetic cause such that gene expression remains under the endogenous control mechanisms. This has been accomplished to date only in transformed cells or their reprogrammed induced pluripotent stem cell counterparts; however, it has not yet been reported in primary patient cells. Here we show the ability to correct a mutation in Fanconi anemia D1 (FANCD1 primary patient fibroblasts. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 system was employed to target and correct a FANCD1 gene deletion. Homologous recombination using an oligonucleotide donor was achieved and a pure population of modified cells was obtained by using inhibitors of poly adenosine diphosphate-ribose polymerase (poly ADP-ribose polymerase. FANCD1 function was restored and we did not observe any promiscuous cutting of the CRISPR/Cas9 at off target sites. This consideration is crucial in the context of the pre-malignant FA phenotype. Altogether we show the ability to correct a patient mutation in primary FANCD1 cells in a precise manner. These proof of principle studies support expanded application of gene editing for FA.

An orally available, small-molecule polymerase inhibitor shows efficacy against a lethal morbillivirus infection in a large animal model.

Science.gov (United States)

Krumm, Stefanie A; Yan, Dan; Hovingh, Elise S; Evers, Taylor J; Enkirch, Theresa; Reddy, G Prabhakar; Sun, Aiming; Saindane, Manohar T; Arrendale, Richard F; Painter, George; Liotta, Dennis C; Natchus, Michael G; von Messling, Veronika; Plemper, Richard K

2014-04-16

Measles virus is a highly infectious morbillivirus responsible for major morbidity and mortality in unvaccinated humans. The related, zoonotic canine distemper virus (CDV) induces morbillivirus disease in ferrets with 100% lethality. We report an orally available, shelf-stable pan-morbillivirus inhibitor that targets the viral RNA polymerase. Prophylactic oral treatment of ferrets infected intranasally with a lethal CDV dose reduced viremia and prolonged survival. Ferrets infected with the same dose of virus that received post-infection treatment at the onset of viremia showed low-grade viral loads, remained asymptomatic, and recovered from infection, whereas control animals succumbed to the disease. Animals that recovered also mounted a robust immune response and were protected against rechallenge with a lethal CDV dose. Drug-resistant viral recombinants were generated and found to be attenuated and transmission-impaired compared to the genetic parent virus. These findings may pioneer a path toward an effective morbillivirus therapy that could aid measles eradication by synergizing with vaccination to close gaps in herd immunity due to vaccine refusal.

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

International Nuclear Information System (INIS)

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

2009-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2009-08-14

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

The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism

International Nuclear Information System (INIS)

Lakatos, Petra; Hegedűs, Csaba; Salazar Ayestarán, Nerea; Juarranz, Ángeles; Kövér, Katalin E.; Szabó, Éva; Virág, László

2016-01-01

Highlights: • PARP-1 is not a key regulator of photochemotherapy. • The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism. • Photosensitization by PJ-34 is associated with increased ROS production and DNA damage. • Cells sensitized by PJ-34 undergo caspase-mediated apoptosis. - Abstract: A combination of a photosensitizer with light of matching wavelength is a common treatment modality in various diseases including psoriasis, atopic dermatitis and tumors. DNA damage and production of reactive oxygen intermediates may impact pathological cellular functions and viability. Here we set out to investigate the role of the nuclear DNA nick sensor enzyme poly(ADP-ribose) polymerase 1 in photochemical treatment (PCT)-induced tumor cell killing. We found that silencing PARP-1 or inhibition of its enzymatic activity with Veliparib had no significant effect on the viability of A431 cells exposed to 8-methoxypsoralen (8-MOP) and UVA (2.5 J/cm"2) indicating that PARP-1 is not likely to be a key player in either cell survival or cell death of PCT-exposed cells. Interestingly, however, another commonly used PARP inhibitor PJ-34 proved to be a photosensitizer with potency equal to 8-MOP. Irradiation of PJ-34 with UVA caused changes both in the UV absorption and in the 1H NMR spectra of the compound with the latter suggesting UVA-induced formation of tautomeric forms of the compound. Characterization of the photosensitizing effect revealed that PJ–34 + UVA triggers overproduction of reactive oxygen species, induces DNA damage, activation of caspase 3 and caspase 8 and internucleosomal DNA fragmentation. Cell death in this model could not be prevented by antioxidants (ascorbic acid, trolox, glutathione, gallotannin or cell permeable superoxide dismutase or catalase) but could be suppressed by inhibitors of caspase-3 and −8. In conclusion, PJ-34 is a photosensitizer and PJ–34 + UVA causes DNA damage and caspase

The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism

Energy Technology Data Exchange (ETDEWEB)

Lakatos, Petra; Hegedűs, Csaba [Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); Salazar Ayestarán, Nerea; Juarranz, Ángeles [Department of Biology, Faculty of Sciences, Universidad Autónoma of Madrid, 28049-Madrid (Spain); Kövér, Katalin E. [Department of Inorganic and Analytical Chemistry, Faculty of Sciences, University of Debrecen, Debrecen (Hungary); Szabó, Éva [Department of Dermatology, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); Virág, László, E-mail: lvirag@med.unideb.hu [Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Debrecen (Hungary); MTA-DE Cell Biology and Signaling Research Group, Debrecen (Hungary)

2016-08-15

Highlights: • PARP-1 is not a key regulator of photochemotherapy. • The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism. • Photosensitization by PJ-34 is associated with increased ROS production and DNA damage. • Cells sensitized by PJ-34 undergo caspase-mediated apoptosis. - Abstract: A combination of a photosensitizer with light of matching wavelength is a common treatment modality in various diseases including psoriasis, atopic dermatitis and tumors. DNA damage and production of reactive oxygen intermediates may impact pathological cellular functions and viability. Here we set out to investigate the role of the nuclear DNA nick sensor enzyme poly(ADP-ribose) polymerase 1 in photochemical treatment (PCT)-induced tumor cell killing. We found that silencing PARP-1 or inhibition of its enzymatic activity with Veliparib had no significant effect on the viability of A431 cells exposed to 8-methoxypsoralen (8-MOP) and UVA (2.5 J/cm{sup 2}) indicating that PARP-1 is not likely to be a key player in either cell survival or cell death of PCT-exposed cells. Interestingly, however, another commonly used PARP inhibitor PJ-34 proved to be a photosensitizer with potency equal to 8-MOP. Irradiation of PJ-34 with UVA caused changes both in the UV absorption and in the 1H NMR spectra of the compound with the latter suggesting UVA-induced formation of tautomeric forms of the compound. Characterization of the photosensitizing effect revealed that PJ–34 + UVA triggers overproduction of reactive oxygen species, induces DNA damage, activation of caspase 3 and caspase 8 and internucleosomal DNA fragmentation. Cell death in this model could not be prevented by antioxidants (ascorbic acid, trolox, glutathione, gallotannin or cell permeable superoxide dismutase or catalase) but could be suppressed by inhibitors of caspase-3 and −8. In conclusion, PJ-34 is a photosensitizer and PJ–34 + UVA causes DNA damage and caspase

Mw Spectroscopy Coupled with Ultrafast UV Laser Vaporization: {RIBOSE} Found in the Gas Phase

Science.gov (United States)

Cocinero, Emilio J.; Ecija, Patricia; Basterretxea, Francisco J.; Fernandez, Jose A.; Castano, Fernando; Lesarri, Alberto; Grabow, Jens-Uwe

2012-06-01

Sugars are aldoses or ketoses with multiple hydroxy groups which have been elusive to spectroscopic studies. Here we report a rotational study of the aldopentose ribose. According to any standard textbook aldopentoses can exhibit either linear forms, cyclic five-membered (furanose) structures or six-membered (pyranose) rings, occurring either as α- or β- anomers depending on the orientation of the hydroxy group at C-1 (anomeric carbon). β-Furanose is predominant in ribonucleosides, RNA, ATP and other biochemically relevant derivatives, but is β-furanose the native form also of free ribose? Recent condensed-phase X-ray and older NMR studies delivered conflicting results. In order to solve this question we conducted a microwave study on D-ribose that, owing to ultrafast UV laser vaporization, has become the first C-5 sugar observed with rotational resolution. The spectrum revealed six conformations of free ribose, preferentially adopting β-pyranose chairs as well as higher-energy α-pyranose forms. The method also allowed for unambiguous distinction between different orientations of the hydroxy groups, which stabilize the structures by cooperative hydrogen-bond networks. No evidence was observed of the α-/β-furanoses or linear forms found in the biochemical derivatives. i) D. Šišak, L. B. McCusker, G. Zandomeneghi, B. H. Meier, D. Bläser, R. Boese, W. B. Schweizer, R. Gylmour and J. D. Dunitz Angew. Chem. Int. Ed. 49, 4503, 2010. ii) W. Saenger Angew. Chem. Int. Ed. 49, 6487, 2010. i) M. Rudrum, and D. F. Shaw, J. Chem. Soc. 52, 1965. ii) R. U. Lemieux and J. D. Stevens Can. J. Chem. 44, 249, 1966. iii) E. Breitmaier and U. Hollstein Org. Magn. Reson. 8, 573, 1976. E. J. Cocinero, A. Lesarri, P. Écija, F. J. Basterretxea, J. U. Grabow, J. A. Fernández and F. Castaño Angew. Chem. Int. Ed. in press: DOI: 10.1002/anie.201107973, 2012.

Reaction of Br/sub 3/. /sup 2 -/ with 2-deoxy-D-ribose. A preferred attack at C-1

Energy Technology Data Exchange (ETDEWEB)

Parsons, B J; Schulte-Frohlinde, D; von Sonntag, C [Max-Planck-Institut fuer Kohlenforschung, Muelheim an der Ruhr (Germany, F.R.). Inst. fuer Strahlenchemie

1978-06-01

In the photolysis of 5-bromouracil containing DNA Br atoms are expected intermediates. In order to evaluate the possible site of attack of the Br atom at the sugar moiety of DNA the reaction of 2-deoxy-D-Ribose with the Br atom (complexed with two bromide ions) was investigated. Hydroxyl radicals generated by the radiolysis of N/sub 2/O saturated aqueous solutions were converted into Br/sub 3/./sup 2 -/-radicals by 1 M bromide ions. Br/sub 3/./sup 2 -/-reacts with 2-deoxy-D-ribose (k = 3.7 x 10/sup 4/M/sup -1/s/sup -1/, pulse radiolysis). The major product is 2-deoxy-D-erythro-pentonic acid (G = 2.4, ..gamma..-radiolysis). It is formed by hydrogen abstraction from C-1 and oxidation of this radical by other radicals. An alternative route via the radical at C-2 is neglible. It follows that Br/sub 3/./sup 2 -/ reacts preferentially at C-1 of 2-deoxy-D-ribose.

Characteristic antioxidant activity and comprehensive flavor compound profile of scallop (Chlamys farreri) mantle hydrolysates-ribose Maillard reaction products.

Science.gov (United States)

Han, Jia-Run; Yan, Jia-Nan; Sun, Shi-Guang; Tang, Yue; Shang, Wen-Hui; Li, Ao-Ting; Guo, Xiao-Kun; Du, Yi-Nan; Wu, Hai-Tao; Zhu, Bei-Wei; Xiong, Youling L

2018-09-30

The objective of the present study was to improve the utilization of scallop (Chlamys farreri) byproducts by using Maillard reaction. Scallop mantle hydrolysates (SMHs) were prepared using neutrase then reacted with ribose. Thirty-four peptides were identified from SMHs by UPLC-Q-TOF-MS, and the abundance of Asp and Lys suggested the strong Maillard reactivity. The formation of Schiff's base as well as modification of amide I, II and III bands in Maillard reaction products (MRPs) was confirmed by ultraviolet-visible, fluorescence, and Fourier transform infrared spectroscopy. Thirty volatile compounds were produced by the reaction of SMHs with ribose. Moreover, MRPs with enhanced radical scavenging and anti-linoleic acid peroxidation activities over SMHs promoted the survival and reduced the DNA damage of HepG2 cells treated with hydrogen peroxide. These results suggest that SMHs-ribose MRPs can be potentially used as food antioxidant for suppressing of lipid oxidation or protecting of cell from oxidative damage. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Novel Mechanism of Sugar Selection Utilized by a Human X-family DNA Polymerase†

OpenAIRE

Brown, Jessica A.; Fiala, Kevin A.; Fowler, Jason D.; Sherrer, Shanen M.; Newmister, Sean A.; Dyum, Wade W.; Suo, Zucai

2009-01-01

During DNA synthesis, most DNA polymerases and reverse transcriptases select against ribonucleotides via a steric clash between the ribose 2′-hydroxyl group and the bulky side chain of an active site residue. Here, we demonstrated that human DNA polymerase λ used a novel sugar selection mechanism to discriminate against ribonucleotides, whereby the ribose 2′-hydroxyl group was excluded mostly by a backbone segment and slightly by the side chain of Y505. Such a steric clash was further demonst...

The PARP inhibitor PJ-34 sensitizes cells to UVA-induced phototoxicity by a PARP independent mechanism.

Science.gov (United States)

Lakatos, Petra; Hegedűs, Csaba; Salazar Ayestarán, Nerea; Juarranz, Ángeles; Kövér, Katalin E; Szabó, Éva; Virág, László

2016-08-01

A combination of a photosensitizer with light of matching wavelength is a common treatment modality in various diseases including psoriasis, atopic dermatitis and tumors. DNA damage and production of reactive oxygen intermediates may impact pathological cellular functions and viability. Here we set out to investigate the role of the nuclear DNA nick sensor enzyme poly(ADP-ribose) polymerase 1 in photochemical treatment (PCT)-induced tumor cell killing. We found that silencing PARP-1 or inhibition of its enzymatic activity with Veliparib had no significant effect on the viability of A431 cells exposed to 8-methoxypsoralen (8-MOP) and UVA (2.5J/cm(2)) indicating that PARP-1 is not likely to be a key player in either cell survival or cell death of PCT-exposed cells. Interestingly, however, another commonly used PARP inhibitor PJ-34 proved to be a photosensitizer with potency equal to 8-MOP. Irradiation of PJ-34 with UVA caused changes both in the UV absorption and in the 1H NMR spectra of the compound with the latter suggesting UVA-induced formation of tautomeric forms of the compound. Characterization of the photosensitizing effect revealed that PJ-34+UVA triggers overproduction of reactive oxygen species, induces DNA damage, activation of caspase 3 and caspase 8 and internucleosomal DNA fragmentation. Cell death in this model could not be prevented by antioxidants (ascorbic acid, trolox, glutathione, gallotannin or cell permeable superoxide dismutase or catalase) but could be suppressed by inhibitors of caspase-3 and -8. In conclusion, PJ-34 is a photosensitizer and PJ-34+UVA causes DNA damage and caspase-mediated cell death independently of PARP-1 inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

Microglial activation induced by brain trauma is suppressed by post-injury treatment with a PARP inhibitor

Directory of Open Access Journals (Sweden)

d'Avila Joana C

2012-02-01

Full Text Available Abstract Background Traumatic brain injury (TBI induces activation of microglia. Activated microglia can in turn increase secondary injury and impair recovery. This innate immune response requires hours to days to become fully manifest, thus providing a clinically relevant window of opportunity for therapeutic intervention. Microglial activation is regulated in part by poly(ADP-ribose polymerase-1 (PARP-1. Inhibition of PARP-1 activity suppresses NF-kB-dependent gene transcription and thereby blocks several aspects of microglial activation. Here we evaluated the efficacy of a PARP inhibitor, INO-1001, in suppressing microglial activation after cortical impact in the rat. Methods Rats were subjected to controlled cortical impact and subsequently treated with 10 mg/kg of INO-1001 (or vehicle alone beginning 20 - 24 hours after the TBI. Brains were harvested at several time points for histological evaluation of inflammation and neuronal survival, using markers for microglial activation (morphology and CD11b expression, astrocyte activation (GFAP, and neuronal survival (NeuN. Rats were also evaluated at 8 weeks after TBI using measures of forelimb dexterity: the sticky tape test, cylinder test, and vermicelli test. Results Peak microglial and astrocyte activation was observed 5 to 7 days after this injury. INO-1001 significantly reduced microglial activation in the peri-lesion cortex and ipsilateral hippocampus. No rebound inflammation was observed in rats that were treated with INO-1001 or vehicle for 12 days followed by 4 days without drug. The reduced inflammation was associated with increased neuronal survival in the peri-lesion cortex and improved performance on tests of forelimb dexterity conducted 8 weeks after TBI. Conclusions Treatment with a PARP inhibitor for 12 days after TBI, with the first dose given as long as 20 hours after injury, can reduce inflammation and improve histological and functional outcomes.

Effects of PARP-1 inhibitors AG-014699 and AZD2281 on proliferation and apoptosis of human hepatoma cell line HepG2

Directory of Open Access Journals (Sweden)

DU Senrong

2015-06-01

Full Text Available ObjectiveTo observe the inhibitory and pro-apoptotic effects of two poly(ADP-ribose polymerase (PARP-1 inhibitors, AG-014699 and AZD2281, on human hepatoma HepG2 cells and preliminarily explore the mechanism by which AG-014699 induces HepG2 cell apoptosis, and to provide a new therapeutic target for hepatoma. MethodsThe effects of different concentrations of AG-014699 and AZD2281 on HepG2 cell proliferation were determined by MTT assay. The cell apoptosis rate was measured by flow cytometry. The expression levels of caspase-3 and caspase-8 were measured by Western Blot. Inter-group comparison was made by t test. ResultsBoth AG-014699 and AZD2281 suppressed HepG2 cell proliferation in a time- and dose-dependent manner. However, the sensitivity of HepG2 cells to the two PARP-1 inhibitors was different. The half-maximal inhibitory concentrations of AG-014699 and AZD2281 at 48 h determined by MTT assay were about 20 μmol/L and 400 μmol/L, respectively. Flow cytometry and Western blot were not used to evaluate the apoptosis of HepG2 cells exposed to AZD2281 to which these cells were not sensitive. HepG2 cell apoptosis could be induced by 10, 30, and 50 μmol/L AG-014699, and the highest apoptosis rate at 48 h was significantly higher than that of the control group (3100%±2.13% vs 09%±0013%, P＜0.01. Compared with those in the control group, the protein levels of caspase-3 and caspase-8 in HepG2 cells after 48-h exposure to 30, and 50 μmol/L AG-014699 increased. ConclusionThe two PARP-1 inhibitors AG-014699 and AZD2281 can inhibit the proliferation of HepG2 cells, which showed different sensitivities to the two inhibitors. AG-014699 can induce HepG2 cell apoptosis by up-regulating the protein expression of caspase-3 and caspase-8.

Structural Implications for Selective Targeting of PARPs.

Science.gov (United States)

Steffen, Jamin D; Brody, Jonathan R; Armen, Roger S; Pascal, John M

2013-12-20

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD(+) as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed "synthetic lethality." In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients.

Concurrent targeting of nitrosative stress-PARP pathway corrects functional, behavioral and biochemical deficits in experimental diabetic neuropathy

Energy Technology Data Exchange (ETDEWEB)

Negi, Geeta; Kumar, Ashutosh [Molecular Neuropharmacology Laboratory, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar, Punjab 160062 (India); Sharma, Shyam S., E-mail: sssharma@niper.ac.in [Molecular Neuropharmacology Laboratory, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar, Punjab 160062 (India)

2010-01-01

Peroxynitrite mediated nitrosative stress, an indisputable initiator of DNA damage and overactivation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated after sensing DNA damage, are two crucial pathogenetic mechanisms in diabetic neuropathy. The intent of the present study was to investigate the effect of combination of a peroxynitrite decomposition catalyst (PDC), FeTMPyP and a PARP inhibitor, 4-ANI against diabetic peripheral neuropathy. The end points of evaluation of the study included motor nerve conduction velocity (MNCV) and nerve blood flow (NBF) for evaluating nerve functions; thermal hyperalgesia and mechanical allodynia for assessing nociceptive alterations, malondialdehyde and peroxynitrite levels to detect oxidative stress-nitrosative stress; NAD concentration in sciatic nerve to assess overactivation of PARP. Additionally immunohistochemical studies for nitrotyrosine and Poly(ADP-ribose) (PAR) was also performed. Treatment with the combination of FeTMPyP and 4-ANI led to significant improvement in nerve functions and pain parameters and also attenuated the oxidative-nitrosative stress markers. Further, the combination also reduced the overactivation of PARP as evident from increased NAD levels and decreased PAR immunopositivity in sciatic nerve microsections. Thus, it can be concluded that treatment with the combination of a PDC and PARP inhibitor attenuates alteration in peripheral nerves in diabetic neuropathy (DN).

Kinetics of Glycoxidation of Bovine Serum Albumin by Glucose, Fructose and Ribose and Its Prevention by Food Components

Directory of Open Access Journals (Sweden)

Izabela Sadowska-Bartosz

2014-11-01

Full Text Available The aim of this study was to compare the kinetics of the glycoxidation of bovine serum albumin (BSA as a model protein by three sugars: glucose, fructose and ribose, using fluorometric measurements of the content of advanced glycation end products (AGEs, protein-bound fructosamine, dityrosine, N'-formylkynurenine, kynurenine, tryptophan, the content of advanced oxidation protein products (AOPP, protein carbonyl groups, as well as thiol groups. Moreover, the levels of glycoalbumin and AGEs were determined by using an enzyme-linked immunosorbent assay. Based on the kinetic results, the optimal incubation time for studies of the modification of the glycoxidation rate by additives was chosen, and the effects of 25 compounds of natural origin on the glycoxidation of BSA induced by various sugars were examined. The same compounds were found to have different effects on glycoxidation induced by various sugars, which suggests caution in extrapolation from experiments based on one sugar to other sugars. From among the compounds tested, the most effective inhibitors of glycoxidation were: polyphenols, pyridoxine and 1-cyano-4-hydroxycinnamic acid.

Novel histone deacetylase 8-selective inhibitor 1,3,4-oxadiazole-alanine hybrid induces apoptosis in breast cancer cells.

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Pidugu, Vijaya Rao; Yarla, Nagendra Sastry; Bishayee, Anupam; Kalle, Arunasree M; Satya, Alapati Krishna

2017-11-01

Identification of isoform-specific histone deacetylase inhibitors (HDACi) is a significant advantage to overcome the adverse side effects of pan-HDACi for the treatment of various diseases, including cancer. We have designed, and synthesized novel 1,3,4 oxadiazole with glycine/alanine hybrids as HDAC8-specific inhibitors and preliminary evaluation has indicated that 1,3,4 oxadiazole with alanine hybrid [(R)-2-amino-N-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)propanamide (10b)] to be a potent HDAC8 inhibitor. In the present study, the in vitro efficacy of the molecule in inhibiting the cancer cell proliferation and the underlying molecular mechanism was studied. 10b inhibited the growth of MDA-MB-231 and MCF7 breast cancer cells, with a lower IC 50 of 230 and 1000 nM, respectively, compared to K562, COLO-205 and HepG2 cells and was not cytotoxic to normal breast epithelial cells, MCF10A. 10b was specific to HDAC8 and did not affect the expression of other class I HDACs. Further, a dose-dependent increase in H3K9 acetylation levels demonstrated the HDAC-inhibitory activity of 10b in MDA-MB-231 cells. Flow cytometric analysis indicated a dose-dependent increase and decrease in the percent apoptotic cells and mitochondrial membrane potential, respectively, when treated with 10b. Immunoblot analysis showed a modulation of Bax/Bcl2 ratio with a decrease in Bcl2 expression and no change in Bax expression. 10b treatment resulted in induction of p21 and inhibition of CDK1 proteins along with cytochrome c release from mitochondria, activation of caspases-3 and -9 and cleavage of poly ADP-ribose polymerase leading to apoptotic death of MDA-MB-231 and MCF7 cells. In conclusion, our results clearly demonstrated the efficacy of 10b as an anticancer agent against breast cancer.

Generation and Comprehensive Analysis of an Influenza Virus Polymerase Cellular Interaction Network▿†§

Science.gov (United States)

Tafforeau, Lionel; Chantier, Thibault; Pradezynski, Fabrine; Pellet, Johann; Mangeot, Philippe E.; Vidalain, Pierre-Olivier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent

2011-01-01

The influenza virus transcribes and replicates its genome inside the nucleus of infected cells. Both activities are performed by the viral RNA-dependent RNA polymerase that is composed of the three subunits PA, PB1, and PB2, and recent studies have shown that it requires host cell factors to transcribe and replicate the viral genome. To identify these cellular partners, we generated a comprehensive physical interaction map between each polymerase subunit and the host cellular proteome. A total of 109 human interactors were identified by yeast two-hybrid screens, whereas 90 were retrieved by literature mining. We built the FluPol interactome network composed of the influenza virus polymerase (PA, PB1, and PB2) and the nucleoprotein NP and 234 human proteins that are connected through 279 viral-cellular protein interactions. Analysis of this interactome map revealed enriched cellular functions associated with the influenza virus polymerase, including host factors involved in RNA polymerase II-dependent transcription and mRNA processing. We confirmed that eight influenza virus polymerase-interacting proteins are required for virus replication and transcriptional activity of the viral polymerase. These are involved in cellular transcription (C14orf166, COPS5, MNAT1, NMI, and POLR2A), translation (EIF3S6IP), nuclear transport (NUP54), and DNA repair (FANCG). Conversely, we identified PRKRA, which acts as an inhibitor of the viral polymerase transcriptional activity and thus is required for the cellular antiviral response. PMID:21994455

Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.

Science.gov (United States)

Appleby, Todd C; Perry, Jason K; Murakami, Eisuke; Barauskas, Ona; Feng, Joy; Cho, Aesop; Fox, David; Wetmore, Diana R; McGrath, Mary E; Ray, Adrian S; Sofia, Michael J; Swaminathan, S; Edwards, Thomas E

2015-02-13

Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site. Copyright © 2015, American Association for the Advancement of Science.

Promoter analysis of the Chilo iridescent virus DNA polymerase and major capsid protein genes

NARCIS (Netherlands)

Nalcacioglu, R.; Marks, H.; Vlak, J.M.; Demirbag, Z.; Oers, van M.M.

2003-01-01

The DNA polymerase (DNApol) and major capsid protein (MCP) genes were used as models to study promoter activity in Chilo iridescent virus (CIV). Infection of Bombyx mori SPC-BM-36 cells in the presence of inhibitors of DNA or protein synthesis showed that DNApol, as well as helicase, is an

Postnatal changes of gene expression for tissue inhibitors of metalloproteinase-1 and -2 and cystatins S and C, in rat submandibular gland demonstrated by quantitative reverse transcription-polymerase chain reaction.

Science.gov (United States)

Nishiura, T; Abe, K

1999-01-01

The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.

A serine palmitoyltransferase inhibitor blocks hepatitis C virus replication in human hepatocytes.

Science.gov (United States)

Katsume, Asao; Tokunaga, Yuko; Hirata, Yuichi; Munakata, Tsubasa; Saito, Makoto; Hayashi, Hitohisa; Okamoto, Koichi; Ohmori, Yusuke; Kusanagi, Isamu; Fujiwara, Shinya; Tsukuda, Takuo; Aoki, Yuko; Klumpp, Klaus; Tsukiyama-Kohara, Kyoko; El-Gohary, Ahmed; Sudoh, Masayuki; Kohara, Michinori

2013-10-01

Host cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice. We tested the ability of NA808 to inhibit SPT's enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors. NA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors. The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor.

Science.gov (United States)

Gong, Qingguo; Menon, Lakshmi; Ilina, Tatiana; Miller, Lena G; Ahn, Jinwoo; Parniak, Michael A; Ishima, Rieko

2011-01-01

HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors. © 2010 John Wiley & Sons A/S.

Spirulina maxima extract prevents cell death through BDNF activation against amyloid beta 1-42 (Aβ1-42) induced neurotoxicity in PC12 cells.

Science.gov (United States)

Koh, Eun-Jeong; Kim, Kui-Jin; Choi, Jia; Kang, Do-Hyung; Lee, Boo-Yong

2018-04-23

Spirulina maxima is a blue-green micro alga that contains abundant amounts of proteins (60-70%), vitamins, chlorophyll a, and C-phycocyanin (C-PC). It has been shown to reduce oxidative stress, and prevent diabetes and non-alcoholic fatty liver disease. However, it is unclear whether Spirulina maxima 70% ethanol extract (SM70EE), chlorophyll a, and C-PC prevent Aβ 1-42 -induced neurotoxicity in PC12 cells. The aim of this study was to investigate whether SM70EE, chlorophyll a, and C-PC prevent Aβ 1-42 -induced cell death. SM70EE, chlorophyll a, and C-PC suppressed the Aβ 1-42 -induced increase in poly-ADP ribose polymerase-1 (PARP-1) cleavage and reduced Aβ 1-42 -induced decreases in glutathione and its associated factors. The level of brain-derived neurotrophic factor (BDNF), which plays a critical role in neuronal survival and neuroprotection, was increased by SM70EE, chlorophyll a, and C-PC in Aβ 1-42 -treated cells. SM70EE treatment decreased oxidative stress and cell death in response to Aβ 1-42 treatment, while simultaneously suppressing PARP cleavage and increasing the levels of glutathione (GSH) and its associated factors. Moreover, SM70EE lowered the levels of APP and BACE1, two major factors involved in APP processing, and increased BDNF expression during Aβ 1-42 -induced neurotoxicity in PC12 cells. We suggest that SM70EE prevents cell death caused by Aβ 1-42 -induced neurotoxicity via the activation of BDNF signaling. Copyright © 2018 Elsevier B.V. All rights reserved.

Isorhynchophylline, a Potent Plant Alkaloid, Induces Apoptotic and Anti-Metastatic Effects in Human Hepatocellular Carcinoma Cells through the Modulation of Diverse Cell Signaling Cascades

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Hanwool Lee

2017-05-01

Full Text Available Isorhynchophylline (Rhy is an active pharmacological component of Uncaria rhynchophylla that has been reported previously to exert significant antihypertensive and neuroprotective effects. However, very little is known about its potential anti-cancer activities. This study was carried out to evaluate the anticancer effects of Rhy against various human carcinoma cell lines. We found that Rhy exhibited substantial cytotoxic effect against human hepatocellular carcinoma HepG2 cells when compared with other human carcinoma cell lines including those of lung, pancreas, prostate, head and neck, breast, multiple myeloma, brain and renal cell carcinoma. Rhy induced apoptosis as characterized by accumulation of cells in sub G1 phase; positive Annexin V binding; activation of caspase-8, -9, and -3; and cleavage of PARP (poly-ADP ribose polymerase. This effect of Rhy correlated with the down-regulation of various proteins that mediated cell proliferation, cell survival, metastasis, and angiogenesis. Moreover, cell proliferation, migration, and constitutive CXCR4 (C-X-C chemokine receptor type 4, MMP-9 (Matrix metallopeptidase-9, and MMP-2 expression were inhibited upon Rhy treatment. We further investigated the effect of Rhy on the oncogenic cell signaling cascades through phospho-kinase array profiling assay. Rhy was found to abrogate phospho-p38, ERK, JNK, CREB, c-Jun, Akt, and STAT3 signals, but interestingly enhanced phospho-p53 signal. Overall, our results indicate, for the first time, that Rhy could exert anticancer and anti-metastatic effects through regulation of multiple signaling cascades in hepatocellular carcinoma cells.

Isorhynchophylline, a Potent Plant Alkaloid, Induces Apoptotic and Anti-Metastatic Effects in Human Hepatocellular Carcinoma Cells through the Modulation of Diverse Cell Signaling Cascades

Science.gov (United States)

Lee, Hanwool; Baek, Seung Ho; Lee, Jong Hyun; Kim, Chulwon; Ko, Jeong-Hyeon; Lee, Seok-Geun; Chinnathambi, Arunachalam; Alharbi, Sulaiman Ali; Yang, Woong Mo; Um, Jae-Young; Sethi, Gautam; Ahn, Kwang Seok

2017-01-01

Isorhynchophylline (Rhy) is an active pharmacological component of Uncaria rhynchophylla that has been reported previously to exert significant antihypertensive and neuroprotective effects. However, very little is known about its potential anti-cancer activities. This study was carried out to evaluate the anticancer effects of Rhy against various human carcinoma cell lines. We found that Rhy exhibited substantial cytotoxic effect against human hepatocellular carcinoma HepG2 cells when compared with other human carcinoma cell lines including those of lung, pancreas, prostate, head and neck, breast, multiple myeloma, brain and renal cell carcinoma. Rhy induced apoptosis as characterized by accumulation of cells in sub G1 phase; positive Annexin V binding; activation of caspase-8, -9, and -3; and cleavage of PARP (poly-ADP ribose polymerase). This effect of Rhy correlated with the down-regulation of various proteins that mediated cell proliferation, cell survival, metastasis, and angiogenesis. Moreover, cell proliferation, migration, and constitutive CXCR4 (C-X-C chemokine receptor type 4), MMP-9 (Matrix metallopeptidase-9), and MMP-2 expression were inhibited upon Rhy treatment. We further investigated the effect of Rhy on the oncogenic cell signaling cascades through phospho-kinase array profiling assay. Rhy was found to abrogate phospho-p38, ERK, JNK, CREB, c-Jun, Akt, and STAT3 signals, but interestingly enhanced phospho-p53 signal. Overall, our results indicate, for the first time, that Rhy could exert anticancer and anti-metastatic effects through regulation of multiple signaling cascades in hepatocellular carcinoma cells. PMID:28534824

Hsp90 inhibitors reduce influenza virus replication in cell culture

International Nuclear Information System (INIS)

Chase, Geoffrey; Deng, Tao; Fodor, Ervin; Leung, B.W.; Mayer, Daniel; Schwemmle, Martin; Brownlee, George

2008-01-01

The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses

New and convenient synthesis of 2-deoxy-D-ribose from 2,4-O-ethylidene-D-erythrose

Energy Technology Data Exchange (ETDEWEB)

Hauske, J.R.; Rapoport, H.

1979-01-01

A new synthesis is described of 2-deoxy-D-erythro-pentose (2-deoxy-D-ribose,2-deoxy-D-arabinose (1)), starting from D-glucose. The synthesis proceeds through direct olefination of 2,4-O-ethylidene-D-erythrose (2) by addition of the stabilized ylides generated from dimethylphosphorylmethyl phenyl sulfide (4) and the corresponding sulfoxide 5. These afford the key intermediates, thio-enol ether 7 and ..cap alpha..,..beta..-unsaturated sulfoxide 8, which when subjected to mercuric ion assisted hydrolysis gave high yields of 2-deoxy-D-ribose (1). This facile chain extension of 2 required its existance as a monomer, and conditions effective for obtaining the monomer have been developed. Detailed /sup 1/H and /sup 13/C NMR studies of these compounds are presented.

Inhibition of post-transcriptional RNA processing by CDK inhibitors and its implication in anti-viral therapy.

Directory of Open Access Journals (Sweden)

Jitka Holcakova

Full Text Available Cyclin-dependent kinases (CDKs are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional

The HSP90 inhibitor 17-N-allylamino-17-demethoxy geldanamycin (17-AAG) synergizes with cisplatin and induces apoptosis in cisplatin-resistant esophageal squamous cell carcinoma cell lines via the Akt/XIAP pathway.

Science.gov (United States)

Ui, Takashi; Morishima, Kazue; Saito, Shin; Sakuma, Yuji; Fujii, Hirofumi; Hosoya, Yoshinori; Ishikawa, Shumpei; Aburatani, Hiroyuki; Fukayama, Masashi; Niki, Toshiro; Yasuda, Yoshikazu

2014-02-01

Although cisplatin (CDDP) is a key drug in the treatment of esophageal squamous cell carcinoma (ESCC), acquired chemoresistance remains a major problem. Combination therapy may represent one strategy to overcome this resistance. Heat shock protein 90 (HSP90) is known to be overexpressed in several types of cancer cells, and its inhibition by small molecules, either alone or in combination, has shown promise in the treatment of solid malignancies. In the present study, we evaluated the synergistic effects of combining CDDP with the HSP90 inhibitor 17-N-allylamino-17-demethoxy geldanamycin (17-AAG) on two CDDP-resistant human esophageal squamous cancer cell lines, KYSE30 and KYSE150. The results obtained demonstrated the synergistic inhibitory effects of CDDP and 17-AAG on the growth of KYSE30 and KYSE150 cells. Cell growth and cell number were more effectively reduced by the combined treatment with CDDP and 17-AAG than by the treatment with either CDDP or 17-AAG alone. Western blotting revealed that the combined action of CDDP and 17-AAG cleaved poly (ADP-ribose) polymerase (PARP) and caspase-3, which demonstrated that the reduction in both cell growth and cell number was mediated by apoptosis. Time-course experiments showed that reduction in X-linked inhibitor of apoptosis protein (XIAP) and phosphorylated Akt were concomitant with apoptosis. The results of the present study demonstrate that 17-AAG synergizes with CDDP and induces apoptosis in CDDP-resistant ESCC cell lines, and also that modulation of the Akt/XIAP pathway may underlie this synergistic effect. Combination therapy with CDDP and an HSP90 inhibitor may represent a promising strategy to overcome CDDP resistance in ESCC.

Whole Blood PCR Amplification with Pfu DNA Polymerase and Its Application in Single-Nucleotide Polymorphism Analysis.

Science.gov (United States)

Liu, Er-Ping; Wang, Yan; He, Xiao-Hui; Guan, Jun-Jie; Wang, Jin; Qin, Zheng-Hong; Sun, Wan-Ping

2015-11-01

Point-of-care genetic analysis may require polymerase chain reaction (PCR) to be carried out on whole blood. However, human blood contains natural inhibitors of PCR such as hemoglobin, immunoglobulin G, lactoferrin, and proteases, as well as anticoagulant agents, including EDTA and heparin that can reduce whole blood PCR efficiency. Our purpose was to develop a highly specific, direct whole blood single-nucleotide polymorphism (SNP) analysis method based on allele-specific (AS) PCR that is mediated by Pfu DNA polymerase and phosphorothioate-modified AS primers. At high Mg(2+) concentrations, Pfu DNA polymerase efficiently amplified genomic DNA in a reaction solution containing up to 14% whole blood. Among the three anticoagulants tested, Pfu DNA polymerase showed the highest activity with sodium citrate. Meanwhile, Triton X-100 and betaine inhibited Pfu DNA polymerase activity in whole blood PCR, whereas trehalose had virtually no effect. These findings provided for the development of a low-cost, simple, and fast direct whole blood genotyping method that uses Pfu DNA polymerase combined with phosphorothioate AS primers for CYP2C9*3 and VKORC1(-1639) loci. With its high DNA amplification efficiency and tolerance of various blood conditions, Pfu DNA polymerase can be used in clinical laboratories to analyze SNPs in whole blood samples.

Preferential uptake of ribose by primitive cells might explain why RNA was favored over its analogs

Science.gov (United States)

Pohorille, Andrew; Wei, Chenyu

Permeation of molecules through membranes is a fundamental process in biological systems, which not only involves mass and signal transfers between the interior of a contemporary cell and its environment, but was also of crucial importance in the origin of life. In the absence of complex protein transporters, nutrients and building blocks of biopolymers must have been able to permeate membranes at sufficient rates to support primordial metabolism and cel-lular reproduction. From this perspective one class of solutes that is of special interest are monosaccharides, which serve not only as nutritional molecules but also as building blocks for information molecules. In particular, ribose is a part of the RNA backbone, but RNA analogs containing a number of other sugars have also been shown to form stable duplexes. Why, among these possibilities, ribose (and, subsequently, deoxyribose) was selected for the backbone of information polymers is still poorly understood. It was recently found that ribose permeates membranes an order of magnitude faster than its diastereomers, arabinose and xylose [1]. On this basis it was hypothesized that differences in membrane permeability to aldopentoses provide a mechanism for preferential delivery of ribose to primitive cells for subsequent, selective incorporation into nucleotides and their polymers. However, the origins of these unusually large differences had not been well understood. We addressed this issue in molecular dynamics simulations combined with free energy calculations. It was found that the free energy barrier for transferring ribose from water to the bilayer is lower by 1.5-2 kcal/mol than the barrier for transferring the other two aldopentoses. The calculated [2] and measured [1] permeability coefficients are in an excellent agreement. The sugar structures that permeate the membrane are -pyranoses, with a possible contribution of the -anomer for arabinose. The furanoid form of ribose is not substantially involved in

Replication fork stability confers chemoresistance in BRCA-deficient cells

DEFF Research Database (Denmark)

Chaudhuri, Arnab Ray; Callen, Elsa; Ding, Xia

2016-01-01

/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11......Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3...... nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations...

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

Science.gov (United States)

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

2007-01-01

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

Role of DNA polymerase α in chromosomal aberration production by ionizing radiation

International Nuclear Information System (INIS)

Bender, M.A.

1983-01-01

Aphidicolin is a tetracyclic diterpinoid fungal antibiotic which inhibits DNA synthesis in eukaryotic cells by interfering specifically with DNA polymerase α, apparently by binding to and inactivating the DNA-polymerase α complex. We have shown that aphidicolin, like other inhibitors of DNA synthesis, both induces chromosomal aberrations in human peripheral lymphocytes, and, as a post-treatment, interacts synergistically with x rays to produce greatly enhanced aberration yields. The present experiments explore the effects of aphidicolin in human lymphocytes in the post-DNA-synthetic G 2 phase of the cell cycle. These experiments utilized labeling with tritiated thymidine to positively identify cells in the S phase at the time of treatment, and used serial colcemid collections and fixations to determine aberration yields over as much of the G 2 phase as feasible. Because DNA polymerase α is the only DNA synthetic or repair enzyme known to be affected by aphidicolin, we infer that this enzyme is directly involved in the repair of DNA lesions which can result in visible chromosomal aberrations. (DT)

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

Science.gov (United States)

Cline, J; Braman, J C; Hogrefe, H H

1996-09-15

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.

A Novel Mechanism of Sugar Selection Utilized by a Human X-family DNA Polymerase†

Science.gov (United States)

Brown, Jessica A.; Fiala, Kevin A.; Fowler, Jason D.; Sherrer, Shanen M.; Newmister, Sean A.; Dyum, Wade W.; Suo, Zucai

2009-01-01

During DNA synthesis, most DNA polymerases and reverse transcriptases select against ribonucleotides via a steric clash between the ribose 2′-hydroxyl group and the bulky side chain of an active site residue. Here, we demonstrated that human DNA polymerase λ used a novel sugar selection mechanism to discriminate against ribonucleotides, whereby the ribose 2′-hydroxyl group was excluded mostly by a backbone segment and slightly by the side chain of Y505. Such a steric clash was further demonstrated to be dependent on the size and orientation of the substituent covalently attached at the ribonucleotide C2′ position. PMID:19900463

Synthesis of Gabosine A and N from Ribose by the Use of Ring-Closing Metathesis

DEFF Research Database (Denmark)

Monrad, Rune Nygaard; Fanefjord, Mette; Hansen, Flemming Gundorph

2009-01-01

-methylallyl bromide. The functionalized octa-1,7-diene, thus obtained, is converted into the six-membered gabosine skeleton by ring-closing olefin metathesis. Subsequent protective group manipulations and oxidation gives rise to gabosine N in a total of 8 steps from ribose while the synthesis of gabosine...

FRET Response of a Modified Ribose Receptor Expressed in the Diatom Thalassiosira pseudonana

Energy Technology Data Exchange (ETDEWEB)

Miller, Hanna

2011-08-26

The ability to insert complex proteins into silica has many applications including biosensing. Previous research has demonstrated how to direct proteins to the biosilica of diatoms [1]. Here, we show that a complex fusion protein that includes an enzyme, a bacterial ribose periplasmic binding protein, flanked by fluorescent proteins constituting a FRET pair can remain functional in the frustules of living diatoms. A Sil3 tag is attached to the N-terminal end to localize the fusion protein to frustules of the diatom Thalassiosira pseudonana. When ribose was applied, a larger decrease in FRET response was seen in transformed cells than in untransformed cells. Multiple forms of the expression vector were tested to find the optimal system; specifically, a one-vector system was compared to a two-vector system and the gDNA version of the Sil3 localization tag was compared to the cDNA version. The optimal system was found to be a one-vector system with the genomic version of the Sil3 tag to direct the protein to the frustules. Localization of the enzyme to the frustules was further confirmed through cell fluorescence imaging.

Possible involvement of 12-lipoxygenase activation in glucose-deprivation/reload-treated neurons.

Science.gov (United States)

Nagasawa, Kazuki; Kakuda, Taichi; Higashi, Youichirou; Fujimoto, Sadaki

2007-12-18

The aim of this study was to clarify whether 12-lipoxygenase (12-LOX) activation was involved in reactive oxygen species (ROS) generation, extensive poly(ADP-ribose) polymerase (PARP) activation and neuronal death induced by glucose-deprivation, followed by glucose-reload (GD/R). The decrease of neuronal viability and accumulation of poly(ADP-ribose) induced by GD/R were prevented 3-aminobenzamide, a representative PARP inhibitor, demonstrating this treatment protocol caused the same oxidative stress with the previously reported one. The PARP activation, ROS generation and decrease of neuron viability induced by GD/R treatment were almost completely abolished by an extracellular zinc chelator, CaEDTA. p47(phox), a cytosolic component of NADPH oxidase was translocated the membrane fraction by GD/R, indicating its activation, but it did not generate detectable ROS. Surprisingly, pharmacological inhibition of NADPH oxidase with apocynin and AEBSF further decreased the decreased neuron viability induced by GD/R. On the other hand, AA861, a 12-LOX inhibitor, prevented ROS generation and decrease of neuron viability caused by GD/R. Interestingly, an antioxidant, N-acetyl-l-cysteine rescued the neurons from GD/R-induced oxidative stress, implying effectiveness of antioxidant administration. These findings suggested that activation of 12-LOX, but not NADPH oxidase, following to zinc release might play an important role in ROS generation and decrease of viability in GD/R-treated neurons.

The inhibition of nitric oxide-activated poly(ADP-ribose) synthetase attenuates transsynaptic alteration of spinal cord dorsal horn neurons and neuropathic pain in the rat.

Science.gov (United States)

Mao, J; Price, D D; Zhu, J; Lu, J; Mayer, D J

1997-09-01

Transsynaptic alteration of spinal cord dorsal horn neurons characterized by hyperchromatosis of cytoplasm and nucleoplasm (so-called 'dark' neurons) occurs in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the common sciatic nerve. The incidence of dark neurons in CCI rats has been proposed to be mediated by glutamate-induced neurotoxicity. In the present study, we examined whether the inhibition of the nitric oxide (NO)-activated poly(ADP-ribose) synthetase (PARS), a nuclear enzyme critical to glutamate-induced neurotoxicity, would both reduce the incidence of dark neurons and attenuate behavioral manifestations of neuropathic pain in CCI rats. Dark neurons were observed bilaterally (with ipsilateral predominance) within the spinal cord dorsal horn, particularly in laminae I-II, of rats 8 days after unilateral sciatic nerve ligation as compared to sham operated rats. The number of dark neurons in the dorsal horn was dose-dependently reduced in CCI rats receiving once daily intrathecal (i.t.) treatment with the PARS inhibitor benzamide (200 or 400 nmol, but not 100 nmol benzamide or saline) for 7 days. Consistent with the histological improvement, thermal hyperalgesia, mechanical hyperalgesia, and low threshold mechano-allodynia also were reliably reduced in CCI rats treated with either 200 or 400 nmol benzamide. Neither dark neurons nor neuropathic pain behaviors were reliably affected by i.t. administration of either 800 nmol novobiocin (a mono(ADP-ribose) synthetase) or 800 nmol benzoic acid (the backbone structure of benzamide), indicating a selective effect of benzamide. Intrathecal treatment with an NO synthase inhibitor NG-nitro-L-arginine methyl ester (40 nmol, but not its inactive D-isomer) utilizing the same benzamide treatment regimen resulted in similar reductions of both dark neurons and neuropathic pain behaviors in CCI rats. These results provide, for the first time, in vivo evidence indicating that benzamide is

Adaptive changes in NAD+ metabolism in ultraviolet light-irradiated murine lymphoma cells

International Nuclear Information System (INIS)

Kleczkowska, H.E.; Szumiel, I.; Althaus, F.R.

1990-01-01

We have determined the ability of UV254nm-irradiated murine lymphoma cells to adapt their NAD+ metabolism to the increased NAD+ consumption for the poly ADP-ribosylation of chromatin proteins. Two murine lymphoma sublines with differential UV-sensitivity and poly(ADP-ribose) turnover were used as a model system. The first subline, designated LY-R is UV254nm-sensitive and tumorigenic in DBA/2 mice. The second subline, LY-S is UV254nm-resistant and nontumorigenic. Following treatment of these cells with 2 mM benzamide, an inhibitor of the NAD(+)-utilizing enzyme poly(ADP-ribose) polymerase, NAD+ levels slowly increased up to about 160% of control levels after 3 hours. When benzamide was added to these cultures 20 min after UV254nm irradiation, a dramatic transient increase of NAD+ levels was observed within 4 min in LY-R cells and more moderately in LY-S cells. At later times after UV254nm irradiation, the NAD+ levels increased in both sublines reaching up to 200% of the concentrations prior to benzamide treatment. These results demonstrate an adaptative response of NAD+ metabolism to UV254nm irradiation. In parallel, we observed a differential repartitioning of ADP-ribosyl residues between the NAD+ and poly(ADP-ribose) pools of LY-R and LY-S cells that correlates with the differential UV sensitivity of these cells

Genistein, a tyrosine kinase inhibitor, enhanced radiosensitivity in human esophageal cancer cell lines in vitro: Possible involvement of inhibition of survival signal transduction pathways

International Nuclear Information System (INIS)

Akimoto, Tetsuo; Nonaka, Tetsuo; Ishikawa, Hitoshi; Sakurai, Hideyuki; Saitoh, Jun-ichi; Takahashi, Takeo; Mitsuhashi, Norio

2001-01-01

Purpose: The effect of genistein, a tyrosine kinase inhibitor, on radiosensitivity was examined, especially focusing on 'survival signal transduction pathways'. Methods and Materials: Two human esophageal squamous cell cancer cell lines, TE-1 (p53, mutant) and TE-2 (p53, wild), were used. Radiosensitivity was determined by clonogenic assay, and activation of survival signals was examined by Western blot. Results: Genistein (30 μM) greatly enhanced radiosensitivity in these cell lines by suppressing radiation-induced activation of survival signals, p42/p44 extracellular signal-regulated kinase and AKT/PKB. Significant increase in the percentage of apoptotic cells and increased poly[ADP-ribose] polymerase cleavage were observed in TE-2, but not in TE-1 even after combination of genistein with irradiation. In terms of changes in expression of p53-related proteins, increase in expression of Bax and decrease in that of Bcl-2 were observed in TE-2 but not in TE-1, suggesting that the main mode of cell death induced by genistein in a cell line with wild type p53 differed from that with mutant p53. Conclusions: This study suggested that survival signals, including p42/p44 ERK and AKT/PKB, may be involved in determining radiosensitivity, and genistein would be a potent therapeutic agent that has an enhancing effect on radiation

The association of TP53 mutations with the resistance of colorectal carcinoma to the insulin-like growth factor-1 receptor inhibitor picropodophyllin

International Nuclear Information System (INIS)

Wang, Quan; Wei, Feng; Lv, Guoyue; Li, Chunsheng; Liu, Tongjun; Hadjipanayis, Costas G; Zhang, Guikai; Hao, Chunhai; Bellail, Anita C

2013-01-01

There is growing evidence indicating the insulin-like growth factor 1 receptor (IGF-1R) plays a critical role in the progression of human colorectal carcinomas. IGF-1R is an attractive drug target for the treatment of colon cancer. Picropodophyllin (PPP), of the cyclolignan family, has recently been identified as an IGF-1R inhibitor. The aim of this study is to determine the therapeutic response and mechanism after colorectal carcinoma treatment with PPP. Seven colorectal carcinoma cell lines were treated with PPP. Following treatment, cells were analyzed for growth by a cell viability assay, sub-G1 apoptosis by flow cytometry, caspase cleavage and activation of AKT and extracellular signal-regulated kinase (ERK) by western blot analysis. To examine the in vivo therapeutic efficacy of PPP, mice implanted with human colorectal carcinoma xenografts underwent PPP treatment. PPP treatment blocked the phosphorylation of IGF-1R, AKT and ERK and inhibited the growth of TP53 wild-type but not mutated colorectal carcinoma cell lines. The treatment of PPP also induced apoptosis in TP53 wild-type cells as evident by the presence of sub-G1 cells and the cleavage of caspase-9, caspase-3, DNA fragmentation factor-45 (DFF45), poly (ADP-ribose) polymerase (PARP), and X-linked inhibitor of apoptosis protein (XIAP). The loss of BAD phosphorylation in the PPP-treated TP53 wild type cells further suggested that the treatment induced apoptosis through the BAD-mediated mitochondrial pathway. In contrast, PPP treatment failed to induce the phosphorylation of AKT and ERK and caspase cleavage in TP53 mutated colorectal carcinoma cell lines. Finally, PPP treatment suppressed the growth of xenografts derived from TP53 wild type but not mutated colorectal carcinoma cells. We report the association of TP53 mutations with the resistance of treatment of colorectal carcinoma cells in culture and in a xenograft mouse model with the IGF-1R inhibitor PPP. TP53 mutations often occur in colorectal

Mitochondrial and Nuclear Cross Talk in Cell Death: Parthanatos

OpenAIRE

Andrabi, Shaida A.; Dawson, Ted M.; Dawson, Valina L.

2008-01-01

Poly(ADP-ribose) polymerase-1 (PARP-1) PARP-1 is an abundant nuclear protein first described to facilitate DNA base excision repair. Recent work has expanded the physiologic functions of PARP-1 and it is clear that the full range of biologic actions of this important protein are not yet fully understood. Regulation of the product of PARP-1, poly(ADP-ribose) (PAR), is a dynamic process with poly(ADP-ribose) glycohydrolase (PARG) playing a major role in the degradation of the polymer. Under pat...

Novel Neuroprotective Strategies in Ischemic Retinal Lesions

Directory of Open Access Journals (Sweden)

Robert Gabriel

2010-02-01

Full Text Available Retinal ischemia can be effectively modeled by permanent bilateral common carotid artery occlusion, which leads to chronic hypoperfusion-induced degeneration in the entire rat retina. The complex pathways leading to retinal cell death offer a complex approach of neuroprotective strategies. In the present review we summarize recent findings with different neuroprotective candidate molecules. We describe the protective effects of intravitreal treatment with: (i urocortin 2; (ii a mitochondrial ATP-sensitive K+ channel opener, diazoxide; (iii a neurotrophic factor, pituitary adenylate cyclase activating polypeptide; and (iv a novel poly(ADP-ribose polymerase inhibitor (HO3089. The retinoprotective effects are demonstrated with morphological description and effects on apoptotic pathways using molecular biological techniques.

Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

Science.gov (United States)

Pavlov, Andrey R; Pavlova, Nadejda V; Kozyavkin, Sergei A; Slesarev, Alexei I

2012-03-13

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

Science.gov (United States)

Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

2012-01-01

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

Inhibition of matrix metalloproteinase-2 by PARP inhibitors

International Nuclear Information System (INIS)

Nicolescu, Adrian C.; Holt, Andrew; Kandasamy, Arulmozhi D.; Pacher, Pal; Schulz, Richard

2009-01-01

Matrix metalloproteinase-2 (MMP-2), a ubiquitously expressed zinc-dependent endopeptidase, and poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme regulating DNA repair, are activated by nitroxidative stress associated with various pathologies. As MMP-2 plays a detrimental role in heart injuries resulting from enhanced nitroxidative stress, where PARP and MMP inhibitors are beneficial, we hypothesized that PARP inhibitors may affect MMP-2 activity. Using substrate degradation assays to determine MMP-2 activity we found that four PARP inhibitors (3-AB, PJ-34, 5-AIQ, and EB-47) inhibited 64 kDa MMP-2 in a concentration-dependent manner. The IC 50 values of PJ-34 and 5-AIQ were in the high micromolar range and comparable to those of known MMP-2 inhibitors doxycycline, minocycline or o-phenanthroline, whereas those for 3-AB and EB-47 were in the millimolar range. Co-incubation of PARP inhibitors with doxycycline showed an additive inhibition of MMP-2 that was significant for 3-AB alone. These data demonstrate that the protective effects of some PARP inhibitors may include inhibition of MMP-2 activity.

Inhibition of matrix metalloproteinase-2 by PARP inhibitors

Energy Technology Data Exchange (ETDEWEB)

Nicolescu, Adrian C.; Holt, Andrew; Kandasamy, Arulmozhi D. [Departments of Pharmacology and Pediatrics, Cardiovascular Research Centre, University of Alberta, Edmonton, Alta., Canada T6G 2S2 (Canada); Pacher, Pal [National Institutes of Health, NIAAA, Laboratory of Physiologic Studies, Bethesda, MD (United States); Schulz, Richard, E-mail: richard.schulz@ualberta.ca [Departments of Pharmacology and Pediatrics, Cardiovascular Research Centre, University of Alberta, Edmonton, Alta., Canada T6G 2S2 (Canada)

2009-10-02

Matrix metalloproteinase-2 (MMP-2), a ubiquitously expressed zinc-dependent endopeptidase, and poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme regulating DNA repair, are activated by nitroxidative stress associated with various pathologies. As MMP-2 plays a detrimental role in heart injuries resulting from enhanced nitroxidative stress, where PARP and MMP inhibitors are beneficial, we hypothesized that PARP inhibitors may affect MMP-2 activity. Using substrate degradation assays to determine MMP-2 activity we found that four PARP inhibitors (3-AB, PJ-34, 5-AIQ, and EB-47) inhibited 64 kDa MMP-2 in a concentration-dependent manner. The IC{sub 50} values of PJ-34 and 5-AIQ were in the high micromolar range and comparable to those of known MMP-2 inhibitors doxycycline, minocycline or o-phenanthroline, whereas those for 3-AB and EB-47 were in the millimolar range. Co-incubation of PARP inhibitors with doxycycline showed an additive inhibition of MMP-2 that was significant for 3-AB alone. These data demonstrate that the protective effects of some PARP inhibitors may include inhibition of MMP-2 activity.

Low ATM protein expression and depletion of p53 correlates with olaparib sensitivity in gastric cancer cell lines

Science.gov (United States)

Kubota, Eiji; Williamson, Christopher T; Ye, Ruiqiong; Elegbede, Anifat; Peterson, Lars; Lees-Miller, Susan P; Bebb, D Gwyn

2014-01-01

Small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP) have shown considerable promise in the treatment of homologous recombination (HR)-defective tumors, such as BRCA1- and BRCA2-deficient breast and ovarian cancers. We previously reported that mantle cell lymphoma cells with deficiency in ataxia telangiectasia mutated (ATM) are sensitive to PARP-1 inhibitors in vitro and in vivo. Here, we report that PARP inhibitors can potentially target ATM deficiency arising in a solid malignancy. We show that ATM protein expression varies between gastric cancer cell lines, with NUGC4 having significantly reduced protein levels. Significant correlation was found between ATM protein expression and sensitivity to the PARP inhibitor olaparib, with NUGC4 being the most sensitive. Moreover, reducing ATM kinase activity using a small-molecule inhibitor (KU55933) or shRNA-mediated depletion of ATM protein enhanced olaparib sensitivity in gastric cancer cell lines with depletion or inactivation of p53. Our results demonstrate that ATM is a potential predictive biomarker for PARP-1 inhibitor activity in gastric cancer harboring disruption of p53, and that combined inhibition of ATM and PARP-1 is a rational strategy for expanding the utility of PARP-1 inhibitors to gastric cancer with p53 disruption. PMID:24841718

Verdazyl-ribose: A new radical for solid-state dynamic nuclear polarization at high magnetic field

Science.gov (United States)

Thurber, Kent R.; Le, Thanh-Ngoc; Changcoco, Victor; Brook, David J. R.

2018-04-01

Solid-state dynamic nuclear polarization (DNP) using the cross-effect relies on radical pairs whose electron spin resonance (ESR) frequencies differ by the nuclear magnetic resonance (NMR) frequency. We measure the DNP provided by a new water-soluble verdazyl radical, verdazyl-ribose, under both magic-angle spinning (MAS) and static sample conditions at 9.4 T, and compare it to a nitroxide radical, 4-hydroxy-TEMPO. We find that verdazyl-ribose is an effective radical for cross-effect DNP, with the best relative results for a non-spinning sample. Under non-spinning conditions, verdazyl-ribose provides roughly 2× larger 13C cross-polarized (CP) NMR signal than the nitroxide, with similar polarization buildup times, at both 29 K and 76 K. With MAS at 7 kHz and 1.5 W microwave power, the verdazyl-ribose does not provide as much DNP as the nitroxide, with the verdazyl providing less NMR signal and a longer polarization buildup time. When the microwave power is decreased to 30 mW with 5 kHz MAS, the two types of radical are comparable, with the verdazyl-doped sample having a larger NMR signal which compensates for its longer polarization buildup time. We also present electron spin relaxation measurements at Q-band (1.2 T) and ESR lineshapes at 1.2 and 9.4 T. Most notably, the verdazyl radical has a longer T1e than the nitroxide (9.9 ms and 1.3 ms, respectively, at 50 K and 1.2 T). The verdazyl electron spin lineshape is significantly affected by the hyperfine coupling to four 14N nuclei, even at 9.4 T. We also describe 3000-spin calculations to illustrate the DNP potential of possible radical pairs: verdazyl-verdazyl, verdazyl-nitroxide, or nitroxide-nitroxide pairs. These calculations suggest that the verdazyl radical at 9.4 T has a narrower linewidth than optimal for cross-effect DNP using verdazyl-verdazyl pairs. Because of the hyperfine coupling contribution to the electron spin linewidth, this implies that DNP using the verdazyl radical would improve at lower

Verdazyl-ribose: A new radical for solid-state dynamic nuclear polarization at high magnetic field.

Science.gov (United States)

Thurber, Kent R; Le, Thanh-Ngoc; Changcoco, Victor; Brook, David J R

2018-04-01

Solid-state dynamic nuclear polarization (DNP) using the cross-effect relies on radical pairs whose electron spin resonance (ESR) frequencies differ by the nuclear magnetic resonance (NMR) frequency. We measure the DNP provided by a new water-soluble verdazyl radical, verdazyl-ribose, under both magic-angle spinning (MAS) and static sample conditions at 9.4 T, and compare it to a nitroxide radical, 4-hydroxy-TEMPO. We find that verdazyl-ribose is an effective radical for cross-effect DNP, with the best relative results for a non-spinning sample. Under non-spinning conditions, verdazyl-ribose provides roughly 2× larger 13 C cross-polarized (CP) NMR signal than the nitroxide, with similar polarization buildup times, at both 29 K and 76 K. With MAS at 7 kHz and 1.5 W microwave power, the verdazyl-ribose does not provide as much DNP as the nitroxide, with the verdazyl providing less NMR signal and a longer polarization buildup time. When the microwave power is decreased to 30 mW with 5 kHz MAS, the two types of radical are comparable, with the verdazyl-doped sample having a larger NMR signal which compensates for its longer polarization buildup time. We also present electron spin relaxation measurements at Q-band (1.2 T) and ESR lineshapes at 1.2 and 9.4 T. Most notably, the verdazyl radical has a longer T 1e than the nitroxide (9.9 ms and 1.3 ms, respectively, at 50 K and 1.2 T). The verdazyl electron spin lineshape is significantly affected by the hyperfine coupling to four 14 N nuclei, even at 9.4 T. We also describe 3000-spin calculations to illustrate the DNP potential of possible radical pairs: verdazyl-verdazyl, verdazyl-nitroxide, or nitroxide-nitroxide pairs. These calculations suggest that the verdazyl radical at 9.4 T has a narrower linewidth than optimal for cross-effect DNP using verdazyl-verdazyl pairs. Because of the hyperfine coupling contribution to the electron spin linewidth, this implies that DNP using the verdazyl

Interaction of gold nanoparticles with Pfu DNA polymerase and effect on polymerase chain reaction.

Science.gov (United States)

Sun, L-P; Wang, S; Zhang, Z-W; Ma, Y-Y; Lai, Y-Q; Weng, J; Zhang, Q-Q

2011-03-01

The interaction of gold nanoparticles with Pfu DNA polymerase has been investigated by a number of biological, optical and electronic spectroscopic techniques. Polymerase chain reaction was performed to show gold nanoparticles' biological effect. Ultraviolet-visible and circular dichroism spectra analysis were applied to character the structure of Pfu DNA polymerase after conjugation with gold nanoparticles. X-ray photoelectron spectroscopy was used to investigate the bond properties of the polymerase-gold nanoparticles complex. The authors demonstrate that gold nanoparticles do not affect the amplification efficiency of polymerase chain reaction using Pfu DNA polymerase, and Pfu DNA polymerase displays no significant changes of the secondary structure upon interaction with gold nanoparticles. The adsorption of Pfu DNA polymerase to gold nanoparticles is mainly through Au-NH(2) bond and electrostatic interaction. These findings may have important implications regarding the safety issue as gold nanoparticles are widely used in biomedical applications.

Identification of poly(ADP-ribose) polymerase-1 as the OXPHOS-generated ATP sensor of nuclei of animal cells

International Nuclear Information System (INIS)

Kun, Ernest; Kirsten, Eva; Hakam, Alaeddin; Bauer, Pal I.; Mendeleyev, Jerome

2008-01-01

Our results show that in the intact normal animal cell mitochondrial ATP is directly connected to nuclear PARP-1 by way of a specific adenylate kinase enzymatic path. This mechanism is demonstrated in two models: (a) by its inhibition with a specific inhibitor of adenylate kinase, and (b) by disruption of ATP synthesis through uncoupling of OXPHOS. In each instance the de-inhibited PARP-1 is quantitatively determined by enzyme kinetics. The nuclear binding site of PARP-1 is Topo I, and is identified as a critical 'switchpoint' indicating the nuclear element that connects OXPHOS with mRNA synthesis in real time. The mitochondrial-nuclear PARP-1 pathway is not operative in cancer cells

Antitumor efficacy profile of PKI-402, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor.

Science.gov (United States)

Mallon, Robert; Hollander, Irwin; Feldberg, Larry; Lucas, Judy; Soloveva, Veronica; Venkatesan, Aranapakam; Dehnhardt, Christoph; Delos Santos, Efren; Chen, Zecheng; Dos Santos, Osvaldo; Ayral-Kaloustian, Semiramis; Gibbons, Jay

2010-04-01

PKI-402 is a selective, reversible, ATP-competitive, equipotent inhibitor of class I phosphatidylinositol 3-kinases (PI3K), including PI3K-alpha mutants, and mammalian target of rapamycin (mTOR; IC(50) versus PI3K-alpha = 2 nmol/L). PKI-402 inhibited growth of human tumor cell lines derived from breast, brain (glioma), pancreas, and non-small cell lung cancer tissue and suppressed phosphorylation of PI3K and mTOR effector proteins (e.g., Akt at T308) at concentrations that matched those that inhibited cell growth. In MDA-MB-361 [breast: Her2(+) and PIK3CA mutant (E545K)], 30 nmol/L PKI-402 induced cleaved poly(ADP-ribose) polymerase (PARP), a marker for apoptosis. In vivo, PKI-402 inhibited tumor growth in MDA-MB-361, glioma (U87MG), and lung (A549) xenograft models. In MDA-MB-361, PKI-402 at 100 mg/kg (daily for 5 days, one round) reduced initial tumor volume of 260 mm(3) to 129 mm(3) and prevented tumor regrowth for 70 days. In MDA-MB-361 tumors, PKI-402 (100 mg/kg, single dose) suppressed Akt phosphorylation (at T308) and induced cleaved PARP. Suppression of phosphorylated Akt (p-Akt) was complete at 8 hours and still evident at 24 hours. Cleaved PARP was evident at 8 and 24 hours. In normal tissue (heart and lung), PKI-402 (100 mg/kg) had minimal effect on p-Akt, with no detectable cleaved PARP. Preferential accumulation of PKI-402 in tumor tissue was observed. Complete, sustained suppression of Akt phosphorylation may cause tumor regression in MDA-MB-361 and other xenograft models. We are testing whether dual PI3K/mTOR inhibitors can durably suppress p-Akt, induce cleaved PARP, and cause tumor regression in a diverse set of human tumor xenograft models. Mol Cancer Ther; 9(4); 976-84. (c)2010 AACR.

Archaeal DNA Polymerase-B as a DNA Template Guardian: Links between Polymerases and Base/Alternative Excision Repair Enzymes in Handling the Deaminated Bases Uracil and Hypoxanthine

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Javier Abellón-Ruiz

2016-01-01

Full Text Available In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil; Endonuclease V (EndoV, which recognises hypoxanthine; and Endonuclease Q (EndoQ, (which recognises both uracil and hypoxanthine. Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by deaminated bases in template strands, a feature unique to this domain. Thus the three repair enzymes and the two polymerases show overlapping specificity for uracil and hypoxanthine. Here it is demonstrated that binding of Pol-D to primer-templates containing deaminated bases inhibits the activity of UDG, EndoV, and EndoQ. Similarly Pol-B almost completely turns off EndoQ, extending earlier work that demonstrated that Pol-B reduces catalysis by UDG and EndoV. Pol-B was observed to be a more potent inhibitor of the enzymes compared to Pol-D. Although Pol-D is directly inhibited by template strand uracil, the presence of Pol-B further suppresses any residual activity of Pol-D, to near-zero levels. The results are compatible with Pol-D acting as the replicative polymerase and Pol-B functioning primarily as a guardian preventing deaminated base-induced DNA mutations.

Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication.

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Aye, Seaim Lwin; Fujiwara, Kei; Ueki, Asuka; Doi, Nobuhide

2018-05-05

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5' to 3' exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol. Copyright © 2018 Elsevier Inc. All rights reserved.

Hepatitis C Virus NS3 Inhibitors: Current and Future Perspectives

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Kazi Abdus Salam

2013-01-01

Full Text Available Currently, hepatitis C virus (HCV infection is considered a serious health-care problem all over the world. A good number of direct-acting antivirals (DAAs against HCV infection are in clinical progress including NS3-4A protease inhibitors, RNA-dependent RNA polymerase inhibitors, and NS5A inhibitors as well as host targeted inhibitors. Two NS3-4A protease inhibitors (telaprevir and boceprevir have been recently approved for the treatment of hepatitis C in combination with standard of care (pegylated interferon plus ribavirin. The new therapy has significantly improved sustained virologic response (SVR; however, the adverse effects associated with this therapy are still the main concern. In addition to the emergence of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase portion of the HCV NS3 protein. This review article summarizes our current understanding of HCV treatment, particularly with those of NS3 inhibitors.

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

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

2017-12-07

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

Evaluation of Concurrent Radiation, Temozolomide and ABT-888 Treatment Followed by Maintenance Therapy with Temozolomide and ABT-888 in a Genetically Engineered Glioblastoma Mouse Model.

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Lemasson, Benjamin; Wang, Hanxiao; Galbán, Stefanie; Li, Yinghua; Zhu, Yuan; Heist, Kevin A; Tsein, Christina; Chenevert, Thomas L; Rehemtulla, Alnawaz; Galbán, Craig J; Holland, Eric C; Ross, Brian D

2016-02-01

Despite the use of ionizing radiation (IR) and temozolomide (TMZ), outcome for glioblastoma (GBM) patients remains dismal. Poly (ADP-ribose) polymerase (PARP) is important in repair pathways for IR-induced DNA damage and TMZ-induced alkylation at N7-methylguanine and N3-methyldenine. However, optimized protocols for administration of PARP inhibitors have not been delineated. In this study, the PARP inhibitor ABT-888 was evaluated in combination with and compared to current standard-of-care in a genetically engineered mouse GBM model. Results demonstrated that concomitant TMZ/IR/ABT-888 with adjuvant TMZ/ABT-888 was more effective in inducing apoptosis and reducing proliferation with significant tumor growth delay and improved overall survival over concomitant TMZ/IR with adjuvant TMZ. Diffusion-weighted MRI, an early translatable response biomarker detected changes in tumors reflecting response at 1 day post TMZ/IR/ABT-888 treatment. This study provides strong scientific rationale for the development of an optimized dosing regimen for a PARP inhibitor with TMZ/IR for upfront treatment of GBM. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation of Concurrent Radiation, Temozolomide and ABT-888 Treatment Followed by Maintenance Therapy with Temozolomide and ABT-888 in a Genetically Engineered Glioblastoma Mouse Model

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Benjamin Lemasson

2016-02-01

Full Text Available Despite the use of ionizing radiation (IR and temozolomide (TMZ, outcome for glioblastoma (GBM patients remains dismal. Poly (ADP-ribose polymerase (PARP is important in repair pathways for IR-induced DNA damage and TMZ-induced alkylation at N7-methylguanine and N3-methyldenine. However, optimized protocols for administration of PARP inhibitors have not been delineated. In this study, the PARP inhibitor ABT-888 was evaluated in combination with and compared to current standard-of-care in a genetically engineered mouse GBM model. Results demonstrated that concomitant TMZ/IR/ABT-888 with adjuvant TMZ/ABT-888 was more effective in inducing apoptosis and reducing proliferation with significant tumor growth delay and improved overall survival over concomitant TMZ/IR with adjuvant TMZ. Diffusion-weighted MRI, an early translatable response biomarker detected changes in tumors reflecting response at 1 day post TMZ/IR/ABT-888 treatment. This study provides strong scientific rationale for the development of an optimized dosing regimen for a PARP inhibitor with TMZ/IR for upfront treatment of GBM.

Analysis of redox relationships in the plant cell cycle: determinations of ascorbate, glutathione and poly (ADPribose)polymerase (PARP) in plant cell cultures.

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Foyer, Christine H; Pellny, Till K; Locato, Vittoria; De Gara, Laura

2008-01-01

Reactive oxygen species (ROS) and low molecular weight antioxidants, such as glutathione and ascorbate, are powerful signaling molecules that participate in the control of plant growth and development, and modulate progression through the mitotic cell cycle. Enhanced reactive oxygen species accumulation or low levels of ascorbate or glutathione cause the cell cycle to arrest and halt progression especially through the G1 checkpoint. Plant cell suspension cultures have proved to be particularly useful tools for the study of cell cycle regulation. Here we provide effective and accurate methods for the measurement of changes in the cellular ascorbate and glutathione pools and the activities of related enzymes such poly (ADP-ribose) polymerase during mitosis and cell expansion, particularly in cell suspension cultures. These methods can be used in studies seeking to improve current understanding of the roles of redox controls on cell division and cell expansion.

Are There Mutator Polymerases?

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Miguel Garcia-Diaz

2003-01-01

Full Text Available DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.

Ascorbic acid and a cytostatic inhibitor of glycolysis synergistically induce apoptosis in non-small cell lung cancer cells.

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Saleha B Vuyyuri

Full Text Available Ascorbic acid (AA exhibits significant anticancer activity at pharmacologic doses achievable by parenteral administration that have minimal effects on normal cells. Thus, AA has potential uses as a chemotherapeutic agent alone or in combination with other therapeutics that specifically target cancer-cell metabolism. We compared the effects of AA and combinations of AA with the glycolysis inhibitor 3-(3-pyridinyl-1-(4-pyridinyl-2-propen-1-one (3-PO on the viability of three non-small cell lung cancer (NSCLC cell lines to the effects on an immortalized lung epithelial cell line. AA concentrations of 0.5 to 5 mM caused a complete loss of viability in all NSCLC lines compared to a <10% loss of viability in the lung epithelial cell line. Combinations of AA and 3-PO synergistically enhanced cell death in all NSCLC cell lines at concentrations well below the IC50 concentrations for each compound alone. A synergistic interaction was not observed in combination treatments of lung epithelial cells and combination treatments that caused a complete loss of viability in NSCLC cells had modest effects on normal lung cell viability and reactive oxygen species (ROS levels. Combination treatments induced dramatically higher ROS levels compared to treatment with AA and 3-PO alone in NSCLC cells and combination-induced cell death was inhibited by addition of catalase to the medium. Analyses of DNA fragmentation, poly (ADP-ribose polymerase cleavage, annexin V-binding, and caspase activity demonstrated that AA-induced cell death is caused via the activation of apoptosis and that the combination treatments caused a synergistic induction of apoptosis. These results demonstrate the effectiveness of AA against NSCLC cells and that combinations of AA with 3-PO synergistically induce apoptosis via a ROS-dependent mechanism. These results support further evaluation of pharmacologic concentrations of AA as an adjuvant treatment for NSCLC and that combination of AA with

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

International Nuclear Information System (INIS)

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

1981-01-01

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

Preliminary crystallographic analysis of two hypothetical ribose-5-phosphate isomerases from Streptococcus mutans

International Nuclear Information System (INIS)

Wang, Chen; Fan, Xuexin; Cao, Xiaofang; Liu, Xiang; Li, Lanfen; Su, Xiaodong

2012-01-01

Two hypothetical ribose-5-phosphate isomerases from S. mutans have been produced in E. coli and crystallized. The crystals diffracted to high resolutions suitable for crystallographic analyses. Study of the enzymes from sugar metabolic pathways may provide a better understanding of the pathogenesis of the human oral pathogen Streptococcus mutans. Bioinformatics, biochemical and crystallization methods were used to characterize and understand the function of two putative ribose-5-phosphate isomerases: SMU1234 and SMU2142. The proteins were cloned and constructed with N-terminal His tags. Protein purification was performed by Ni 2+ -chelating and size-exclusion chromatography. The crystals of SUM1234 diffracted to 1.9 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 48.97, b = 98.27, c = 101.09 Å, α = β = γ = 90°. The optimized SMU2142 crystals diffracted to 2.7 Å resolution and belonged to space group P1, with unit-cell parameters a = 53.7, b = 54.1, c = 86.5 Å, α = 74.2, β = 73.5, γ = 83.7°. Initial phasing of both proteins was attempted by molecular replacement; the structure of SMU1234 could easily be solved, but no useful results were obtained for SMU2142. Therefore, SeMet-labelled SMU2142 will be prepared for phasing

[The effect of 3-aminobenzamide on the mitotic cycle of Chinese hamster cells cultured on a medium with 5-bromodeoxyuridine following ionizing radiation action].

Science.gov (United States)

Kirillova, T V; Rozanov, Iu M; Spivak, I M

1992-01-01

A specific inhibitor of poly(ADP-ribose)polymerase-3-aminobenzamide (6 mM) has been shown to: 1) reduce survival of non-irradiated CHO-K1 cells, cultivated in medium containing 5-bromodeoxyuridine (10 mkM, BDU cells), and increase their radiosensitivity; 2) induce G2 delay in BDU cells while progressing through the cell cycle as analysed by the DNA flow cytometry; 3) increase to a great degree G2 delay in X-irradiated BDU cells. 3-Aminobenzamide is primarily effective when it is present during the first or two first cell cycles after the initial addition of BDU. The above data confirm the involvement, presumably an indirect one, of ADP-ribosylation in the DNA repair through affecting the chromatin structure.

Resistance Patterns Associated with HCV NS5A Inhibitors Provide Limited Insight into Drug Binding

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Moheshwarnath Issur

2014-11-01

Full Text Available Direct-acting antivirals (DAAs have significantly improved the treatment of infection with the hepatitis C virus. A promising class of novel antiviral agents targets the HCV NS5A protein. The high potency and broad genotypic coverage are favorable properties. NS5A inhibitors are currently assessed in advanced clinical trials in combination with viral polymerase inhibitors and/or viral protease inhibitors. However, the clinical use of NS5A inhibitors is also associated with new challenges. HCV variants with decreased susceptibility to these drugs can emerge and compromise therapy. In this review, we discuss resistance patterns in NS5A with focus prevalence and implications for inhibitor binding.

The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

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Lonneke van der Linden

2015-03-01

Full Text Available The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71 for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylenebis(oxy]bis(5-nitro-benzonitrile with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family. Surprisingly, coxsackievirus B3 (CVB3 and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight

Identification of DNA polymerase molecules repairing DNA irradiated damage and molecular biological study on modified factors of mutation rate

Energy Technology Data Exchange (ETDEWEB)

Yamada, Koichi; Inoue, Shuji [National Inst. of Healthand Nutrition, Tokyo (Japan)

1999-02-01

DNA repairing polymerase has not been identified in human culture cells because the specificities of enzyme inhibitors used in previous studies were not so high. In this study, anti-sense oligonucleotides were transfected into human fibroblast cells by electroporation and several clones selected by geneticin treatment were found to express the RNA of the incorporated DNA. However, the expression was not significant and its reproducibility was poor. Then, a study on repairing mechanism was made using XP30 RO and XP 115 LO cells which are variant cells of xeroderma pigmentosum, a human hereditary disease aiming to identify the DNA polymerase related to the disease. There were abnormalities in DNA polymerase subunit {delta} or {epsilon} which consists DNA replication complex. Thus, it was suggested that the DNA replication of these mutant cells might terminate at the site containing such abnormality. (M.N.)

Higher number of pentosidine cross-links induced by ribose does not alter tissue stiffness of cancellous bone

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Willems, Nop M.B.K., E-mail: n.willems@acta.nl [Dept. of Orthodontics, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Langenbach, Geerling E.J. [Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Stoop, Reinout [Dept. of Metabolic Health Research, TNO, P.O. Box 2215, 2301 CE Leiden (Netherlands); Toonder, Jaap M.J. den [Dept. of Mechanical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven (Netherlands); Mulder, Lars [Dept. of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven (Netherlands); Zentner, Andrej [Dept. of Orthodontics, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Everts, Vincent [Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands)

2014-09-01

The role of mature collagen cross-links, pentosidine (Pen) cross-links in particular, in the micromechanical properties of cancellous bone is unknown. The aim of this study was to examine nonenzymatic glycation effects on tissue stiffness of demineralized and non-demineralized cancellous bone. A total of 60 bone samples were derived from mandibular condyles of six pigs, and assigned to either control or experimental groups. Experimental handling included incubation in phosphate buffered saline alone or with 0.2 M ribose at 37 °C for 15 days and, in some of the samples, subsequent complete demineralization of the sample surface using 8% EDTA. Before and after experimental handling, bone microarchitecture and tissue mineral density were examined by means of microcomputed tomography. After experimental handling, the collagen content and the number of Pen, hydroxylysylpyridinoline (HP), and lysylpyridinoline (LP) cross-links were estimated using HPLC, and tissue stiffness was assessed by means of nanoindentation. Ribose treatment caused an up to 300-fold increase in the number of Pen cross-links compared to nonribose-incubated controls, but did not affect the number of HP and LP cross-links. This increase in the number of Pen cross-links had no influence on tissue stiffness of both demineralized and nondemineralized bone samples. These findings suggest that Pen cross-links do not play a significant role in bone tissue stiffness. - Highlights: • The assessment of effects of glycation in bone using HPLC, microCT, and nanoindentation • Ribose incubation: 300‐fold increase in the number of pentosidine cross-links • 300‐fold increase in the number of pentosidine cross-links: no changes in bone tissue stiffness.

Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag-Pol.

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Anna Figueiredo

2006-11-01

Full Text Available Nonnucleoside reverse transcriptase inhibitors (NNRTIs target HIV-1 reverse transcriptase (RT by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation.

A heterogeneous Pd-Bi/C catalyst in the synthesis of L-lyxose and L-ribose from naturally occurring D-sugars.

Science.gov (United States)

Fan, Ao; Jaenicke, Stephan; Chuah, Gaik-Khuan

2011-10-26

A critical step in the synthesis of the rare sugars, L-lyxose and L-ribose, from the corresponding D-sugars is the oxidation to the lactone. Instead of conventional oxidizing agents like bromine or pyridinium dichromate, it was found that a heterogeneous catalyst, Pd-Bi/C, could be used for the direct oxidation with molecular oxygen. The composition of the catalyst was optimized and the best results were obtained with 5 : 1 atomic ratio of Pd : Bi. The overall yields of the five-step procedure to L-ribose and L-lyxose were 47% and 50%, respectively. The synthetic procedure is advantageous from the viewpoint of overall yield, reduced number of steps, and mild reaction conditions. Furthermore, the heterogeneous oxidation catalyst can be easily separated from the reaction mixture and reused with no loss of activity.

Synthesis of deleobuvir, a potent hepatitis C virus polymerase inhibitor, and its major metabolites labeled with carbon-13 and carbon-14.

Science.gov (United States)

Latli, Bachir; Hrapchak, Matt; Chevliakov, Maxim; Li, Guisheng; Campbell, Scot; Busacca, Carl A; Senanayake, Chris H

2015-05-30

Deleobuvir, (2E)-3-(2-{1-[2-(5-bromopyrimidin-2-yl)-3-cyclopentyl-1-methyl-1H-indole-6-carboxamido]cyclobutyl}-1-methyl-1H-benzimidazol-6-yl)prop-2-enoic acid (1), is a non-nucleoside, potent, and selective inhibitor of hepatitis C virus NS5B polymerase. Herein, we describe the detailed synthesis of this compound labeled with carbon-13 and carbon-14. The synthesis of its three major metabolites, namely, the reduced double bond metabolite (2) and the acyl glucuronide derivatives of (1) and (2), is also reported. Aniline-(13) C6 was the starting material to prepare butyl (E)-3-(3-methylamino-4-nitrophenyl-(13) C6 )acrylate [(13) C6 ]-(11) in six steps. This intermediate was then used to obtain [(13) C6 ]-(1) and [(13) C6 ]-(2) in five and four more steps, respectively. For the radioactive synthesis, potassium cyanide-(14) C was used to prepare 1-cylobutylaminoacid [(14) C]-(23) via Buchrer-Bergs reaction. The carbonyl chloride of this acid was then used to access both [(14) C]-(1) and [(14) C]-(2) in four steps. The acyl glucuronide derivatives [(13) C6 ]-(3), [(13) C6 ]-(4) and [(14) C]-(3) were synthesized in three steps from the acids [(13) C6 ]-(1), [(13) C6 ]-(2) and [(14) C]-(1) using known procedures. Copyright © 2015 John Wiley & Sons, Ltd.

Substituted 2,6-bis(benzimidazol-2-yl)pyridines: a novel chemical class of pestivirus inhibitors that targets a hot spot for inhibition of pestivirus replication in the RNA-dependent RNA polymerase.

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Musiu, Simone; Pürstinger, Gerhard; Stallinger, Sylvia; Vrancken, Robert; Haegeman, Andy; Koenen, Frank; Leyssen, Pieter; Froeyen, Mathy; Neyts, Johan; Paeshuyse, Jan

2014-06-01

2,6-Bis(benzimidazol-2-yl)pyridine (BBP/CSFA-0) was identified in a CPE-based screening as a selective inhibitor of the in vitro bovine viral diarrhea virus (BVDV) replication. The EC50-values for the inhibition of BVDV-induced cytopathic (CPE) effect, viral RNA synthesis and the production of infectious virus were 0.3±0.1μM, 0.05±0.01μM and 0.3±0.04μM, respectively. Furthermore, BBP/CSFA-0 inhibits the in vitro replication of the classical swine fever virus (CSFV) with an EC50 of 0.33±0.25μM. BBP/CSFA-0 proved in vitro inactive against the hepatitis C virus, that belongs like BVDV and CSFV to the family of Flaviviridae. Modification of the substituents on the two 1H-benzimidazole groups of BBP resulted in analogues equipotent in anti-BVDV activity (EC50=0.7±0.1μM), devoid of cytotoxicity (S.I.=142). BBP resistant BVDV was selected for and was found to carry the I261M mutation in the viral RNA-dependent RNA polymerase (RdRp). Likewise, BBP-resistant CSFV was selected for; this variant carries either an I261N or a P262A mutation in NS5B. Molecular modeling revealed that I261 and P262 are located in a small cavity near the fingertip domain of the pestivirus polymerase. BBP-resistant BVDV and CSFV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110 and LZ37) that are known to target the same region of the RdRp. BBP did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). BBP interacts likely with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37. This indicates that this region is a "hot spot" for inhibition of pestivirus replication. Copyright © 2014 Elsevier B.V. All rights reserved.

Smoking History Predicts Sensitivity to PARP Inhibitor Veliparib in Patients with Advanced Non-Small Cell Lung Cancer.

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Reck, Martin; Blais, Normand; Juhasz, Erzsebet; Gorbunova, Vera; Jones, C Michael; Urban, Laszlo; Orlov, Sergey; Barlesi, Fabrice; Kio, Ebenezer; Keilholz, Ulrich; Qin, Qin; Qian, Jiang; Nickner, Caroline; Dziubinski, Juliann; Xiong, Hao; Mittapalli, Rajendar K; Dunbar, Martin; Ansell, Peter; He, Lei; McKee, Mark; Giranda, Vincent; Ramalingam, Suresh S

2017-07-01

Tobacco-related NSCLC is associated with reduced survival and greater genomic instability. Veliparib, a potent poly(adenosine diphosphate-ribose) polymerase inhibitor, augments platinum-induced DNA damage. A phase 2 trial of untreated advanced NSCLC showed a trend for improved outcomes (hazard ratio [HR] = 0.80, 95% confidence interval: 0.54-1.18, p = 0.27 for overall survival and HR = 0.72, 95% CI: 0.45-1.15, p = 0.17 for progression-free survival) when veliparib was added to carboplatin/paclitaxel. Here we report an exploratory analysis by smoking history. Patients were randomized 2:1 to receive carboplatin/paclitaxel with veliparib, 120 mg (n = 105), or placebo (n = 53). Patients were stratified by histologic subtype and smoking history (recent smokers [n = 95], former smokers [n = 42], and never-smokers [n = 21]). Plasma cotinine level was measured as a chemical index of smoking. Mutation status was assessed by whole exome sequencing (n = 38). Smoking history, histologic subtype, age, Eastern Cooperative Oncology Group performance status, sex, and geographic region predicted veliparib benefit in univariate analyses. In multivariate analysis, history of recent smoking was most predictive for veliparib benefit. Recent smokers treated with veliparib derived significantly greater progression-free survival and overall survival benefits (HR = 0.38 [p Smoking history predicted for efficacy with a veliparib-chemotherapy combination; toxicity was acceptable regardless of smoking history. A prespecified analysis of recent smokers is planned for ongoing phase 3 studies of veliparib in NSCLC. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Modulation of energy homeostasis in maize and Arabidopsis to develop lines tolerant to drought, genotoxic and oxidative stresses

Directory of Open Access Journals (Sweden)

Elizabeth Njuguna

2018-02-01

Full Text Available Abiotic stresses cause crop losses worldwide that reduce the average yield by more than 50%. Due to the high energy consumed to enhance the respiration rates, the excessive reactive oxygen species release provokes cell death and, ultimately, whole plant decay. A metabolic engineering approach in maize (Zea mays altered the expression of two poly(ADP-ribosylation metabolic pathway proteins, poly(ADP-ribose polymerase (PARP and ADP-ribose-specifIc Nudix hydrolase (NUDX genes that play a role in the maintenance of the energy homeostasis during stresses. By means of RNAi hairpin silencing and CRISPR/Cas9 gene editing strategies, the PARP expression in maize was downregulated or knocked down. The Arabidopsis NUDX7 gene and its two maize homologs, ZmNUDX2 and ZmNUDX8, were overexpressed in maize and Arabidopsis. Novel phenotypes were observed, such as significant tolerance to oxidative stress and improved yield in Arabidopsis and a trend of tolerance to mild drought stress in maize and in Arabidopsis. Key words: poly(ADP-ribose polymerase, Nudix hydrolase, CRISPR/Cas9, maize, oxidative stress, drought stress

Raddeanin A induces human gastric cancer cells apoptosis and inhibits their invasion in vitro

International Nuclear Information System (INIS)

Xue, Gang; Zou, Xi; Zhou, Jin-Yong; Sun, Wei; Wu, Jian; Xu, Jia-Li; Wang, Rui-Ping

2013-01-01

Highlights: •Raddeanin A is a triterpenoid saponin in herb medicine Anemone raddeana Regel. •Raddeanin A can inhibit 3 kinds of gastric cancer cells’ proliferation and invasion. •Caspase-cascades’ activation indicates apoptosis induced by Raddeanin A. •MMPs, RECK, Rhoc and E-cad are involved in Raddeanin A-induced invasion inhibition. -- Abstract: Raddeanin A is one of the triterpenoid saponins in herbal medicine Anemone raddeana Regel which was reported to suppress the growth of liver and lung cancer cells. However, little was known about its effect on gastric cancer (GC) cells. This study aimed to investigate its inhibitory effect on three kinds of different differentiation stage GC cells (BGC-823, SGC-7901 and MKN-28) in vitro and the possible mechanisms. Proliferation assay and flow cytometry demonstrated Raddeanin A’s dose-dependent inhibitory effect and determined its induction of cells apoptosis, respectively. Transwell assay, wounding heal assay and cell matrix adhesion assay showed that Raddeanin A significantly inhibited the abilities of the invasion, migration and adhesion of the BGC-823 cells. Moreover, quantitative real time PCR and Western blot analysis found that Raddeanin A increased Bax expression while reduced Bcl-2, Bcl-xL and Survivin expressions and significantly activated caspase-3, caspase-8, caspase-9 and poly-ADP ribose polymerase (PARP). Besides, Raddeanin A could also up-regulate the expression of reversion inducing cysteine rich protein with Kazal motifs (RECK), E-cadherin (E-cad) and down-regulate the expression of matrix metalloproteinases-2 (MMP-2), MMP-9, MMP-14 and Rhoc. In conclusion, Raddeanin A inhibits proliferation of human GC cells, induces their apoptosis and inhibits the abilities of invasion, migration and adhesion, exhibiting potential to become antitumor drug

The Fanconi anemia pathway sensitizes to DNA alkylating agents by inducing JNK-p53-dependent mitochondrial apoptosis in breast cancer cells.

Science.gov (United States)

Zhao, Lin; Li, Yanlin; He, Miao; Song, Zhiguo; Lin, Shu; Yu, Zhaojin; Bai, Xuefeng; Wang, Enhua; Wei, Minjie

2014-07-01

The Fanconi anemia/BRCA (FA/BRCA) DNA damage repair pathway plays a pivotal role in the cellular response to DNA alkylating agents and greatly influences drug response in cancer treatment. However, the molecular mechanisms underlying the FA/BRCA pathway reversed resistance have received limited attention. In the present study, we investigated the effect of Fanconi anemia complementation group F protein (FANCF), a critical factor of the FA/BRCA pathway, on cancer cell apoptosis induced by DNA alkylating agents such as mitomycin c (MMC). We found that FANCF shRNA potentiated MMC-induced cytotoxicity and apoptosis in MCF-7 and MDA-MB-231 breast cancer cells. At a mechanistic level, FANCF shRNA downregulated the anti-apoptotic protein Bcl-2 and upregulated the pro-apoptotic protein Bax, accompanied by release of cyt-c and smac into the cytosol in MMC-treated cells. Furthermore, activation of caspase-3 and -9, other than caspase-8, cleavage of poly(ADP ribose) polymerase (PARP), and a decrease of mitochondrial membrane potential (MMP) indicated that involvement of the mitochondrial apoptotic pathway in FANCF silencing of MMC-treated breast cancer cells. A decrease in IAP family proteins XIAP and survivin were also observed following FANCF silencing in MMC-treated breast cancer cells. Notably, FANCF shRNA was able to increase p53 levels through activation of the JNK pathway in MMC-treated breast cancer cells. Furthermore, p53 inhibition using pifithrin-α abolished the induction of caspase-3 and PARP by FANCF shRNA and MMC, indicating that MMC-induced apoptosis is substantially enhanced by FANCF shRNA via p53-dependent mechanisms. To our knowledge, we provide new evidence for the potential application of FANCF as a chemosensitizer in breast cancer therapy.

Two modes of cell death caused by exposure to nanosecond pulsed electric field.

Directory of Open Access Journals (Sweden)

Olga N Pakhomova

Full Text Available High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF, are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity ("nanoelectroporation", leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1-2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6-24 hr post nsPEF. These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.

The microRNA-302b-inhibited insulin-like growth factor-binding protein 2 signaling pathway induces glioma cell apoptosis by targeting nuclear factor IA.

Directory of Open Access Journals (Sweden)

Chin-Cheng Lee

Full Text Available MicroRNAs are small noncoding RNAs that post-transcriptionally control the expression of genes involved in glioblastoma multiforme (GBM development. Although miR-302b functions as a tumor suppressor, its role in GBM is still unclear. Therefore, this study comprehensively explored the roles of miR-302b-mediated gene networks in GBM cell death. We found that miR-302b levels were significantly higher in primary astrocytes than in GBM cell lines. miR-302b overexpression dose dependently reduced U87-MG cell viability and induced apoptosis through caspase-3 activation and poly(ADP ribose polymerase degradation. A transcriptome microarray revealed 150 downregulated genes and 380 upregulated genes in miR-302b-overexpressing cells. Nuclear factor IA (NFIA, higher levels of which were significantly related to poor survival, was identified as a direct target gene of miR-302b and was involved in miR-302b-induced glioma cell death. Higher NFIA levels were observed in GBM cell lines and human tumor sections compared with astrocytes and non-tumor tissues, respectively. NFIA knockdown significantly enhanced apoptosis. We found high levels of insulin-like growth factor-binding protein 2 (IGFBP2, another miR-302b-downregulated gene, in patients with poor survival. We verified that NFIA binds to the IGFBP2 promoter and transcriptionally enhances IGFBP2 expression levels. We identified that NFIA-mediated IGFBP2 signaling pathways are involved in miR-302b-induced glioma cell death. The identification of a regulatory loop whereby miR-302b inhibits NFIA, leading to a decrease in expression of IGFBP-2, may provide novel directions for developing therapies to target glioblastoma tumorigenesis.

Raddeanin A induces human gastric cancer cells apoptosis and inhibits their invasion in vitro

Energy Technology Data Exchange (ETDEWEB)

Xue, Gang [Department of Oncology, Nanjing University of Chinese Medicine, Nanjing (China); Zou, Xi [Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing (China); Zhou, Jin-Yong [Laboratory Center, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing (China); Sun, Wei [Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing (China); Wu, Jian [Laboratory Center, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing (China); Xu, Jia-Li [Department of Oncology, Nanjing University of Chinese Medicine, Nanjing (China); Wang, Rui-Ping, E-mail: ruipingwang61@hotmail.com [Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing (China)

2013-09-20

Highlights: •Raddeanin A is a triterpenoid saponin in herb medicine Anemone raddeana Regel. •Raddeanin A can inhibit 3 kinds of gastric cancer cells’ proliferation and invasion. •Caspase-cascades’ activation indicates apoptosis induced by Raddeanin A. •MMPs, RECK, Rhoc and E-cad are involved in Raddeanin A-induced invasion inhibition. -- Abstract: Raddeanin A is one of the triterpenoid saponins in herbal medicine Anemone raddeana Regel which was reported to suppress the growth of liver and lung cancer cells. However, little was known about its effect on gastric cancer (GC) cells. This study aimed to investigate its inhibitory effect on three kinds of different differentiation stage GC cells (BGC-823, SGC-7901 and MKN-28) in vitro and the possible mechanisms. Proliferation assay and flow cytometry demonstrated Raddeanin A’s dose-dependent inhibitory effect and determined its induction of cells apoptosis, respectively. Transwell assay, wounding heal assay and cell matrix adhesion assay showed that Raddeanin A significantly inhibited the abilities of the invasion, migration and adhesion of the BGC-823 cells. Moreover, quantitative real time PCR and Western blot analysis found that Raddeanin A increased Bax expression while reduced Bcl-2, Bcl-xL and Survivin expressions and significantly activated caspase-3, caspase-8, caspase-9 and poly-ADP ribose polymerase (PARP). Besides, Raddeanin A could also up-regulate the expression of reversion inducing cysteine rich protein with Kazal motifs (RECK), E-cadherin (E-cad) and down-regulate the expression of matrix metalloproteinases-2 (MMP-2), MMP-9, MMP-14 and Rhoc. In conclusion, Raddeanin A inhibits proliferation of human GC cells, induces their apoptosis and inhibits the abilities of invasion, migration and adhesion, exhibiting potential to become antitumor drug.

Nicotinamide starvation and inhibition of poly(ADP-Ribose) synthesis enhance the induced mutation in Chinese hamster V79 cells

International Nuclear Information System (INIS)

Okada, Gensaku; Kaneko, Ichiro; Mitsui, Hideki.

1987-01-01

The effects of nicotinamide (NA) deficiency and added NA and 3-aminobenzamide (3AB) on the cytotoxicity and the induction of mutations in Chinese hamster V79-14 cells were investigated. In NA deficiency the addition of NA (up to 4 mM) and 3AB (up to 7.5 mM) was not cytotoxic. The presence of NA prior to exposure to mitomycin C (MMC) or γ-rays produced a dose-dependent increase in the relative cloning ability of DNA-damaged cells. The lethality of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was significantly potentiated by pre-treatment with 5 mM 3AB, but no potentiation by 3AB was observed for MMC, ultraviolet (UV)-B light, or γ-rays. Among cells pre-cultured in NA-free medium there were increased frequencies of mutations at both the hypoxanthineguanine phosphoribosyltransferase (HGPRT) and the adenine phosphoribosyltransferase (APRT) loci following DNA damage. The enhancing effect by NA deficiency was time-dependent. Incubation with NA prior to DNA damage produced a significant reduction in the frequency of mutations. The addition of 3AB to the nicotinamide adenine dinucleotide (NAD + )-depleted cell cultures before or after the DNA damage also strongly increased the frequency of induced mutations, with increasing concentrations of 3AB up to 5 mM, but the frequency was reduced at higher concentrations. The interaction between NA deficiency and the addition of 3AB appears to act synergistically on mutation induction. A correlation was observed between the potential of inhibiting poly (ADP-ribose) polymerase and the enhancement of mutation frequency. (author)

Ribose catabolism of Escherichia coli: characterization of the rpiB gene encoding ribose phosphate isomerase B and of the rpiR gene, which is involved in regulation of rpiB expression

DEFF Research Database (Denmark)

Sørensen, Kim I.; Hove-Jensen, Bjarne

1996-01-01

. The rpiB gene resided on a 4.6-kbp HindIII-EcoRV DNA fragment from phage lambda 10H5 (642) of the Kohara gene library and mapped at 92.85 min. Consistent with this map position, the cloned DNA fragment contained two divergent open reading frames of 149 and 296 codons, encoding ribose phosphate isomerase B...

A Novel, Highly Selective Inhibitor of Pestivirus Replication That Targets the Viral RNA-Dependent RNA Polymerase

Science.gov (United States)

Paeshuyse, Jan; Leyssen, Pieter; Mabery, Eric; Boddeker, Nina; Vrancken, Robert; Froeyen, Matheus; Ansari, Israrul H.; Dutartre, Hélène; Rozenski, Jef; Gil, Laura H. V. G.; Letellier, Carine; Lanford, Robert; Canard, Bruno; Koenen, Frank; Kerkhofs, Pierre; Donis, Ruben O.; Herdewijn, Piet; Watson, Julia; De Clercq, Erik; Puerstinger, Gerhard; Neyts, Johan

2006-01-01

We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 ± 0.01 μM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 ± 0.02 μM) and production of infectious virus (EC50 = 0.074 ± 0.003 μM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was ∼2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed. PMID:16352539

Apoptotic cell death through inhibition of protein kinase CKII activity by 3,4-dihydroxybenzaldehyde purified from Xanthium strumarium.

Science.gov (United States)

Lee, Bang Hyo; Yoon, Soo-Hyun; Kim, Yun-Sook; Kim, Sang Kook; Moon, Byong Jo; Bae, Young-Seuk

2008-01-01

The CKII inhibitory compound was purified from the fruit of Xanthium strumarium by organic solvent extraction and silica gel chromatography. The inhibitory compound was identified as 3,4-dihydroxybenzaldehyde by analysis with FT-IR, FAB-Mass, EI-Mass, (1)H-NMR and (13)C-NMR. 3,4-dihydroxybenzaldehyde inhibited the phosphotransferase activity of CKII with IC(50) of about 783 microM. Steady-state studies revealed that the inhibitor acts as a competitive inhibitor with respect to the substrate ATP. A value of 138.6 microM was obtained for the apparent K(i). Concentration of 300 microM 3,4-dihydroxybenzaldehyde caused 50% growth inhibition of human cancer cell U937. 3,4-dihydroxybenzaldehyde-induced cell death was characterised with the cleavage of poly(ADP-ribose) polymerase and procaspase-3. Furthermore, the inhibitor induced the fragmentation of DNA into multiples of 180 bp, indicating that it triggered apoptosis. This induction of apoptosis by 3,4-dihydroxybenzaldehyde was also confirmed by using flow cytometry analysis. Since CKII is involved in cell proliferation and oncogenesis, these results suggest that 3,4-dihydroxybenzaldehyde may function by inhibiting oncogenic disease, at least in part, through the inhibition of CKII activity.

NAD+-Dependent Deacetylase Hst1p Controls Biosynthesis and Cellular NAD+ Levels in Saccharomyces cerevisiae

OpenAIRE

Bedalov, Antonio; Hirao, Maki; Posakony, Jeffrey; Nelson, Melisa; Simon, Julian A.

2003-01-01

Nicotine adenine dinucleotide (NAD+) performs key roles in electron transport reactions, as a substrate for poly(ADP-ribose) polymerase and NAD+-dependent protein deacetylases. In the latter two processes, NAD+ is consumed and converted to ADP-ribose and nicotinamide. NAD+ levels can be maintained by regeneration of NAD+ from nicotinamide via a salvage pathway or by de novo synthesis of NAD+ from tryptophan. Both pathways are conserved from yeast to humans. We describe a critical role of the ...

Inhibition of Fatty Acid Synthesis Induces Apoptosis of Human Pancreatic Cancer Cells.

Science.gov (United States)

Nishi, Koji; Suzuki, Kenta; Sawamoto, Junpei; Tokizawa, Yuma; Iwase, Yumiko; Yumita, Nagahiko; Ikeda, Toshihiko

2016-09-01

Cancer cells tend to have a high requirement for lipids, including fatty acids, cholesterol and triglyceride, because of their rapid proliferative rate compared to normal cells. In this study, we investigated the effects of inhibition of lipid synthesis on the proliferation and viability of human pancreatic cancer cells. Of the inhibitors of lipid synthesis that were tested, 5-(tetradecyloxy)-2-furoic acid (TOFA), which is an inhibitor of acetyl-CoA carboxylase, and the fatty acid synthase (FAS) inhibitors cerulenin and irgasan, significantly suppressed the proliferation of MiaPaCa-2 and AsPC-1 cells. Treatment of MiaPaCa-2 cells with these inhibitors significantly increased the number of apoptotic cells. In addition, TOFA increased caspase-3 activity and induced cleavage of poly (ADP-ribose) polymerase in MiaPaCa-2 cells. Moreover, addition of palmitate to MiaPaCa-2 cells treated with TOFA rescued cells from apoptotic cell death. These results suggest that TOFA induces apoptosis via depletion of fatty acids and that, among the various aspects of lipid metabolism, inhibition of fatty acid synthesis may be a notable target for the treatment of human pancreatic cancer cells. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells

Energy Technology Data Exchange (ETDEWEB)

Jones, J.C.; Stevsner, Tinna; Bohr, Vilhelm A. (National Cancer Institute, NIH, Bethesda, MD (USA). Division of Cancer Treatment, Laboratory of Molecular Pharmacology); Mattern, M.R. (Smith Kline Beecham Pharmaceuticals, King of Prussia, PA (USA). Department of Biomolecular Discovery)

1991-09-01

The effects were studied of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. The inhibitors were tested of DNA poly-merase {alpha} and {delta} (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topo-isomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, the effects were tested of the potential topoisomerase I activator, {beta}-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; {beta}-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair. (author). 36 refs.; 3 figs.; 2 tabs.

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

Science.gov (United States)

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

2013-01-01

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

Functional roles of DNA polymerases β and γ

International Nuclear Information System (INIS)

Huebscher, U.; Kuenzle, C.C.; Spadari, S.

1979-01-01

The physiological functions of DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC2.7.7.7)β and γ were investigated by using neuronal nuclei and synaptosomes isolated from rat brain. uv irradiation of neuronal nuclei from 60-day-old rats resulted in a 7- to 10-fold stimulation of DNA repair synthesis attributable to DNA polymerase β which, at this developmental stage, is virtually the only DNA polymerase present in the nuclei. No repair synthesis could be elicited by treating the nuclei with N-methyl-N-nitrosourea, but this was probably due to the inability of brain tissue to excise alkylated bases from DNA. The role of DNA polymerase γ was studied in synaptosomes by using a system mimicking in vivo mitochondrial DNA synthesis. By showing that under these conditions, DNA replication occurs in miatochondria, and exploiting the fact that DNA polymerase γ is the only DNA polymerase present in mitochondria, evidence was obtained for a role of DNA polymerase γ in mitochondrial DNA replication. Based on these results and on the wealth of literature on DNA polymerase α, we conclude that DNA polymerase α is mainly responsible for DNA replication in nuclei, DNA polymerase β is involved in nuclear DNA repair, and DNA polymerase γ is the mitochondrial replicating enzyme. However, minor roles for DNA polymerase α in DNA repair or for DNA polymerase β in DNA replication cannot be excluded

Reference quantum chemical calculations on RNA base pairs directly involving the 2'-OH group of ribose

Czech Academy of Sciences Publication Activity Database

Šponer, Jiří; Zgarbová, M.; Jurečka, Petr; Riley, K.E.; Šponer, Judit E.; Hobza, Pavel

2009-01-01

Roč. 5, č. 4 (2009), s. 1166-1179 ISSN 1549-9618 R&D Projects: GA AV ČR(CZ) IAA400040802; GA AV ČR(CZ) IAA400550701; GA MŠk(CZ) LC06030; GA MŠk(CZ) LC512 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702; CEZ:AV0Z40550506 Keywords : RNA * ribose * quantum calculations Subject RIV: BO - Biophysics Impact factor: 4.804, year: 2009

DNA polymerase preference determines PCR priming efficiency.

Science.gov (United States)

Pan, Wenjing; Byrne-Steele, Miranda; Wang, Chunlin; Lu, Stanley; Clemmons, Scott; Zahorchak, Robert J; Han, Jian

2014-01-30

Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially

Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

OpenAIRE

Davidson, John F.; Fox, Richard; Harris, Dawn D.; Lyons-Abbott, Sally; Loeb, Lawrence A.

2003-01-01

Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20–50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq...

Synthesis, conformational analysis, and biological activity of new analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) as IMP dehydrogenase inhibitors.

Science.gov (United States)

Franchetti, Palmarisa; Cappellacci, Loredana; Pasqualini, Michela; Petrelli, Riccardo; Jayaprakasan, Vetrichelvan; Jayaram, Hiremagalur N; Boyd, Donald B; Jain, Manojkumar D; Grifantini, Mario

2005-03-15

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogues T-2'-MeAD (1) and T-3'-MeAD (2) containing, respectively, a methyl group at the ribose 2'-C-, and 3'-C-position of the adenosine moiety, were prepared as potential selective human inosine monophosphate dehydrogenase (IMPDH) type II inhibitors. The synthesis of heterodinucleotides was carried out by CDI-catalyzed coupling reaction of unprotected 2'-C-methyl- or 3'-C-methyl-adenosine 5'-monophosphate with 2',3'-O-isopropylidene-tiazofurin 5'-monophosphate, and then deisopropylidenation. Biological evaluation of dinucleotides 1 and 2 as inhibitors of recombinant human IMPDH type I and type II resulted in a good activity. Inhibition of both isoenzymes by T-2'-MeAD and T-3'-MeAD was noncompetitive with respect to NAD substrate. Binding of T-3'-MeAD was comparable to that of parent compound TAD, while T-2'-MeAD proved to be a weaker inhibitor. However, no significant difference was found in inhibition of the IMPDH isoenzymes. T-2'-MeAD and T-3'-MeAD were found to inhibit the growth of K562 cells (IC(50) 30.7 and 65.0muM, respectively).

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

Energy Technology Data Exchange (ETDEWEB)

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

1983-08-10

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

Lifelong endurance training attenuates age-related genotoxic stress in human skeletal muscle

OpenAIRE

Cobley, James N; Sakellariou, George K; Murray, Scott; Waldron, Sarah; Gregson, Warren; Burniston, Jatin G; Morton, James P; Iwanejko, Lesley A; Close, Graeme L

2013-01-01

Background The aim of the present study was to determine the influence of age and habitual activity level, at rest and following a single bout of high-intensity exercise, on the levels of three proteins poly(ADP-ribose) polymerase-1 (PARP-1), cleaved-PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), involved in the DNA repair and cell death responses to stress and genotoxic insults. Muscle biopsies were obtained from the vastus lateralis of young trained (22 ± 3 years, n = 6), young untraine...

Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

DEFF Research Database (Denmark)

Willemoës, Martin; Hove-Jensen, Bjarne

1997-01-01

The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir

NARCIS (Netherlands)

Sarrazin, Christoph; Kieffer, Tara L.; Bartels, Doug; Hanzelka, Brian; Müh, Ute; Welker, Martin; Wincheringer, Dennis; Zhou, Yi; Chu, Hui-May; Lin, Chao; Weegink, Christine; Reesink, Henk; Zeuzem, Stefan; Kwong, Ann D.

2007-01-01

BACKGROUND & AIMS: Telaprevir (VX-950), a hepatitis C virus (HCV) NS3.4A protease inhibitor, has shown strong antiviral activity in phase 1 clinical studies. Because of high levels of HCV replication and the low fidelity of HCV polymerase, selection of resistant isolates during therapy may occur.

Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta.

Science.gov (United States)

Sarkar, Sailendra Nath; Bakshi, Sankar; Mokkapati, Sanath K; Roy, Sujit; Sengupta, Dibyendu N

2004-07-16

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.

Toward a unified nomenclature for mammalian ADP-ribosyltransferases.

Science.gov (United States)

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

2010-04-01

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

Towards producing novel fish gelatin films by combination treatments of ultraviolet radiation and sugars (ribose and lactose) as cross-linking agents.

Science.gov (United States)

Bhat, Rajeev; Karim, A A

2014-07-01

Developing novel fish gelatin films with better mechanical properties than mammalian gelatin is a challenging but promising endeavor. Studies were undertaken to produce fish gelatin films by combining treatments with different sugars (ribose and lactose) followed 'by' 'and' ultraviolet (UV) radiation, as possible cross-linking agents. Increase in tensile strength and percent elongation at break was recorded, which was more significant in films without sugars that were exposed to UV radiation. Films with added ribose showed decreased solubility after UV treatment and exhibited higher swelling percentage than films with added lactose, which readily dissolved in water. FTIR spectra of all the films showed identical patterns, which indicated no major changes to have occurred in the functional groups as a result of interaction between gelatin, sugars and UV irradiation. The results of this study could be explored for commercial use, depending on industrial needs for either production of edible films or for food packaging purposes.

High-efficiency liposomal encapsulation of a tyrosine kinase inhibitor leads to improved in vivo toxicity and tumor response profile

Directory of Open Access Journals (Sweden)

Mukthavaram R

2013-10-01

Full Text Available Rajesh Mukthavaram,1 Pengfei Jiang,1 Rohit Saklecha,1 Dmitri Simberg,3,4 Ila Sri Bharati,1 Natsuko Nomura,1 Ying Chao,1 Sandra Pastorino,1 Sandeep C Pingle,1 Valentina Fogal,1 Wolf Wrasidlo,1,2 Milan Makale,1,2 Santosh Kesari1,21Translational Neuro-Oncology Laboratories, 2Department of Neurosciences, 3Solid Tumor Therapeutics Program, Moores Cancer Center, UC San Diego, La Jolla, CA, 4Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Denver, CO, USAAbstract: Staurosporine (STS is a potent pan-kinase inhibitor with marked activity against several chemotherapy-resistant tumor types in vitro. The translational progress of this compound has been hindered by poor pharmacokinetics and toxicity. We sought to determine whether liposomal encapsulation of STS would enhance antitumor efficacy and reduce toxicity, thereby supporting the feasibility of further preclinical development. We developed a novel reverse pH gradient liposomal loading method for STS, with an optimal buffer type and drug-to-lipid ratio. Our approach produced 70% loading efficiency with good retention, and we provide, for the first time, an assessment of the in vivo antitumor activity of STS. A low intravenous dose (0.8 mg/kg inhibited U87 tumors in a murine flank model. Biodistribution showed preferential tumor accumulation, and body weight data, a sensitive index of STS toxicity, was unaffected by liposomal STS, but did decline with the free compound. In vitro experiments revealed that liposomal STS blocked Akt phosphorylation, induced poly(ADP-ribose polymerase cleavage, and produced cell death via apoptosis. This study provides a basis to explore further the feasibility of liposomally encapsulated STS, and potentially related compounds for the management of resistant solid tumors.Keywords: liposomes, staurosporine, glioblastoma, biodistribution, efficacy

In Vitro and In Vivo Enhancement of Chemoradiation Using the Oral PARP Inhibitor ABT-888 in Colorectal Cancer Cells

Energy Technology Data Exchange (ETDEWEB)

Shelton, Joseph W., E-mail: jwshelt@emory.edu [Department of Radiation Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Waxweiler, Timothy V.; Landry, Jerome; Gao, Huiying; Xu, Yanbo; Wang, Lanfang [Department of Radiation Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); El-Rayes, Bassel [Department of Hematology and Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Shu, Hui-Kuo G. [Department of Radiation Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States)

2013-07-01

Purpose: Poly(ADP-ribose) polymerase plays a critical role in the recognition and repair of DNA single-strand breaks and double-strand breaks (DSBs). ABT-888 is an orally available inhibitor of this enzyme. This study seeks to evaluate the use of ABT-888 combined with chemotherapy and radiation therapy (RT) in colorectal carcinoma models. Methods and Materials: RT clonogenic assays were performed on HCT116 and HT29 cells treated with 5-fluorouracil, irinotecan, or oxaliplatin with or without ABT. The surviving fraction at 2 Gy and dose-modifying factor at 10% survival were analyzed. Synergism was assessed by isobologram analysis for combination therapies. γH2AX and neutral comet assays were performed to assess the effect of therapy on DSB formation/repair. In vivo assessments were made by use of HCT116 cells in a xenograft mouse model. Tumor growth delay was measured at a volume of 500 mm{sup 3}. Results: Both lines were radiosensitized by ABT alone, and ABT further increased chemotherapy dose-modifying factors to the 1.6 to 1.8 range. All combinations were synergistic (combination indices <0.9). ABT treatment significantly increased DSB after RT (γH2AX, 69% vs 43%; P=.017) and delayed repair. We found tumor growth delays of 7.22 days for RT; 11.90 days for RT and ABT; 13.5 days for oxaliplatin, RT, and ABT; 14.17 days for 5-fluorouracil, RT, and ABT; and 23.81 days for irinotecan, RT, and ABT. Conclusion: ABT-888 radiosensitizes at similar or higher levels compared with classic chemotherapies and acts synergistically with these chemotherapies to enhance RT effects. In vivo confirmation of these results indicates a potential role for combining its use with existing chemoradiation regimens.

SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

Science.gov (United States)

Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J.; Cowling, Victoria H.; Cairns, Bradley R.; White, Robert J.

2015-01-01

Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription. PMID:25798578

Evaluation of Apoptotic and Growth Inhibitory Activity of Phloretin in ...

African Journals Online (AJOL)

Nuclear Magnetic Resonance (NMR), 13C-NMR and electrospray ionization tandem ... this effectively induced cleavage of anti-poly (ADP-ribose) polymerase (PARP) as well as downregulation of Bcl2 protein expression in BGC823 cells after 24 h ...

Alteration in the Nuclear Structure of Breast Cancer Cells in Response to ECM Signaling

National Research Council Canada - National Science Library

Han, Hye-Jung

2001-01-01

.... We have previously identified a MAR binding protein of 1 14kDa from malignant breast carcinomas. The p114 MAR-binding activity was found to be attributed to two separate proteins, poly(ADP-ribose) polymerase (PARP) and SAF...

The expanding polymerase universe.

Science.gov (United States)

Goodman, M F; Tippin, B

2000-11-01

Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded. Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread. The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.

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.

The Development of FVIII Inhibitors in Relation to IL10 Gene Polymorphism in Hemophilia A Egyptian Pediatric Patients.

Science.gov (United States)

Sadek, Hoda; Youssry, Ilham; Ibrahim, Nihal Salah Eldeen; Abou-Elalla, Amany Ahmed; Atef, Gehad; Mousa, Somaia Mohammed

2017-06-01

Development of inhibitors against Factor VIII (FVIII) in hemophilia A patients is a serious complication of therapy. Many cytokines, including interleukin-10 (IL10), may affect inhibitor development; however, literature data are not sufficient to prove this association. The aim of this study was to investigate the relation between FVIII inhibitor formation and IL10-1082A/G polymorphism among Egyptian hemophiliacs. Patients were screened for FVIII inhibitors using the Bethesda method. IL10-1082A/G polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism. Six patients (12%) developed inhibitors. No statistically significant difference was found between inhibitor positive and negative patients regarding IL10-1082A/G genotypes, disease severity, or treatment-related variables (type of FVIII received, treatment regimen, age at first exposure to FVIII, and frequency of replacement therapy). FVIII inhibitor formation in this group of Egyptian hemophiliacs was not correlated to IL10-1082A/G polymorphism, disease severity, or any of the treatment variables.

Inhibitory effect of gold nanoparticles on the D-ribose glycation of bovine serum albumin

Directory of Open Access Journals (Sweden)

Liu W

2014-11-01

Full Text Available Weixi Liu,1 Menashi A Cohenford,1–3 Leslie Frost,3 Champika Seneviratne,4 Joel A Dain1 1Department of Chemistry, University of Rhode Island, Kingston, RI, USA; 2Department of Integrated Science and Technology, 3Department of Chemistry, Marshall University, Huntington, WV, USA; 4Department of Chemistry, College of the North Atlantic, Labrador, NL, Canada Abstract: Formation of advanced glycation end products (AGEs by nonenzymatic glycation of proteins is a major contributory factor to the pathophysiology of diabetic conditions including senile dementia and atherosclerosis. This study describes the inhibitory effect of gold nanoparticles (GNPs on the D-ribose glycation of bovine serum albumin (BSA. A combination of analytical methods including ultraviolet–visible spectrometry, high performance liquid chromatography, circular dichroism, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometry were used to determine the extent of BSA glycation in the presence of citrate reduced spherical GNPs of various sizes and concentrations. GNPs of particle diameters ranging from 2 nm to 20 nm inhibited BSA’s AGE formation. The extent of inhibition correlated with the total surface area of the nanoparticles. GNPs of highest total surface area yielded the most inhibition whereas those with the lowest total surface area inhibited the formation of AGEs the least. Additionally, when GNPs’ total surface areas were set the same, their antiglycation activities were similar. This inhibitory effect of GNPs on BSA’s glycation by D-ribose suggests that colloidal particles may have a therapeutic application for the treatment of diabetes and conditions that promote hyperglycemia. Keywords: gold nanoparticles, glycation, AGEs, GNPs, BSA

Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols.

Science.gov (United States)

Angers, M; Cloutier, J F; Castonguay, A; Drouin, R

2001-08-15

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relative efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo(-) proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo(-), while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo(-) at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.

Optimization of Maillard reaction with ribose for enhancing anti-allergy effect of fish protein hydrolysates using response surface methodology.

Science.gov (United States)

Yang, Sung-Yong; Kim, Se-Wook; Kim, Yoonsook; Lee, Sang-Hoon; Jeon, Hyeonjin; Lee, Kwang-Won

2015-06-01

Halibut is served on sushi and as sliced raw fish fillets. We investigated the optimal conditions of the Maillard reaction (MR) with ribose using response surface methodology to reduce the allergenicity of its protein. A 3-factored and 5-leveled central composite design was used, where the independent variables were substrate (ribose) concentration (X1, %), reaction time (X2, min), and pH (X3), while the dependent variables were browning index (Y1, absorbance at 420nm), DPPH scavenging (Y2, EC50 mg/mL), FRAP (Y3, mM FeSO4/mg extract) and β-hexosaminidase release (Y4, %). The optimal conditions were obtained as follows: X1, 28.36%; X2, 38.09min; X3, 8.26. Maillard reaction products of fish protein hydrolysate (MFPH) reduced the amount of nitric oxide synthesis compared to the untreated FPH, and had a significant anti-allergy effect on β-hexosaminidase and histamine release, compared with that of the FPH control. We concluded that MFPH, which had better antioxidant and anti-allergy activities than untreated FPH, can be used as an improved dietary source. Copyright © 2014 Elsevier Ltd. All rights reserved.

Advancing Polymerase Ribozymes Towards Self-Replication

Science.gov (United States)

Tjhung, K. F.; Joyce, G. F.

2017-07-01

Autocatalytic replication and evolution in vitro by (i) a cross-chiral RNA polymerase catalyzing polymerization of mononucleotides of the opposite handedness; (ii) non-covalent assembly of component fragments of an existing RNA polymerase ribozyme.

Auranofin induces apoptosis and necrosis in HeLa cells via oxidative stress and glutathione depletion.

Science.gov (United States)

You, Bo Ra; Shin, Hye Rim; Han, Bo Ram; Kim, Suhn Hee; Park, Woo Hyun

2015-02-01

Auranofin (Au), an inhibitor of thioredoxin reductase, is a known anti‑cancer drug. In the present study, the anti‑growth effect of Au on HeLa cervical cancer cells was examined in association with levels of reactive oxygen species (ROS) and glutathione (GSH). Au inhibited the growth of HeLa cells with an IC50 of ~2 µM at 24 h. This agent induced apoptosis and necrosis, accompanied by the cleavage of poly (ADP‑ribose) polymerase and loss of mitochondrial membrane potential. The pan‑caspase inhibitor, benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone, prevented apoptotic cell death and each of the assessed caspase inhibitors inhibited necrotic cell death induced by Au. With respect to the levels of ROS and GSH, Au increased intracellular O2•- in the HeLa cells and induced GSH depletion. The pan‑caspase inhibitor reduced the levels of O2•- and GSH depletion in Au‑treated HeLa cells. The antioxidant, N‑acetyl cysteine, not only attenuated apoptosis and necrosis in the Au‑treated HeLa cells, but also decreased the levels of O2•- and GSH depletion in the cells. By contrast, L‑buthionine sulfoximine, a GSH synthesis inhibitor, intensified cell death O2•- and GSH depletion in the Au‑treated HeLa cells. In conclusion, Au induced apoptosis and necrosis in HeLa cells via the induction of oxidative stress and the depletion of GSH.

The HRD Decision-Which PARP Inhibitor to Use for Whom and When.

Science.gov (United States)

Kohn, Elise C; Lee, Jung-Min; Ivy, S Percy

2017-12-01

Rucaparib, a polyADPribose polymerase inhibitor (PARPi), was approved recently for use in women with high-grade serous ovarian cancer (HGSOC). It is now one of three approved PARPi for use in recurrent ovarian cancer, a family of agents that has changed the HGSOC treatment landscape and outcome. Clin Cancer Res; 23(23); 7155-7. ©2017 AACR See related article by Balasubramaniam et al., p. 7165 . ©2017 American Association for Cancer Research.

Regulation of chromatin structure by poly(ADP-ribosylation

Directory of Open Access Journals (Sweden)

Sascha eBeneke

2012-09-01

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

DNA polymerase zeta cooperates with polymerases kappa and iota in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients.

Science.gov (United States)

Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

2009-07-14

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase eta, the POLH gene product. A deficiency in DNA polymerase eta due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase zeta cooperates with DNA polymerases kappa and iota to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases zeta and kappa, but not iota, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.

DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

Science.gov (United States)

Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

2009-01-01

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase η, the POLH gene product. A deficiency in DNA polymerase η due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase ζ cooperates with DNA polymerases κ and ι to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases ζ and κ, but not ι, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load. PMID:19564618

RNA binding and replication by the poliovirus RNA polymerase

International Nuclear Information System (INIS)

Oberste, M.S.

1988-01-01

RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to 32 P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K a for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 x 10 9 M -1 . The polymerase binds to a subgenomic RNAs which contain the 3' end of the genome with a K a similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3' noncoding region

Synthesis of Nucleosides through Direct Glycosylation of Nucleobases with 5-O-Monoprotected or 5-Modified Ribose: Improved Protocol, Scope, and Mechanism

Czech Academy of Sciences Publication Activity Database

Downey, Alan Michael; Pohl, Radek; Roithová, J.; Hocek, Michal

2017-01-01

Roč. 23, č. 16 (2017), s. 3910-3917 ISSN 0947-6539 R&D Projects: GA TA ČR(CZ) TE01020028; GA ČR(CZ) GA16-00178S Institutional support: RVO:61388963 Keywords : epoxides * glycosylation * nucleosides * riboses * synthesis design Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 5.317, year: 2016

Prediction of Active Site and Distal Residues in E. coli DNA Polymerase III alpha Polymerase Activity.

Science.gov (United States)

Parasuram, Ramya; Coulther, Timothy A; Hollander, Judith M; Keston-Smith, Elise; Ondrechen, Mary Jo; Beuning, Penny J

2018-02-20

The process of DNA replication is carried out with high efficiency and accuracy by DNA polymerases. The replicative polymerase in E. coli is DNA Pol III, which is a complex of 10 different subunits that coordinates simultaneous replication on the leading and lagging strands. The 1160-residue Pol III alpha subunit is responsible for the polymerase activity and copies DNA accurately, making one error per 10 5 nucleotide incorporations. The goal of this research is to determine the residues that contribute to the activity of the polymerase subunit. Homology modeling and the computational methods of THEMATICS and POOL were used to predict functionally important amino acid residues through their computed chemical properties. Site-directed mutagenesis and biochemical assays were used to validate these predictions. Primer extension, steady-state single-nucleotide incorporation kinetics, and thermal denaturation assays were performed to understand the contribution of these residues to the function of the polymerase. This work shows that the top 15 residues predicted by POOL, a set that includes the three previously known catalytic aspartate residues, seven remote residues, plus five previously unexplored first-layer residues, are important for function. Six previously unidentified residues, R362, D405, K553, Y686, E688, and H760, are each essential to Pol III activity; three additional residues, Y340, R390, and K758, play important roles in activity.

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.

Science.gov (United States)

Freemont, P S; Ollis, D L; Steitz, T A; Joyce, C M

1986-09-01

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

Induction of intrachromosomal homologous recombination in whole plants

International Nuclear Information System (INIS)

Puchta, H.; Swoboda, P.; Hohn, B.

1995-01-01

The influence of different factors on frequencies of intrachromosomal homologous recombination in whole Arabidopsis thaliana and tobacco plants was analyzed using a disrupted β-glucuronidase marker gene. Recombination frequencies were enhanced several fold by DNA damaging agents like UV-light or MMS (methyl methanesulfonate). Applying 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP)ribose polymerase (PARP), an enzyme that is postulated to be involved in DNA repair, enhanced homologous recombination frequencies strongly. These findings indicate that homologous recombination is involved in DNA repair and can (at least partially) compensate for other DNA repair pathways. Indications that recombination in plants can be induced by environmental stress factors that are not likely to be involved in DNA metabolism were also found; Arabidopsis plants growing in a medium containing 0.1 M NaCl exhibited elevated recombination frequencies. The possible general effects of ‘environmental’ challenges on genome flexibility are discussed. (author)

Ubiquitin-dependent system controls radiation induced apoptosis

International Nuclear Information System (INIS)

Delic, J.; Magdelenat, H.; Glaisner, S.; Magdelenat, H.; Maciorowski, Z.

1997-01-01

The selective proteolytic pathway, dependent upon 'N-end rule' protein recognition/ubiquitination and on the subsequent proteasome dependent processing of ubiquitin conjugates, operates in apoptosis induced by γ-irradiation. The proteasome inhibitor peptide aldehyde, MG132, efficiently induced apoptosis and was also able (at doses lower than those required for apoptosis induction) to potentiate apoptosis induced by DNA damage. Its specificity is suggested by the induction of the ubiquitin (UbB and UbC) and E1 (ubiquitin activating enzyme) genes and by an altered ubiquitination pattern. More selectively, a di-peptide competitor of the 'N-end rule' of ubiquitin dependent protein processing inhibited radiation induced apoptosis. This inhibition is also followed by an altered ubiquitination pattern and by activation of Poly (ADP-ribose) polymerase (PARP). These data strongly suggest that early apoptosis radiation induced events are controlled by ubiquitin-dependent proteolytic processing. (author)

Evolving a polymerase for hydrophobic base analogues.

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Loakes, David; Gallego, José; Pinheiro, Vitor B; Kool, Eric T; Holliger, Philipp

2009-10-21

Hydrophobic base analogues (HBAs) have shown great promise for the expansion of the chemical and coding potential of nucleic acids but are generally poor polymerase substrates. While extensive synthetic efforts have yielded examples of HBAs with favorable substrate properties, their discovery has remained challenging. Here we describe a complementary strategy for improving HBA substrate properties by directed evolution of a dedicated polymerase using compartmentalized self-replication (CSR) with the archetypal HBA 5-nitroindole (d5NI) and its derivative 5-nitroindole-3-carboxamide (d5NIC) as selection substrates. Starting from a repertoire of chimeric polymerases generated by molecular breeding of DNA polymerase genes from the genus Thermus, we isolated a polymerase (5D4) with a generically enhanced ability to utilize HBAs. The selected polymerase. 5D4 was able to form and extend d5NI and d5NIC (d5NI(C)) self-pairs as well as d5NI(C) heteropairs with all four bases with efficiencies approaching, or exceeding, those of the cognate Watson-Crick pairs, despite significant distortions caused by the intercalation of the d5NI(C) heterocycles into the opposing strand base stack, as shown by nuclear magnetic resonance spectroscopy (NMR). Unlike Taq polymerase, 5D4 was also able to extend HBA pairs such as Pyrene: varphi (abasic site), d5NI: varphi, and isocarbostyril (ICS): 7-azaindole (7AI), allowed bypass of a chemically diverse spectrum of HBAs, and enabled PCR amplification with primers comprising multiple d5NI(C)-substitutions, while maintaining high levels of catalytic activity and fidelity. The selected polymerase 5D4 promises to expand the range of nucleobase analogues amenable to replication and should find numerous applications, including the synthesis and replication of nucleic acid polymers with expanded chemical and functional diversity.

Transcription Profiling of Bacillus subtilis Cells Infected with AR9, a Giant Phage Encoding Two Multisubunit RNA Polymerases.

Science.gov (United States)

Lavysh, Daria; Sokolova, Maria; Slashcheva, Marina; Förstner, Konrad U; Severinov, Konstantin

2017-02-14

Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5' ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases. IMPORTANCE Phages regulate the timing of the expression of their own genes to coordinate processes in the infected cell and maximize the release of viral progeny. Phages also alter the levels of host transcripts. Here we present the results of a temporal analysis of the host and viral transcriptomes of Bacillus subtilis infected with a giant phage, AR9. We identify viral promoters recognized by two virus-encoded RNA polymerases that are a unique feature of the phiKZ-related group of phages to which AR9 belongs. Our results set the stage for future analyses of highly unusual RNA polymerases encoded by AR9 and other phiKZ-related phages. Copyright © 2017 Lavysh et al.

Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR

DEFF Research Database (Denmark)

Søndergaard, Mette Sofie Rousing; Löfström, Charlotta; Al-Habib, Zahra Fares Sayer

DNA extractions and intermediate or bad with the crude extractions, while TaKaRa ExTaq HS only performed well with the purest extractions of fecal samples and intermediate with semi-automated magnetic beads based extracted fecal samples. In conclusion, our data shows that exchanging the DNA polymerase......Diagnostic analyses of foodborne pathogens are increasingly based on molecular methods such as PCR, which can improve the sensitivity and reduce the analysis time. The core of PCR is the enzyme performing the reaction: the DNA polymerase. Changing the polymerase can influence the sensitivity...... commercially available polymerases and four master mixes in two validated PCR assays, for Campylobacter and Salmonella, respectively, to develop more sensitive, robust and cost effective assays. The polymerases were screened on purified DNA and the five best performing, for each PCR assay, were then applied...

The synthetic inhibitor of Fibroblast Growth Factor Receptor PD166866 controls negatively the growth of tumor cells in culture

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Castelli Mauro

2009-12-01

Full Text Available Abstract Background Many experimental data evidence that over-expression of various growth factors cause disorders in cell proliferation. The role of the Fibroblast Growth Factors (FGF in growth control is indisputable: in particular, FGF1 and its tyrosine kinase receptor (FGFR1 act through a very complex network of mechanisms and pathways. In this work we have evaluated the antiproliferative activity effect of PD166866, a synthetic molecule inhibiting the tyrosin kinase action of FGFR1. Methods Cells were routinely grown in Dulbecco Modified Eagle's medium supplemented with newborn serum and a penicillin-streptomycin mixture. Cell viability was evaluated by Mosmann assay and by trypan blue staining. DNA damage was assessed by in situ fluorescent staining with Terminal Deoxynucleotidyl Transferase dUTP nick end labeling (TUNEL assay. Assessment of oxidative stress at membrane level was measured by quantitative analysis of the intra-cellular formation of malonyl-dialdheyde (MDA deriving from the decomposition of poly-unsaturated fatty acids. The expression of Poly-ADP-Ribose-Polymerase (PARP, consequent to DNA fragmentation, was evidenced by immuno-histochemistry utilizing an antibody directed against an N-terminal fragment of the enzyme. Results The bioactivity of the drug was investigated on Hela cells. Cytoxicity was assessed by the Mosmann assay and by vital staining with trypan blue. The target of the molecule is most likely the cell membrane as shown by the significant increase of the intracellular concentration of malonyl-dihaldheyde. The increase of this compound, as a consequence of the treatment with PD166866, is suggestive of membrane lipoperoxidation. The TUNEL assay gave a qualitative, though clear, indication of DNA damage. Furthermore we demonstrate intracellular accumulation of poly-ADP-ribose polymerase I. This enzyme is a sensor of nicks on the DNA strands and this supports the idea that treatment with the drug induces cell

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

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Gurupreet S. Sethi

2017-09-01

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

Checkpoint inhibitors in endometrial cancer: preclinical rationale and clinical activity.

Science.gov (United States)

Mittica, Gloria; Ghisoni, Eleonora; Giannone, Gaia; Aglietta, Massimo; Genta, Sofia; Valabrega, Giorgio

2017-10-27

Treatment of advanced and recurrent endometrial cancer (EC) is still an unmet need for oncologists and gynecologic oncologists. The Cancer Genome Atlas Research Network (TCGA) recently provided a new genomic classification, dividing EC in four subgroups. Two types of EC, the polymerase epsilon (POLE)-ultra-mutated and the microsatellite instability-hyper-mutated (MSI-H), are characterized by a high mutation rate providing the rationale for a potential activity of checkpoint inhibitors. We analyzed all available evidence supporting the role of tumor microenvironment (TME) in EC development and the therapeutic implications offered by immune checkpoint inhibitors in this setting. We performed a review on Pubmed with Mesh keywords 'endometrial cancer' and the name of each checkpoint inhibitor discussed in the article. The same search was operated on clinicaltrial.gov to identify ongoing clinical trials exploring PD-1/PD-L1 and CTLA-4 axis in EC, particularly focusing on POLE-ultra-muted and MSI-H cancer types. POLE-ultra-mutated and MSI-H ECs showed an active TME expressing high number of neo-antigens and an elevated amount of tumor infiltrating lymphocytes (TILs). Preliminary results from a phase-1 clinical trial (KEYNOTE-028) demonstrated antitumor activity of Pembrolizumab in EC. Moreover, both Pembrolizumab and Nivolumab reported durable clinical responses in POLE-ultra-mutated patients. Immune checkpoint inhibitors are an attractive option in POLE-ultra-mutated and MSI-H ECs. Future investigations in these subgroups include combinations of checkpoints inhibitors with chemotherapy and small tyrosine kinase inhibitors (TKIs) to enhance a more robust intra-tumoral immune response.

Structure and function of DNA polymerase μ

International Nuclear Information System (INIS)

Matsumoto, Takuro; Maezawa, So

2013-01-01

DNA polymerases are enzymes playing the central role in DNA metabolism, including DNA replication, DNA repair and recombination. DNA polymerase μ (pol μ DNA polymerase λ (pol λ) and terminal deoxynucleotidyltransferase (TdT) in X family DNA polymerases function in non-homologous end-joining (NHEJ), which is the predonmiant repair pathway for DNA double-strand breaks (DSBs). NHEJ involves enzymes that capture both ends of the broken DNA strand, bring them together in a synaptic DNA-protein complex, and repair the DSB. Pol μ and pol λ fill in the gaps at the junction to maintain the genomic integrity. TdT synthesizes N region at the junction during V(D)J recombination and promotes diversity of immunoglobulin or T-cell receptor gene. Among these three polymerases, the regulatory mechanisms of pol μ remain rather unclear. We have approached the mechanism of pol μ from both sides of structure and cellular dynamics. Here, we propose some new insights into pol μ and the probable NHEJ model including our findings. (author)

Polymerase Gamma Disease through the Ages

Science.gov (United States)

Saneto, Russell P.; Naviaux, Robert K.

2010-01-01

The most common group of mitochondrial disease is due to mutations within the mitochondrial DNA polymerase, polymerase gamma 1 ("POLG"). This gene product is responsible for replication and repair of the small mitochondrial DNA genome. The structure-function relationship of this gene product produces a wide variety of diseases that at times, seems…

Synthesis of New Acadesine (AICA-riboside Analogues Having Acyclic d-Ribityl or 4-Hydroxybutyl Chains in Place of the Ribose

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Gennaro Piccialli

2013-08-01

Full Text Available The antiviral activity of certain acyclic nucleosides drew our attention to the fact that the replacement of the furanose ring by an alkyl group bearing hydroxyl(s could be a useful structural modification to modulate the biological properties of those nucleosides. Herein, we report on the synthesis of some novel acadesine analogues, where the ribose moiety is mimicked by a d-ribityl or by a hydroxybutyl chain.

Using the Hepatitis C Virus RNA-Dependent RNA Polymerase as a Model to Understand Viral Polymerase Structure, Function and Dynamics

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Ester Sesmero

2015-07-01

Full Text Available Viral polymerases replicate and transcribe the genomes of several viruses of global health concern such as Hepatitis C virus (HCV, human immunodeficiency virus (HIV and Ebola virus. For this reason they are key targets for therapies to treat viral infections. Although there is little sequence similarity across the different types of viral polymerases, all of them present a right-hand shape and certain structural motifs that are highly conserved. These features allow their functional properties to be compared, with the goal of broadly applying the knowledge acquired from studying specific viral polymerases to other viral polymerases about which less is known. Here we review the structural and functional properties of the HCV RNA-dependent RNA polymerase (NS5B in order to understand the fundamental processes underlying the replication of viral genomes. We discuss recent insights into the process by which RNA replication occurs in NS5B as well as the role that conformational changes play in this process.

The Treatment of BRCA1/2 Hereditary BRCA1/2 and Sporadic Breast Cancer with Poly(ADP-Ribose) Polymerase Inhibitors and Chemotherapy

Science.gov (United States)

2009-09-01

pathology, social issues, and healthcare are also covered. Respond to questions from the community and guidance to solve individual health care... Narcissism , the new mental illness? The Journal 29 Dec 2008 C1.    De Soto JA, Treating migraine headaches, The Journal 1 Dec 2008 C1‐C2.    De Soto JA...Anti‐Estrogens.   Doctoral Dissertation, Advisor: Donnell Bowen Ph.D., Professor of Pharmacology &  Oncology. Dec 2005      Media     CNN News, Rob Snyder

Low energy electron-initiated ion-molecule reactions of ribose analogues

International Nuclear Information System (INIS)

Mozejko, P.

2003-01-01

Recent experiments in which plasmid DNA samples were bombarded with low energy ( 2 O, DNA bases, and sugar-phosphate backbone analogues. To this end, the cyclic molecule tetrahydrofuran, and its derivatives, provide useful models for the sugar-like molecules contained in the backbone of DNA. In addition to LEE induced dissociation by processes such as dissociative electron attachment (DEA), molecules may be damaged by ions and neutral species of non-thermal energies created by LEE in the surrounding environment. In this contribution, we investigate with electron stimulated desorption techniques, LEE damage to films of desoxy-ribose analogues in the presence of various molecular coadsorbates, that simulate changes in local molecular environment. In one type of experiments tetrahydrofuran is deposited onto multilayer O2. A desorbed signal of OH - indicates ion-molecule reactions of the type O - + C 4 H 8 O -> OH - + C 4 H 7 O, where the O - was formed initially by DEA to O 2 . Further electron stimulated desorption measurements for tetrahydrofuran and derivatives adsorbed on H 2 O, Kr, N 2 O and CH 3 OH will be presented and discussed

α,β-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction.

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Olivier Martínez

Full Text Available (S(C5', R(P α,β-D- Constrained Nucleic Acids (CNA are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.

The Inhibition of microRNA-128 on IGF-1-Activating mTOR Signaling Involves in Temozolomide-Induced Glioma Cell Apoptotic Death.

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Peng-Hsu Chen

Full Text Available Temozolomide (TMZ, an alkylating agent of the imidazotetrazine series, is a first-line chemotherapeutic drug used in the clinical therapy of glioblastoma multiforme, the most common and high-grade primary glioma in adults. Micro (miRNAs, which are small noncoding RNAs, post-transcriptionally regulate gene expressions and are involved in gliomagenesis. However, no studies have reported relationships between TMZ and miRNA gene regulation. We investigated TMZ-mediated miRNA profiles and its molecular mechanisms underlying the induction of glioma cell death. By performing miRNA microarray and bioinformatics analyses, we observed that expression of 248 miRNAs was altered, including five significantly upregulated and 17 significantly downregulated miRNAs, in TMZ-treated U87MG cells. miR-128 expression levels were lower in different glioma cells and strongly associated with poor survival. TMZ treatment significantly upregulated miR-128 expression. TMZ significantly enhanced miR-128-1 promoter activity and transcriptionally regulated miR-128 levels through c-Jun N-terminal kinase 2/c-Jun pathways. The overexpression and knockdown of miR-128 expression significantly affected TMZ-mediated cell viability and apoptosis-related protein expression. Furthermore, the overexpression of miR-128 alone enhanced apoptotic death of glioma cells through caspase-3/9 activation, poly(ADP ribose polymerase degradation, reactive oxygen species generation, mitochondrial membrane potential loss, and non-protective autophagy formation. Finally, we identified that key members in mammalian target of rapamycin (mTOR signaling including mTOR, rapamycin-insensitive companion of mTOR, insulin-like growth factor 1, and PIK3R1, but not PDK1, were direct target genes of miR-128. TMZ inhibited mTOR signaling through miR-128 regulation. These results indicate that miR-128-inhibited mTOR signaling is involved in TMZ-mediated cytotoxicity. Our findings may provide a better understanding

Disrupting Na⁺,HCO₃^--cotransporter NBCn1 (Slc4a7) delays murine breast cancer development

DEFF Research Database (Denmark)

Lee, S.; Axelsen, T. V.; Andersen, Anne Poder

2016-01-01

of NBCn1 genotype, the cleaved fraction of poly(ADP-ribose) polymerase (PARP)-1 and expression of monocarboxylate transporter (MCT)1 increased while phosphorylation of Akt and ERK1 decreased as functions of tumor volume. Cell proliferation, evaluated from Ki-67 and phospho-histone H3 staining, was ~60...

PARP Inhibition Prevents Ethanol-Induced Neuroinflammatory Signaling and Neurodegeneration in Rat Adult-Age Brain Slice Cultures

Science.gov (United States)

Tajuddin, Nuzhath; Kim, Hee-Yong

2018-01-01

Using rat adult-age hippocampal-entorhinal cortical (HEC) slice cultures, we examined the role of poly [ADP-ribose] polymerase (PARP) in binge ethanol’s brain inflammatory and neurodegenerative mechanisms. Activated by DNA strand breaks, PARP (principally PARP1 in the brain) promotes DNA repair via poly [ADP-ribose] (PAR) products, but PARP overactivation triggers regulated neuronal necrosis (e.g., parthanatos). Previously, we found that brain PARP1 levels were upregulated by neurotoxic ethanol binges in adult rats and HEC slices, and PARP inhibitor PJ34 abrogated slice neurodegeneration. Binged HEC slices also exhibited increased Ca+2-dependent phospholipase A2 (PLA2) isoenzymes (cPLA2 IVA and sPLA2 IIA) that mobilize proinflammatory ω6 arachidonic acid (ARA). We now find in 4-day–binged HEC slice cultures (100 mM ethanol) that PARP1 elevations after two overnight binges precede PAR, cPLA2, and sPLA2 enhancements by 1 day and high-mobility group box-1 (HMGB1), an ethanol-responsive alarmin that augments proinflammatory cytokines via toll-like receptor-4 (TLR4), by 2 days. After verifying that PJ34 effectively blocks PARP activity (↑PAR), we demonstrated that, like PJ34, three other PARP inhibitors—olaparib, veliparib, and 4-aminobenzamide—provided neuroprotection from ethanol. Importantly, PJ34 and olaparib also prevented ethanol’s amplification of the PLA2 isoenzymes, and two PLA2 inhibitors were neuroprotective—thus coupling PARP to PLA2, with PLA2 activity promoting neurodegeneration. Also, PJ34 and olaparib blocked ethanol-induced HMGB1 elevations, linking brain PARP induction to TLR4 activation. The results provide evidence in adult brains that induction of PARP1 may mediate dual neuroinflammatory pathways (PLA2→phospholipid→ARA and HMGB1→TLR4→proinflammatory cytokines) that are complicit in binge ethanol-induced neurodegeneration. PMID:29339456

Quantum dots for a high-throughput Pfu polymerase based multi-round polymerase chain reaction (PCR).

Science.gov (United States)

Sang, Fuming; Zhang, Zhizhou; Yuan, Lin; Liu, Deli

2018-02-26

Multi-round PCR is an important technique for obtaining enough target DNA from rare DNA resources, and is commonly used in many fields including forensic science, ancient DNA analysis and cancer research. However, multi-round PCR is often aborted, largely due to the accumulation of non-specific amplification during repeated amplifications. Here, we developed a Pfu polymerase based multi-round PCR technique assisted by quantum dots (QDs). Different PCR assays, DNA polymerases (Pfu and Taq), DNA sizes and GC amounts were compared in this study. In the presence of QDs, PCR specificity could be retained even in the ninth-round amplification. Moreover, the longer and more complex the targets were, the earlier the abortion happened in multi-round PCR. However, no obvious enhancement of specificity was found in multi-round PCR using Taq DNA polymerase. Significantly, the fidelity of Pfu polymerase based multi-round PCR was not sacrificed in the presence of QDs. Besides, pre-incubation at 50 °C for an hour had no impact on multi-round PCR performance, which further authenticated the hot start effect of QDs modulated in multi-round PCR. The findings of this study demonstrated that a cost-effective and promising multi-round PCR technique for large-scale and high-throughput sample analysis could be established with high specificity, sensibility and accuracy.

Effects of inhibitors of DNA repair on the frequencies of chromosomal aberrations induced by x-rays or alkylating agents in cultured human lymphocytes

International Nuclear Information System (INIS)

Kihlman, B.A.; Andersson, H.C.

1986-01-01

In the first part of this presentation the authors give examples of the synergistic enhancements that are obtained with various inhibitor combinations in G/sub 2/. The second part of the presentation deals with the effects of two agents, also well known for their capacity to potentiate the frequency of chromosomal aberrations induced by physical and chemical agents, but with a different mechanism of action. These agents are caffeine and 3-aminobenzamide (3AB). Caffeine has for decades been used as an inhibitor of DNA repair although its mechanism of action has not been fully understood. 3AB has more recently come into focus as an efficient inhibitor of the synthesis of poly-(ADP-ribose), a substance believed to be of importance in connection with the repair of certain types of DNA damage. The results presented do not quite fit in with the general idea about the mode of action of these agents. All experiments were carried out with whole-blood cultures of human lymphocytes. When inhibitors were used as post-treatments, chromosomal aberrations were induced by X-rays or by the alkylating agents thiotepa (TT) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). X-rays were generated by a Siemens Stabilipan 200 apparatus, at a dose rate of 0.5 Gy/min. The tube (TR 200f) was operated at 180 kV, 10 mA and the radiation filtered through 4 mm Al

Spotlight on olaparib in the treatment of BRCA-mutated ovarian cancer: design, development and place in therapy

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Lorusso D

2018-05-01

Full Text Available Domenica Lorusso, Elisa Tripodi, Giuseppa Maltese, Stefano Lepori, Ilaria Sabatucci, Giorgio Bogani, Francesco Raspagliesi Gynecologic Oncology Unit, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy Abstract: Epithelial ovarian cancer is the sixth most common cancer among women worldwide and the first cause of death among gynecological malignancies. Most of the patients present recurrent disease and unfortunately cannot be cured. The unsatisfactory results obtained with salvage chemotherapy have elicited investigators to search for novel biological agents capable of achieving a better control of the disease. In the setting of homologous recombination deficiency, the DNA errors that occur cannot be accurately repaired, and the treatment with poly(ADP-ribose polymerase (PARP inhibition results in definitive cell death in a process called synthetic lethality. As a result of two positive clinical trials, Olaparib was approved in 2014 by U.S. Food and Drug Administration and European Medicines Agency as the first-in-class PARP inhibitor. Olaparib is effective and well tolerated in homologous recombination deficient patients. Several studies with Olaparib have been conducted in the recurrent setting either as maintenance in platinum-responsive patients or as a single agent. Ongoing trials are focused on the use of olaparib as maintenance in the first-line ovarian cancer setting alone or in combination with antiangiogenic agents. Future perspectives will probably investigate the association of olaparib with novel agents as check-point inhibitors and PI3K-AKT inhibitors. The PARP inhibitor era is just at the beginning. Keywords: olaparib, ovarian cancer, PARP inhibitors, homologous recombination deficiency, BRCA mutation

Damage of DNA and plasma membranes in murine lymphoma cells irradiated under aerobic or hypoxic conditions

International Nuclear Information System (INIS)

Wlodek, D.

1983-01-01

A review of the knowledge of radiation effects on cell membranes and DNA and of repair mechanisms of radiation lesions is given. Investigations of properties of plasma membranes in L5178Y-S and L5178Y-R cells (surface charge, fluidity, transport of amino acids) indicate that there is no direct connection between membrane lesions and reproductive death. It was also found that in irradiated cells of both L5178Y-strains the rate of DNA chain elongation is the same, similarly as the amount of the initial DNA lesions and the rate of repair processes. Difference in the level of DNA synthesis inhibition is not proportional to the lethal effect. The results are also reported point to the difference between L5178Y-S and L5178Y-R cells in susceptibility of post-irradiation DNA synthesis to factors modifying chromatin conformation, such as inhibitors of (ADP-ribose) n polymerase. 221 refs. (author)

Ethanol Extract of Dianthus chinensis L. Induces Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells In Vitro

Science.gov (United States)

Nho, Kyoung Jin; Chun, Jin Mi; Kim, Ho Kyoung

2012-01-01

Dianthus chinensis L. is used to treat various diseases including cancer; however, the molecular mechanism by which the ethanol extract of Dianthus chinensis L. (EDCL) induces apoptosis is unknown. In this study, the apoptotic effects of EDCL were investigated in human HepG2 hepatocellular carcinoma cells. Treatment with EDCL significantly inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis. This induction was associated with chromatin condensation, activation of caspases, and cleavage of poly (ADP-ribose) polymerase protein. However, apoptosis induced by EDCL was attenuated by caspase inhibitor, indicating an important role for caspases in EDCL responses. Furthermore, EDCL did not alter the expression of bax in HepG2 cells but did selectively downregulate the expression of bcl-2 and bcl-xl, resulting in an increase in the ratio of bax:bcl-2 and bax:bcl-xl. These results support a mechanism whereby EDCL induces apoptosis through the mitochondrial pathway and caspase activation in HepG2 cells. PMID:22645629

Molecular mechanism of radioadaptive response: A cross-adaptive response for enhanced repair of DNA damage in adapted cells

International Nuclear Information System (INIS)

Takaji Ikushima

1997-01-01

The radioadaptive response (RAR) has been attributed to the induction of a repair mechanism by low doses of ionizing radiation, but the molecular nature of the mechanism is not yet elucidated. We have characterized RAR in a series of experiments in cultured Chinese hamster V79 cells. A 4-h interval is required for the full expression of RAR, which decays with the progression of cell proliferation. Treatments with inhibitors of poly(ADP-ribose) polymerase, protein- or RNA synthesis, and protein kinase C suppress the RAR expression. The RAR cross-reacts on clastogenic lesions induced by other physical and chemical DNA-damaging agents. The presence of newly synthesised proteins has been detected during the expression period. Experiments performed using single-cell gel electrophoresis provided more direct evidence for a faster and enhaced DNA repair rate in adapted cells. Here, using single-cell gel electrophoresis, a cross-adaptive response has been demonstrated for enhanced repair of DNA damage induced by neocarzinostatin in radio-adapted cells. (author)

Development and validation of a high-performance liquid chromatography method for the quantification of talazoparib in rat plasma: Application to plasma protein binding studies.

Science.gov (United States)

Hidau, Mahendra Kumar; Kolluru, Srikanth; Palakurthi, Srinath

2018-02-01

A sensitive and selective RP-HPLC method has been developed and validated for the quantification of a highly potent poly ADP ribose polymerase inhibitor talazoparib (TZP) in rat plasma. Chromatographic separation was performed with isocratic elution method. Absorbance for TZP was measured with a UV detector (SPD-20A UV-vis) at a λ max of 227 nm. Protein precipitation was used to extract the drug from plasma samples using methanol-acetonitrile (65:35) as the precipitating solvent. The method proved to be sensitive and reproducible over a 100-2000 ng/mL linearity range with a lower limit of quantification (LLQC) of 100 ng/mL. TZP recovery was found to be >85%. Following analytical method development and validation, it was successfully employed to determine the plasma protein binding of TZP. TZP has a high level of protein binding in rat plasma (95.76 ± 0.38%) as determined by dialysis method. Copyright © 2017 John Wiley & Sons, Ltd.

The Paramyxovirus Polymerase Complex as a Target for Next-Generation Anti-Paramyxovirus Therapeutics

Directory of Open Access Journals (Sweden)

Richard K Plemper

2015-05-01

Full Text Available The paramyxovirus family includes major human and animal pathogens, including measles virus, mumps virus, and human respiratory syncytial virus (RSV, as well as the emerging zoonotic Hendra and Nipah viruses. In the United States, RSV is the leading cause of infant hospitalizations due to viral infectious disease. Despite their clinical significance, effective drugs for the improved management of paramyxovirus disease are lacking. The development of novel anti-paramyxovirus therapeutics is therefore urgently needed. Paramyxoviruses contain RNA genomes of negative polarity, necessitating a virus-encoded RNA-dependent RNA polymerase (RdRp complex for replication and transcription. Since an equivalent enzymatic activity is absent in host cells, the RdRp complex represents an attractive druggable target, although structure-guided drug development campaigns are hampered by the lack of high-resolution RdRp crystal structures. Here, we review the current structural and functional insight into the paramyxovirus polymerase complex in conjunction with an evaluation of the mechanism of activity and developmental status of available experimental RdRp inhibitors. Our assessment spotlights the importance of the RdRp complex as a premier target for therapeutic intervention and examines how high-resolution insight into the organization of the complex will pave the path towards the structure-guided design and optimization of much-needed next-generation paramyxovirus RdRp blockers.

An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening.

Science.gov (United States)

Nelson, Emily V; Pacheco, Jennifer R; Hume, Adam J; Cressey, Tessa N; Deflubé, Laure R; Ruedas, John B; Connor, John H; Ebihara, Hideki; Mühlberger, Elke

2017-10-01

Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research. Copyright © 2017 Elsevier B.V. All rights reserved.

Discovery of cyanophage genomes which contain mitochondrial DNA polymerase.

Science.gov (United States)

Chan, Yi-Wah; Mohr, Remus; Millard, Andrew D; Holmes, Antony B; Larkum, Anthony W; Whitworth, Anna L; Mann, Nicholas H; Scanlan, David J; Hess, Wolfgang R; Clokie, Martha R J

2011-08-01

DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on the substantiation of health claims related to ribose and faster recovery from muscle fatigue after exercise (ID 4226) pursuant to Article 13(1) of Regulation (EC) No 1924/2006

DEFF Research Database (Denmark)

Tetens, Inge

Following a request from the European Commission, the Panel on Dietetic Products, Nutrition and Allergies was asked to provide a scientific opinion on a list of health claims pursuant to Article 13 of Regulation (EC) No 1924/2006. This opinion addresses the scientific substantiation of health...... claims in relation to ribose and faster recovery from muscle fatigue after exercise. The scientific substantiation is based on the information provided by the Member States in the consolidated list of Article 13 health claims and references that EFSA has received from Member States or directly from...... stakeholders. The food that is the subject of the health claim is ribose. The Panel considers that ribose is sufficiently characterised. The claimed effect is “maintenance of ATP levels, exercise performance, exercise recovery”. The target population is assumed to be adults performing strenuous exercise...

Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

Science.gov (United States)

Nikiforov, Andrey; Dölle, Christian; Niere, Marc; Ziegler, Mathias

2011-01-01

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule. PMID:21504897

Epigenetic perturbations in the pathogenesis of mustard toxicity; hypothesis and preliminary results

International Nuclear Information System (INIS)

Korkmaz, A.; Yaren, H.; Kunak, I.; Uysal, B.; Kurt, B.; Topal, T.

2009-01-01

The pathogenesis of sulfur mustard (SM) toxicity is not fully understood, although it is related to reactive oxygen and nitrogen species, oxidative stress, DNA damage, poly (ADP-ribose) polymerase activation within the affected cell. We, therefore, made an attempt whether epigenetic aberrations may contribute to pathogenesis of SM poisoning in rats' lung. A total of 40 male SD rats were divided into 4 groups. Group 1 served as control and given 2 ml saline, three groups received single dose of mechlorethamine (MEC) (3.5 mg/kg subcutaneously) with the same time intervals. Group 2 received MEC only; group 3 received histone deacetylase (HDAC) inhibitor (Trichostatine A) (1 mg/kg) and group 4 received DNA methyl transferase (DNMT) inhibitor (5-Azacytidine) (0.02 mg/kg), intraperitoneally. MEC injection resulted in severe lung toxicity with strong interstitial and alveolar edema, hemorrhage, emphysematous changes as well as mild inflammatory cell infiltration and septal thickening. In group 3, the HDAC inhibitor significantly reduced interstitial and alveolar edema, hemorrhage and inflammatory cell infiltration. On the other hand, we have observed severe lung damage by using DNMT inhibitor (group 4). In HDAC inhibitor group, the results were close to sham group. In DNMT inhibitor group, however, lungs were worse than MEC group results. These preliminary results revealed that, SM itself and/or its intracellular metabolites may perturb the epigenetic environment of the affected cell in lung tissue. Hypothetically, MEC may cause HDAC induction leading to a variety of gene silencing. Trichostatine A can reduce the active enzyme level and can reactivate the already silenced genes. Further studies are needed to clarify the involvement of epigenetic perturbations in the pathogenesis of mustard toxicity.(author)

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

International Nuclear Information System (INIS)

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

1986-01-01

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

Interferon antagonist NSs of La Crosse virus triggers a DNA damage response-like degradation of transcribing RNA polymerase II.

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Verbruggen, Paul; Ruf, Marius; Blakqori, Gjon; Överby, Anna K; Heidemann, Martin; Eick, Dirk; Weber, Friedemann

2011-02-04

La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits.

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.

Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase

DEFF Research Database (Denmark)

Willemoës, Martin; Hove-Jensen, Bjarne; Larsen, Sine

2000-01-01

A steady state kinetic investigation of the Pi activation of 5-phospho-D-ribosyl α-1-diphosphate synthase from Escherichia coli suggests that Pi can bind randomly to the enzyme either before or after an ordered addition of free Mg2+ and substrates. Unsaturation with ribose 5-phosphate increased...... the apparent cooperativity of Pi activation. At unsaturating Pi concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with Pi directs the subsequent ordered binding of Mg2+ and substrates via a fast pathway, whereas...... saturation with ribose 5-phosphate leads to the binding of Mg2+ and substrates via a slow pathway where Pi binds to the enzyme last. The random mechanism for Pi binding was further supported by studies with competitive inhibitors of Mg2+, MgATP, and ribose 5-phosphate that all appeared noncompetitive when...

Reverse transcriptase-quantitative polymerase chain reaction (RT ...

African Journals Online (AJOL)

The reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a highly specific polymerase chain reaction (PCR) method that allows one to detect very low transcription levels of functional gene(s) in soil. RT-qPCR helps us to know the active members of the microbial community, and their activities can be ...

Enhancement of DNA polymerase activity in potato tuber slices

International Nuclear Information System (INIS)

Watanabe, Akira; Imaseki, Hidemasa

1977-01-01

DNA polymerase was extracted from potato (Soleum tuberosum L.) tuber discs and the temporal correlation of its activity change to DNA synthesis in vivo was examined during aging of the discs. Most of the DNA polymerase was recovered as a bound form in the 18,000 x g precipitate. Reaction with the bound-form enzyme was dependent on the presence of four deoxynucleoside triphosphates, Mg 2+ , and a template. ''Activated'' DNA and heat-denatured DNA, but not native DNA, were utilized as templates. The polymerase activity was sensitive to SH reagents. Fresh discs, which do not synthesize DNA in vivo, contained a significant amount of DNA polymerase and its activity increased linearly with time until 48 hr after slicing and became four times that of fresh discs after 72 hr, whereas the activity of DNA synthesis in vivo increased with time and decreased after reaching a maximum at 30 hr. Cycloheximide inhibited the enhancement of polymerase activity. DNA polymerase from aged and fresh discs had identical requirements for deoxynucleotides and a template in their reactions, sensitivity to SH reagent, and affinity to thymidine triphosphate. (auth.)

DNA Polymerase Fidelity: Beyond Right and Wrong.

Science.gov (United States)

Washington, M Todd

2016-11-01

Accurate DNA replication depends on the ability of DNA polymerases to discriminate between correctly and incorrectly paired nucleotides. In this issue of Structure, Batra et al. (2016) show the structural basis for why DNA polymerases do not efficiently add correctly paired nucleotides immediately after incorporating incorrectly paired ones. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanisms of Virus-Induced Neural Cell Death

Science.gov (United States)

2002-09-01

Pathol the central nervous system. Lancet 342: 398-401 9:199-208 19. Cinque P, Vago L, Dahl H, Brytting M, Terreni MR, 6. Arribas JR, Clifford DB...methyl coumarin; PARP, poly(ADP-ribose) polymerase; the translocation of cytochrome c and other pro-apoptotic Ac-YVAD-CHO, Ac-Tyr-Val- Ala -Asp-CHO; Ac

Resonance energy transfer study on the proximity relationship between the GTP binding site and the rifampicin binding site of Escherichia coli RNA polymerase

International Nuclear Information System (INIS)

Kumar, K.P.; Chatterji, D.

1990-01-01

Terbium(III) upon complexation with guanosine 5'-triphosphate showed remarkable enhancement of fluorescence emission at 488 and 545 nm when excited at 295 nm. Analysis of the binding data yielded a value for the mean K d between Tb(III) and GTP of 0.2 μM, with three binding sites for TB(III) on GTP. 31 P and 1 H NMR measurements revealed that Tb(III) mainly binds the phosphate moiety of GTP. Fluorescence titration of the emission signals of the TbGTP complex with varying concentrations of Escherichia coli RNA polymerase resulted in a K d values of 4 μM between the TbGTP and the enzyme. It was observed that TbGTP can be incorporated in the place of GTP during E. coli RNA polymerase catalyzed abortive synthesis of dinucleotide tetraphosphate at T7A2 promoter. Both the substrate TbGTP and the inhibitor of the initiation of transcription rifampicin bind to the β-subunit of E. coli RNA polymerase. This allows the measurement of the fluorescence excited-state energy transfer from the donor TbGTP-RNA polymerase to the acceptor rifampicin. Both emission bands of Tb(III) overlap with the rifampicin absorption, and the distances at 50% efficiency of energy transfer were calculated to be 28 and 24 angstrom for the 488- and 545-nm emission bands, respectively. The distance between the substrate binding site and the rifampicin binding site on the β-subunit of E. coli RNA polymerase was measured to be around 30 angstrom. This suggest that the nature of inhibition of transcription by rifampicin is essentially noncompetitive with the substrate

Competition between replicative and translesion polymerases during homologous recombination repair in Drosophila.

Directory of Open Access Journals (Sweden)

Daniel P Kane

Full Text Available In metazoans, the mechanism by which DNA is synthesized during homologous recombination repair of double-strand breaks is poorly understood. Specifically, the identities of the polymerase(s that carry out repair synthesis and how they are recruited to repair sites are unclear. Here, we have investigated the roles of several different polymerases during homologous recombination repair in Drosophila melanogaster. Using a gap repair assay, we found that homologous recombination is impaired in Drosophila lacking DNA polymerase zeta and, to a lesser extent, polymerase eta. In addition, the Pol32 protein, part of the polymerase delta complex, is needed for repair requiring extensive synthesis. Loss of Rev1, which interacts with multiple translesion polymerases, results in increased synthesis during gap repair. Together, our findings support a model in which translesion polymerases and the polymerase delta complex compete during homologous recombination repair. In addition, they establish Rev1 as a crucial factor that regulates the extent of repair synthesis.

Solid-Phase Synthesis of a New Diphosphate 5-Aminoimidazole-4-carboxamide Riboside (AICAR Derivative and Studies toward Cyclic AICAR Diphosphate Ribose

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Gennaro Piccialli

2011-09-01

Full Text Available The solid-phase synthesis of the first example of a new diphosphate AICAR derivative is reported. The new substance is characterized by the presence of a 5'-phosphate group while a second phosphate moiety is installed on a 5-hydroxypentyl chain attached to the 4-N-position of AICAR. Cyclization of the diphosphate derivative by pyrophosphate bond formation allowed for the formation of a novel AICAR-based cyclic ADP-ribose (cADPR mimic.

3-(imidazo[1,2-a:5,4-b']dipyridin-2-yl)aniline inhibits pestivirus replication by targeting a hot spot drug binding pocket in the RNA-dependent RNA polymerase.

Science.gov (United States)

Musiu, Simone; Leyssen, Pieter; Froeyen, Mathy; Chezal, Jean-Michel; Neyts, Johan; Paeshuyse, Jan

2016-05-01

The compound 3-(imidazo[1,2-a:5,4-b']dipyridin-2-yl)aniline (CF02334) was identified as a selective inhibitor of the cytopathic effect (CPE) caused by bovine viral diarrhea virus (BVDV) in a virus-cell-based assay. The EC50-values for inhibition of CPE, viral RNA synthesis and the production of infectious virus progeny were 13.0 ± 0.6 μM, 2.6 ± 0.9 μM and 17.8 ± 0.6 μM, respectively. CF02334 was found to be inactive in the hepatitis C subgenomic replicon system. CF02334-resistant BVDV was obtained and was found to carry the N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). Molecular modeling revealed that N264D is located in a small cavity near the fingertip domain of the pestivirus polymerase. CF02334-resistant BVDV was proven to be cross-resistant to BPIP, AG110 and LZ37, inhibitors that have previously been described to target the same region of the BVDV RdRp. CF02334 did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of BVDV replication complexes. Taken together, these observations indicate that CF02334 likely interacts with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37, which marks this region of the viral polymerase as a "hot spot" for inhibition of pestivirus replication. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of MVP and VPARP promoters indicates a role for chromatin remodeling in the regulation of MVP.

Science.gov (United States)

Emre, Nil; Raval-Fernandes, Sujna; Kickhoefer, Valerie A; Rome, Leonard H

2004-04-16

Multi-drug-resistant cancer cells frequently express elevated levels of ribonucleoprotein complexes termed vaults. The increased expression of vault proteins and their mRNAs has led to the suggestion that vaults may play a direct role in preventing drug toxicity. To further understand vault component up-regulation, the three proteins that comprise the vault, the major vault protein (MVP), vault poly(ADP-ribose) polymerase (VPARP), and telomerase-associated protein-1 (TEP1), were examined with respect to gene amplification and drug-induced chromatin remodeling. Gene amplification was not responsible for increased vault component levels in multi-drug-resistant cancer cell lines. The TATA-less murine MVP and human VPARP promoters were identified and functionally characterized. There was no significant activation of either the MVP or VPARP promoters in drug-resistant cell lines in comparison to their parental, drug-sensitive counterparts. Treatment of various cell lines with sodium butyrate, an inhibitor of histone deacetylase (HDAC), led to an increase in vault component protein levels. Furthermore, treatment with trichostatin A (TSA), a more specific inhibitor of HDAC, caused an increase in MVP protein, mRNA, and promoter activity. These results suggest that up-regulation of MVP in multi-drug resistance (MDR) may involve chromatin remodeling.

Drug therapy for hereditary cancers

Directory of Open Access Journals (Sweden)

Imyanitov Evgeny N

2011-08-01

Full Text Available Abstract Tumors arising in patients with hereditary cancer syndromes may have distinct drug sensitivity as compared to their sporadic counterparts. Breast and ovarian neoplasms from BRCA1 or BRCA2 mutation carriers are characterized by deficient homologous recombination (HR of DNA, that makes them particularly sensitive to platinum compounds or inhibitors of poly (ADP-ribose polymerase (PARP. Outstandingly durable complete responses to high dose chemotherapy have been observed in several cases of BRCA-related metastatic breast cancer (BC. Multiple lines of evidence indicate that women with BRCA1-related BC may derive less benefit from taxane-based treatment than other categories of BC patients. There is virtually no reports directly assessing drug response in hereditary colorectal cancer (CRC patients; studies involving non-selected (i.e., both sporadic and hereditary CRC with high-level microsatellite instability (MSI-H suggest therapeutic advantage of irinotecan. Celecoxib has been approved for the treatment of familial adenomatous polyposis (FAP. Hereditary medullary thyroid cancers (MTC have been shown to be highly responsive to a multitargeted tyrosine kinase inhibitor vandetanib, which exerts specific activity towards mutated RET receptor. Given the rapidly improving accessibility of DNA analysis, it is foreseen that the potential predictive value of cancer-associated germ-line mutations will be increasingly considered in the future studies.

The Potential of Targeting Ribosome Biogenesis in High-Grade Serous Ovarian Cancer

Directory of Open Access Journals (Sweden)

Shunfei Yan

2017-01-01

Full Text Available Overall survival for patients with ovarian cancer (OC has shown little improvement for decades meaning new therapeutic options are critical. OC comprises multiple histological subtypes, of which the most common and aggressive subtype is high-grade serous ovarian cancer (HGSOC. HGSOC is characterized by genomic structural variations with relatively few recurrent somatic mutations or dominantly acting oncogenes that can be targeted for the development of novel therapies. However, deregulation of pathways controlling homologous recombination (HR and ribosome biogenesis has been observed in a high proportion of HGSOC, raising the possibility that targeting these basic cellular processes may provide improved patient outcomes. The poly (ADP-ribose polymerase (PARP inhibitor olaparib has been approved to treat women with defects in HR due to germline BRCA mutations. Recent evidence demonstrated the efficacy of targeting ribosome biogenesis with the specific inhibitor of ribosomal RNA synthesis, CX-5461 in v-myc avian myelocytomatosis viral oncogene homolog (MYC-driven haematological and prostate cancers. CX-5461 has now progressed to a phase I clinical trial in patients with haematological malignancies and phase I/II trial in breast cancer. Here we review the currently available targeted therapies for HGSOC and discuss the potential of targeting ribosome biogenesis as a novel therapeutic approach against HGSOC.

Pharmacological treatment of diabetic neuropathic pain.

Science.gov (United States)

Smith, Howard S; Argoff, Charles E

2011-03-26

Neuropathic pain continues to be a difficult and challenging clinical issue to deal with effectively. Painful diabetic polyneuropathy is a complex pain condition that occurs with reasonable frequency in the population and it may be extremely difficult for clinicians to provide patients with effective analgesia. Chronic neuropathic pain may occur in approximately one of every four diabetic patients. The pain may be described as burning or a deep-seated ache with sporadic paroxysms of lancinating painful exacerbations. The pain is often constant, moderate to severe in intensity, usually primarily involves the feet and generally tends to worsen at night. Treatment may be multimodal but largely involves pharmacological approaches. Pharmacological therapeutic options include antidepressants (tricyclic antidepressants, serotonin-norepinephrine reuptake inhibitors), α2δ ligands and topical (5%) lidocaine patch. Other agents may be different antiepileptic drugs (carbamazepine, lamotrigine, topiramate), topical capsaicin, tramadol and other opioids. Progress continues with respect to understanding various mechanisms that may contribute to painful diabetic neuropathy. Agents that may hold some promise include neurotrophic factors, growth factors, immunomodulators, gene therapy and poly (adenosine diphosphate-ribose) polymerase inhibitors. It is hoped that in the future clinicians will be able to assess patient pathophysiology, which may help them to match optimal therapeutic agents to target individual patient aberrant mechanisms.

Metronomic cyclophosphamide-induced long-term remission after recurrent high-grade serous ovarian cancer: A case study.

Science.gov (United States)

de Boo, Leonora Wijnandina; Vulink, Annelie Johanna Elisabeth; Bos, Monique Elisabeth Martina Maria

2017-12-01

Metronomic oral cyclophosphamide has gained increasing interest in recent years as a promising maintenance therapy in advanced, platinum-sensitive, high-grade serous ovarian cancer (HGSOC). Metronomic treatment with cyclophosphamide refers to the frequent, usually daily, administration of a low (oral) dose of cyclophosphamide with no prolonged drug-free breaks. Main advantages of this treatment are the effective reduction of tumour activity, oral administration in an outpatient setting, low cost and the low toxicity profile. Metronomic oral cyclophosphamide can benefit patients suffering from types of cancer known to be sensitive to alkylating agents, such as platinum-sensitive HGSOC. In recent years, several publications have underlined the advantage of this regimen and possible explanations were explored. We here present a patient with multiple recurrences of metastasized HGSOC, platinum-sensitive, with an on-going complete response to monotherapy with oral cyclophosphamide. This observation supports that patients with relapsing HGSOC who responded to platinum-based chemotherapy and cannot continue platinum-based chemotherapy because of toxicity, can be offered a course of metronomic cyclophosphamide. This case may serve as a reminder that old drugs can be used successfully even in the age of new upcoming therapy such as anti-angiogenic agents (VEGF inhibitors) and poly-ADP-ribose polymerase (PARP) inhibitors.

Trial watch – inhibiting PARP enzymes for anticancer therapy

Science.gov (United States)

Sistigu, Antonella; Manic, Gwenola; Obrist, Florine; Vitale, Ilio

2016-01-01

ABSTRACT Poly(ADP-ribose) polymerases (PARPs) are a members of family of enzymes that catalyze poly(ADP-ribosyl)ation (PARylation) and/or mono(ADP-ribosyl)ation (MARylation), two post-translational protein modifications involved in crucial cellular processes including (but not limited to) the DNA damage response (DDR). PARP1, the most abundant family member, is a nuclear protein that is activated upon sensing distinct types of DNA damage and contributes to their resolution by PARylating multiple DDR players. Recent evidence suggests that, along with DDR, activated PARP1 mediates a series of prosurvival and proapoptotic processes aimed at preserving genomic stability. Despite this potential oncosuppressive role, upregulation and/or overactivation of PARP1 or other PARP enzymes has been reported in a variety of human neoplasms. Over the last few decades, several pharmacologic inhibitors of PARP1 and PARP2 have been assessed in preclinical and clinical studies showing potent antineoplastic activity, particularly against homologous recombination (HR)-deficient ovarian and breast cancers. In this Trial Watch, we describe the impact of PARP enzymes and PARylation in cancer, discuss the mechanism of cancer cell killing by PARP1 inactivation, and summarize the results of recent clinical studies aimed at evaluating the safety and therapeutic profile of PARP inhibitors in cancer patients. PMID:27308587

Ayanin diacetate-induced cell death is amplified by TRAIL in human leukemia cells

International Nuclear Information System (INIS)

Marrero, María Teresa; Estévez, Sara; Negrín, Gledy; Quintana, José; López, Mariana; Pérez, Francisco J.; Triana, Jorge; León, Francisco; Estévez, Francisco

2012-01-01

Highlights: ► Ayanin diacetate as apoptotic inducer in leukemia cells. ► Cell death was prevented by caspase inhibitors and by the overexpression of Bcl-x L . ► The intrinsic and the extrinsic pathways are involved in the mechanism of action. ► Death receptors are up-regulated and TRAIL enhances apoptotic cell death. -- Abstract: Here we demonstrate that the semi-synthetic flavonoid ayanin diacetate induces cell death selectively in leukemia cells without affecting the proliferation of normal lymphocytes. Incubation of human leukemia cells with ayanin diacetate induced G 2 -M phase cell cycle arrest and apoptosis which was prevented by the non-specific caspase inhibitor z-VAD-fmk and reduced by the overexpression of Bcl-x L . Ayanin diacetate-induced cell death was found to be associated with: (i) loss of inner mitochondrial membrane potential, (ii) the release of cytochrome c, (iii) the activation of multiple caspases, (iv) cleavage of poly(ADP-ribose) polymerase and (v) the up-regulation of death receptors for TRAIL, DR4 and DR5. Moreover, the combined treatment with ayanin diacetate and TRAIL amplified cell death, compared to single treatments. These results provide a basis for further exploring the potential applications of this combination for the treatment of cancer.

Cytotoxic Effects of Fascaplysin against Small Cell Lung Cancer Cell Lines

Science.gov (United States)

Hamilton, Gerhard

2014-01-01

Fascaplysin, the natural product of a marine sponge, exhibits anticancer activity against a broad range of tumor cells, presumably through interaction with DNA, and/or as a highly selective cyclin-dependent kinase 4 (CDK4) inhibitor. In this study, cytotoxic activity of fascaplysin against a panel of small cell lung cancer (SCLC) cell lines and putative synergism with chemotherapeutics was investigated. SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent. Fascaplysin was found to show high cytotoxicity against SCLC cells and to induce cell cycle arrest in G1/0 at lower and S-phase at higher concentrations, respectively. The compound generated reactive oxygen species (ROS) and induced apoptotic cell death in the chemoresistant NCI-H417 SCLC cell line. Furthermore, fascaplysin revealed marked synergism with the topoisomerase I-directed camptothecin and 10-hydroxy-camptothecin. The Poly(ADP-ribose)-Polymerase 1 (PARP1) inhibitor BYK 204165 antagonized the cytotoxic activity of fascaplysin, pointing to the involvement of DNA repair in response to the anticancer activity of the drug. In conclusion, fascaplysin seems to be suitable for treatment of SCLC, based on high cytotoxic activity through multiple routes of action, affecting topoisomerase I, integrity of DNA and generation of ROS. PMID:24608973

T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death

Directory of Open Access Journals (Sweden)

Chang-Fang Chiu

2016-08-01

Full Text Available T315, an integrin-linked kinase (ILK inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML cell lines (HL-60 and THP-1 and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy.

Effect of γ-irradiated DNA on the activity of DNA polymerase

International Nuclear Information System (INIS)

Leadon, S.A.; Ward, J.F.

1981-01-01

A cell-free assay was developed to measure the effect of γ-irradiated DNA template on the ability of DNA polymerase to copy unirradiated template. Doses as low as 1 krad were able to decrease (approx. 15%) the activity of both bacterial and mammalian DNA polymerases in the assay. The percentage of polymerase activity decreased as the dose received by the template increased. The reduction in DNA polymerase activity was shown to be due to an inhibition of the enzyme by the irradiated DNA. Irradiated poly(dA-dT) was more effective in reducing polymerase activity than calf thymus DNA. Thus the polymerase-inhibition site(s) appears to be associated with base damage, specifically adenine or thymine. Using a free-radical scavenger, OH radicals were found to be involved in producing the damage sites. The interaction between irradiated DNA and DNA polymerase was found to be specific for the enzyme and not for other proteins present in the assay. The inhibition of DNA polymerase occurred prior to or during the initiation of DNA synthesis rather than after initiation of synthesis, i.e., during elongation

Repair of Clustered Damage and DNA Polymerase Iota.

Science.gov (United States)

Belousova, E A; Lavrik, O I

2015-08-01

Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

Role of polymerase η in mitochondrial mutagenesis of Saccharomyces cerevisiae

Energy Technology Data Exchange (ETDEWEB)

Chatterjee, Nimrat; Pabla, Ritu [Dept. of Cell Biology and Anatomy, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (United States); Siede, Wolfram, E-mail: wolfram.siede@unthsc.edu [Dept. of Cell Biology and Anatomy, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (United States)

2013-02-08

Highlights: ► DNA polymerase η is detectable in mitochondria of budding yeast. ► Pol η reduces UV-induced mitochondrial base pair substitutions and frameshifts. ► For UV-induced base pair substitutions, Pol η and Pol ζ interact epistatically. -- Abstract: DNA polymerase η mostly catalyzes an error-free bypass of the most frequent UV lesions, pyrimidine dimers of the cyclobutane-type. In addition to its nuclear localization, we show here for the first time its mitochondrial localization in budding yeast. In mitochondria, this polymerase improves bypass replication fidelity opposite UV damage as shown in base pair substitution and frameshift assays. For base pair substitutions, polymerase η appears to be related in function and epistatic to DNA polymerase ζ which, however, plays the opposite role in the nucleus.

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

Energy Technology Data Exchange (ETDEWEB)

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

1983-07-14

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

Rev1 contributes to proper mitochondrial function via the PARP-NAD(+)-SIRT1-PGC1 alpha axis

DEFF Research Database (Denmark)

Fakouri, Nima Borhan; Durhuus, Jon Ambaek; Regnell, Christine Elisabeth

2017-01-01

(ADP) ribose polymerase 1 (PARP1) activity, low endogenous NAD+, low expression of SIRT1 and PGC1α and low adenosine monophosphate (AMP)-activated kinase (AMPK) activity. We conclude that replication stress via Rev1-deficiency contributes to metabolic stress caused by compromized mitochondrial function via...... the PARP-NAD+-SIRT1-PGC1α axis....

The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era

Directory of Open Access Journals (Sweden)

Ilana Zalcberg Renault

2011-12-01

Full Text Available Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging.

The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era

Science.gov (United States)

Renault, Ilana Zalcberg; Scholl, Vanesa; Hassan, Rocio; Capelleti, Paola; de Lima, Marcos; Cortes, Jorge

2011-01-01

Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging. PMID:23049363

Nicotine Component of Cigarette Smoke Extract (CSE) Decreases the Cytotoxicity of CSE in BEAS-2B Cells Stably Expressing Human Cytochrome P450 2A13.

Science.gov (United States)

Ji, Minghui; Zhang, Yudong; Li, Na; Wang, Chao; Xia, Rong; Zhang, Zhan; Wang, Shou-Lin

2017-10-13

Cytochrome P450 2A13 (CYP2A13), an extrahepatic enzyme mainly expressed in the human respiratory system, has been reported to mediate the metabolism and toxicity of cigarette smoke. We previously found that nicotine inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism by CYP2A13, but its influence on other components of cigarette smoke remains unclear. The nicotine component of cigarette smoke extract (CSE) was separated, purified, and identified using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), splitting CSE into a nicotine section (CSE-N) and nicotine-free section (CSE-O). Cell viability and apoptosis by Cell Counting Kit-8 (CCK-8) and flow cytometry assays were conducted on immortalized human bronchial epithelial (BEAS-2B) cells stably expressing CYP2A13 (B-2A13) or vector (B-V), respectively. Interestingly, CSE and CSE-O were toxic to BEAS-2B cells whereas CSE-N showed less cytotoxicity. CSE-O was more toxic to B-2A13 cells than to B-V cells (IC 50 of 2.49% vs. 7.06%), which was flatted by 8-methoxypsoralen (8-MOP), a CYP inhibitor. CSE-O rather than CSE or CSE-N increased apoptosis of B-2A13 cells rather than B-V cells. Accordingly, compared to CSE-N and CSE, CSE-O significantly changed the expression of three pairs of pro- and anti-apoptotic proteins, Bcl-2 Associated X Protein/B cell lymphoma-2 (Bax/Bcl-2), Cleaved Poly (Adenosine Diphosphate-Ribose) Polymerase/Poly (Adenosine Diphosphate-Ribose) Polymerase (C-PARP/PARP), and C-caspase-3/caspase-3, in B-2A13 cells. In addition, recombination of CSE-N and CSE-O (CSE-O/N) showed similar cytotoxicity and apoptosis to the original CSE. These results demonstrate that the nicotine component decreases the metabolic activation of CYP2A13 to CSE and aids in understanding the critical role of CYP2A13 in human respiratory diseases caused by cigarette smoking.

Lack of effect of inhibitors of DNA synthesis/repair on the ionizing radiation-induced chromosomal damage in G[sub 2] stage of ataxia telangiectasia cells

Energy Technology Data Exchange (ETDEWEB)

Antoccia, A. (Univ. ' La Sapienza' , Rome (Italy). Dipt. di Genetica e Biologia Molecolare); Palitti, F.; Raggi, T. (Univ. del Tuscia, Viterbo (Italy). Dipt. di Agrobiologia ed Agrochimica); Catena, C. (ENEA, Casaccia (Italy). Centro Ricerche Energia); Tanzarella, C. (Rome Univ. 3 (Italy). Dipt. di Biologia)

1994-09-01

The relationship between the repair processes occurring at the G[sub 2] phase of the cell cycle and cytogenetic damage in ataxia telangiectasia (AT) cells was studied. Lymphoblastoid cells derived from normal, heterozygote AT (HzAT) and three AT patients were exposed to X-rays or fission neutrons and post-treated with inhibitors of DNA synthesis/repair, such as inhibitors of DNA polymerases [alpha], [sigma] and [epsilon] (cytosine arabinoside, ara-C; aphidicolin, APC; buthylphenyl-guanine, BuPdG) or ribonucleotide reductase (hydroxyurea HU). A strong increase of radiation-induced chromosomal aberrations was observed in normal and HzAT cells post-treated with ara-C, APC and HU, but not in the presence of BuPdG. No enhancing effect was observed in cells derived from AT patients, except for HU post-irradiation treatment. These results suggest that the enzymes that can be inhibited by these agents are not directly involved in the repair of radiation damage induced in G[sub 2] cells from AT patients, indicating that probably the AT cells that we used lack the capability to transform the primary DNA lesions into reparable products, or that AT cells might contain a mutated form of DNA polymerase resistant to the inhibitors. (author).

Rapid emergence of hepatitis C virus protease inhibitor resistance is expected

Energy Technology Data Exchange (ETDEWEB)

Rong, Libin [Los Alamos National Laboratory; Perelson, Alan S [Los Alamos National Laboratory; Ribeiro, Ruy M [Los Alamos National Laboratory

2009-01-01

Approximately 170 million people worldwide are infected with hepatitis C virus (HCV). Current therapy, consisting of pegylated interferon (PEG-IFN) and ribavirin (RBV), leads to sustained viral elimination in only about 45% of patients treated. Telaprevir (VX-950), a novel HCV NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients with chronic hepatitis C genotype 1 infection. However, some patients experience viral breakthrough during dosing, with drug resistant variants being 5%-20% of the virus population as early as day 2 after treatment initiation. Why viral variants appear such a short time after the start of dosing is unclear, especially since this has not been seen with monotherapy for either human immunodeficiency virus or hepatitis B virus. Here, using a viral dynamic model, we explain why such rapid emergence of drug resistant variants is expected when potent HCV protease inhibitors are used as monotherapy. Surprisingly, our model also shows that such rapid emergence need not be the case with some potent HCV NS5B polymerase inhibitors. Examining the case of telaprevir therapy in detail, we show the model fits observed dynamics of both wild-type and drug-resistant variants during treatment, and supports combination therapy of direct antiviral drugs with PEG-IFN and/or RBV for hepatitis C.

In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir.

Science.gov (United States)

Cheng, Guofeng; Tian, Yang; Doehle, Brian; Peng, Betty; Corsa, Amoreena; Lee, Yu-Jen; Gong, Ruoyu; Yu, Mei; Han, Bin; Xu, Simin; Dvory-Sobol, Hadas; Perron, Michel; Xu, Yili; Mo, Hongmei; Pagratis, Nikos; Link, John O; Delaney, William

2016-01-11

Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50 values equivalent to those for the wild type. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Resistance analysis and characterization of NITD008 as an adenosine analog inhibitor against hepatitis C virus.

Science.gov (United States)

Qing, Jie; Luo, Rui; Wang, Yaxin; Nong, Junxiu; Wu, Ming; Shao, Yan; Tang, Ruoyi; Yu, Xi; Yin, Zheng; Sun, Yuna

2016-02-01

Hepatitis disease caused by hepatitis C virus (HCV) is a severe threat to global public health, affecting approximately 3% of the world's population. Sofosbuvir (PSI-7977), a uridine nucleotide analog inhibitor targeting the HCV NS5B polymerase, was approved by FDA at the end of 2013 and represents a key step towards a new era in the management of HCV infection. Previous study identified NITD008, an adenosine nucleoside analog, as the specific inhibitor against dengue virus and showed good antiviral effect on other flaviviruses or enteroviruses. In this report, we systematically analyzed the anti-HCV profile of NITD008, which was discovered to effectively suppress the replication of different strains of HCV in human hepatoma cells with a low nanomolar activity. On genotype 2a virus, or 2a, 1a, and 1b replicon cells, EC50 values were 8.7 nM, 93.3 nM, 60.0 nM and 67.2 nM, and selective index values were >2298.9, >214.4, >333.3, >298.5 respectively. We demonstrated that resistance to NITD008 was conferred by mutation in NS5B (S282T) in the HCV infectious virus genotype 2a (JFH-1). Then, we compared the resistant profiles of NITD008 and PSI-7977, and found that the folds change of EC50 of NITD008 to full replicon cells containing mutation S282T was much bigger than PSI-7977(folds 76.50 vs. 4.52). Analysis of NITD008 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of NITD008 was not completely similar to PSI-7977, and meanwhile, S282T resistant mutation to NITD008 emerge more easily in cell culture than PSI-7977. Interestingly, NITD008 displayed significant synergistic effects with the NS5B polymerase inhibitor PSI-7977, however, only additive effects with alpha interferon (IFNα-2b), ribavirin, and an NS3 protease inhibitor. These results verify that NITD008 is an effective analog inhibitor against hepatitis C virus and a good research tool as a supplement to other types of nucleoside analogs. Copyright �

General misincorporation frequency: Re-evaluation of the fidelity of DNA polymerases.

Science.gov (United States)

Yang, Jie; Li, Bianbian; Liu, Xiaoying; Tang, Hong; Zhuang, Xiyao; Yang, Mingqi; Xu, Ying; Zhang, Huidong; Yang, Chun

2018-02-19

DNA replication in cells is performed in the presence of four dNTPs and four rNTPs. In this study, we re-evaluated the fidelity of DNA polymerases using the general misincorporation frequency consisting of three incorrect dNTPs and four rNTPs but not using the traditional special misincorporation frequency with only the three incorrect dNTPs. We analyzed both the general and special misincorporation frequencies of nucleotide incorporation opposite dG, rG, or 8-oxoG by Pseudomonas aeruginosa phage 1 (PaP1) DNA polymerase Gp90 or Sulfolobus solfataricus DNA polymerase Dpo4. Both misincorporation frequencies of other DNA polymerases published were also summarized and analyzed. The general misincorporation frequency is obviously higher than the special misincorporation frequency for many DNA polymerases, indicating the real fidelity of a DNA polymerase should be evaluated using the general misincorporation frequency. Copyright © 2018 Elsevier Inc. All rights reserved.

CDK9-dependent RNA polymerase II pausing controls transcription initiation.

Science.gov (United States)

Gressel, Saskia; Schwalb, Björn; Decker, Tim Michael; Qin, Weihua; Leonhardt, Heinrich; Eick, Dirk; Cramer, Patrick

2017-10-10

Gene transcription can be activated by decreasing the duration of RNA polymerase II pausing in the promoter-proximal region, but how this is achieved remains unclear. Here we use a 'multi-omics' approach to demonstrate that the duration of polymerase pausing generally limits the productive frequency of transcription initiation in human cells ('pause-initiation limit'). We further engineer a human cell line to allow for specific and rapid inhibition of the P-TEFb kinase CDK9, which is implicated in polymerase pause release. CDK9 activity decreases the pause duration but also increases the productive initiation frequency. This shows that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time. CDK9 activity is also associated with long-range chromatin interactions, suggesting that enhancers can influence the pause-initiation limit to regulate transcription.

Dasatinib accelerates valproic acid-induced acute myeloid leukemia cell death by regulation of differentiation capacity.

Directory of Open Access Journals (Sweden)

Sook-Kyoung Heo

Full Text Available Dasatinib is a compound developed for chronic myeloid leukemia as a multi-targeted kinase inhibitor against wild-type BCR-ABL and SRC family kinases. Valproic acid (VPA is an anti-epileptic drug that also acts as a class I histone deacetylase inhibitor. The aim of this research was to determine the anti-leukemic effects of dasatinib and VPA in combination and to identify their mechanism of action in acute myeloid leukemia (AML cells. Dasatinib was found to exert potent synergistic inhibitory effects on VPA-treated AML cells in association with G1 phase cell cycle arrest and apoptosis induction involving the cleavage of poly (ADP-ribose polymerase and caspase-3, -7 and -9. Dasatinib/VPA-induced cell death thus occurred via caspase-dependent apoptosis. Moreover, MEK/ERK and p38 MAPK inhibitors efficiently inhibited dasatinib/VPA-induced apoptosis. The combined effect of dasatinib and VPA on the differentiation capacity of AML cells was more powerful than the effect of each drug alone, being sufficiently strong to promote AML cell death through G1 cell cycle arrest and caspase-dependent apoptosis. MEK/ERK and p38 MAPK were found to control dasatinib/VPA-induced apoptosis as upstream regulators, and co-treatment with dasatinib and VPA to contribute to AML cell death through the regulation of differentiation capacity. Taken together, these results indicate that combined dasatinib and VPA treatment has a potential role in anti-leukemic therapy.

Targeting Signaling Pathways in Epithelial Ovarian Cancer

Directory of Open Access Journals (Sweden)

Johannes Haybaeck

2013-05-01

Full Text Available Ovarian carcinoma (OC is the most lethal gynecological malignancy. Response to platinum-based chemotherapy is poor in some patients and, thus, current research is focusing on new therapy options. The various histological types of OC are characterized by distinctive molecular genetic alterations that are relevant for ovarian tumorigenesis. The understanding of these molecular pathways is essential for the development of novel therapeutic strategies. Purpose: We want to give an overview on the molecular genetic changes of the histopathological types of OC and their role as putative therapeutic targets. In Depth Review of Existing Data: In 2012, the vascular endothelial growth factor (VEGF inhibitor, bevacizumab, was approved for OC treatment. Bevacizumab has shown promising results as single agent and in combination with conventional chemotherapy, but its target is not distinctive when analyzed before treatment. At present, mammalian target of rapamycin (mTOR inhibitors, poly-ADP-ribose polymerase (PARP inhibitors and components of the EGFR pathway are in the focus of clinical research. Interestingly, some phytochemical substances show good synergistic effects when used in combination with chemotherapy. Conclusion: Ongoing studies of targeted agents in conjunction with chemotherapy will show whether there are alternative options to bevacizumab available for OC patients. Novel targets which can be assessed before therapy to predict efficacy are needed. The assessment of therapeutic targets is continuously improved by molecular pathological analyses on tumor tissue. A careful selection of patients for personalized treatment will help to reduce putative side effects and toxicity.

4-arylazo-3,5-diamino-1H-pyrazole CDK inhibitors: SAR study, crystal structure in complex with CDK2, selectivity, and cellular effects

Czech Academy of Sciences Publication Activity Database

Kryštof, Vladimír; Cankař, Petr; Fryšová, I.; Slouka, J.; Kontopidis, G.; Džubák, P.; Hajdúch, M.; Srovnal, J.; de Azevedo Jr., W.F.; Orság, Martin; Paprskářová, Martina; Rolčík, Jakub; Látr, Aleš; Fischer, P.M.; Strnad, Miroslav

2006-01-01

Roč. 49, č. 22 (2006), s. 6500-6509 ISSN 0022-2623 R&D Projects: GA ČR GA301/05/0418 Institutional research plan: CEZ:AV0Z50380511 Source of funding: V - iné verejné zdroje Keywords : DEPENDENT KINASE INHIBITORS * POLYMERASE-II TRANSCRIPTION * TUMOR-NECROSIS-FACTOR Subject RIV: CE - Biochemistry Impact factor: 5.115, year: 2006

File list: Pol.Dig.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

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Comparing the therapeutic efficiency of aminoguanidine and 3-aminobenzamide in lung and intestine toxicity caused by nitrogen mustard in rats

International Nuclear Information System (INIS)

Yaren, H.; Korkmaz, A.; Kunak, Z. I.; Uysal, B.; Topal, T.; Kurt, B; Kenar, L.

2009-01-01

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) and peroxynitrite are responsible for sulfur mustard (SM) induced toxicity. Since endogenous production of peroxynitrite is known to lead to poly(ADP-ribose) polymerase (PARP) activation and sometimes ultimately cell death, in this study, it was aimed to compare the therapeutic efficiencies of aminoguanidine (iNOS inhibitor) and 3 aminobenzamide (PARP inhibitor) in lung and intestine toxicity caused by nitrogen mustard in rats. A total of 40 male Sprague-Dawley rats were divided into 4 groups. Group 1 served as control and given 2 ml saline, three groups received single dose of mechlorethamine (MEC) (3.5 mg/kg subcutaneously) with the same time intervals. Group 2 received MEC only, group 3 received selective iNOS inhibitor aminoguanidine (AG) (100 mg/kg i.p.) and, group 4 received PARP inhibitor 3 aminobenzamide (3-AB) (20 mg/kg i.p.). MEC injection resulted in severe lung toxicity with strong interstitial and alveolar edema, hemorrhage, emphysematous changes, Mild inflammatory cell infiltration and septal thickening. MEC injection also caused mucosal thinning, mild inflammatory cell infiltration, ischemic changes and multifocal, superficial ulcerations (erosions) in small intestine. In AG group, interstitial and alveolar edema, hemorrhage slightly reduced in lung comparing to MEC group. Inflammatory cell infiltration was minimal, septal thickening was similar to MEC group at densely edematous and hemorrhagical areas. In 3 AB group, edematous and hemorrhagic areas were very small, inflammatory cell infiltration was minimal and there were no densly densely edematous and hemorrhagical areas in lung. The results were better than AB group. In intestine, results of AG group were better than MEC group but worse than 3 AB group. These results suggest that both iNOS and PARP inhibitors are effective but PARP inhibitors may be more promising for treatment of SM induced early lung and intestinal toxicity.(author)

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Lifescience Database Archive (English)

Full Text Available Pol.Lar.50.RNA_polymerase_III.AllCell ce10 RNA polymerase RNA polymerase III Larvae... http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Lar.50.RNA_polymerase_III.AllCell.bed ...

File list: Pol.Oth.20.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Oth.20.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Others ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Oth.20.RNA_Polymerase_III.AllCell.bed ...

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

Lifescience Database Archive (English)

Full Text Available Pol.Plc.50.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Placenta... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Plc.50.RNA_polymerase_II.AllCell.bed ...

File list: Pol.Oth.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Oth.05.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Others ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Oth.05.RNA_Polymerase_III.AllCell.bed ...

File list: Pol.Myo.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Myo.05.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Muscle ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Myo.05.RNA_Polymerase_III.AllCell.bed ...

File list: Pol.ALL.20.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.ALL.20.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II All cell ...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.ALL.20.RNA_Polymerase_II.AllCell.bed ...

File list: Pol.Brs.50.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Brs.50.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Breast ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Brs.50.RNA_Polymerase_III.AllCell.bed ...

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

Lifescience Database Archive (English)

Full Text Available Pol.Lar.20.RNA_polymerase_III.AllCell ce10 RNA polymerase RNA polymerase III Larvae... http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Lar.20.RNA_polymerase_III.AllCell.bed ...

File list: Pol.Emb.05.RNA_Polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Emb.05.RNA_Polymerase_III.AllCell mm9 RNA polymerase RNA Polymerase III Embryo ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.05.RNA_Polymerase_III.AllCell.bed ...

File list: Pol.Emb.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Emb.10.RNA_polymerase_III.AllCell ce10 RNA polymerase RNA polymerase III Embryo... http://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Emb.10.RNA_polymerase_III.AllCell.bed ...

File list: Pol.Epd.10.RNA_Polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Epd.10.RNA_Polymerase_II.AllCell mm9 RNA polymerase RNA Polymerase II Epidermis... http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Epd.10.RNA_Polymerase_II.AllCell.bed ...

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

Lifescience Database Archive (English)

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

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

Lifescience Database Archive (English)

Full Text Available Pol.Unc.50.RNA_polymerase_II.AllCell ce10 RNA polymerase RNA polymerase II Unclassi...p://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Unc.50.RNA_polymerase_II.AllCell.bed ...

File list: Pol.Plc.10.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Plc.10.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Placenta... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Plc.10.RNA_polymerase_II.AllCell.bed ...

File list: Pol.Myo.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Myo.10.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Muscle... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Myo.10.RNA_polymerase_III.AllCell.bed ...

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

Lifescience Database Archive (English)

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

File list: Pol.Brs.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Brs.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Breast... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Brs.05.RNA_polymerase_III.AllCell.bed ...

File list: Pol.Plc.05.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Plc.05.RNA_polymerase_II.AllCell hg19 RNA polymerase RNA polymerase II Placenta... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Plc.05.RNA_polymerase_II.AllCell.bed ...

File list: Pol.Unc.20.RNA_polymerase_II.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Unc.20.RNA_polymerase_II.AllCell ce10 RNA polymerase RNA polymerase II Unclassi...p://dbarchive.biosciencedbc.jp/kyushu-u/ce10/assembled/Pol.Unc.20.RNA_polymerase_II.AllCell.bed ...

File list: Pol.Oth.50.RNA_polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Oth.50.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Others... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Oth.50.RNA_polymerase_III.AllCell.bed ...

File list: Pol.Brs.10.RNA_polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Brs.10.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Breast... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Brs.10.RNA_polymerase_III.AllCell.bed ...

File list: Pol.Liv.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Liv.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Liver ...http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Liv.05.RNA_polymerase_III.AllCell.bed ...

File list: Pol.Myo.05.RNA_polymerase_III.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Pol.Myo.05.RNA_polymerase_III.AllCell hg19 RNA polymerase RNA polymerase III Muscle... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Myo.05.RNA_polymerase_III.AllCell.bed ...

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

Lifescience Database Archive (English)