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Sample records for protect cellular dna

  1. Intraspleen DNA inoculation elicits protective cellular immune responses.

    PubMed

    Cano, A; Fragoso, G; Gevorkian, G; Terrazas, L I; Petrossian, P; Govezensky, T; Sciutto, E; Manoutcharian, K

    2001-04-01

    DNA immunization or inoculation is a recent vaccination method that induces both humoral and cellular immune responses in a range of hosts. Independent of the route or site of vaccination, the transfer of antigen-presenting cells (APC) or antigens into lymphoid organs is necessary. The aim of this investigation was to test whether intraspleen (i.s.) DNA inoculation is capable of inducing a protective immune response. We immunized mice by a single i.s. injection of a DNA construct expressing the immunoglobulin (Ig) heavy-chain variable domain (VH) in which the complementarity-determining regions (CDR) had been replaced by a Taenia crassiceps T-cell epitope. In these mice, immune responses and protective effects elicited by the vaccine were measured. We have shown here for the first time that i.s. DNA inoculation can induce protective cellular immune responses and activate CD8(+) T cells. Also, Ig V(H) appeared to be the minimal delivery unit of "antigenized" Ig capable of inducing T-cell activation in a lymphoid organ. The strategy of introducing T-cell epitopes into the molecular context of the V(H) domain in combination with i.s. DNA immunization could have important implications and applications for human immunotherapy.

  2. Polyphenolic compounds from Salvia species protect cellular DNA from oxidation and stimulate DNA repair in cultured human cells.

    PubMed

    Ramos, Alice A; Azqueta, Amaya; Pereira-Wilson, Cristina; Collins, Andrew R

    2010-06-23

    DNA damage can lead to carcinogenesis if replication proceeds without proper repair. This study evaluated the effects of the water extracts of three Salvia sp., Salvia officinalis (SO), Salvia fruticosa (SF), and Salvia lavandulifolia (SL), and of the major phenolic constituents, rosmarinic acid (RA) and luteolin-7-glucoside (L-7-G), on DNA protection in Caco-2 and HeLa cells exposed to oxidative agents and on DNA repair in Caco-2 cells. The comet assay was used to measure DNA damage and repair capacity. The final concentration of each sage extract was 50 microg/mL, and concentrations of RA and L-7-G were 50 and 20 microM, respectively. After a short incubation (2 h), L-7-G protected DNA in Caco-2 cells from damage induced by H(2)O(2) (75 microM); also, after a long incubation (24 h), SF, RA, and L-7-G had protective effects in Caco-2 cells. In HeLa cells, SO, SF, and RA protected against damage induced by H(2)O(2) after 24 h of incubation. Assays of DNA repair show that SO, SF, and L-7-G increased the rate of DNA repair (rejoining of strand breaks) in Caco-2 cells treated with H(2)O(2). The incision activity of a Caco-2 cell extract on a DNA substrate containing specific damage (8-oxoGua) was also measured to evaluate effects on base excision repair (BER) activity. Preincubation for 24 h with SO and L-7-G had a BER inductive effect, increasing incision activity in Caco-2 cells. In conclusion, SO, SF, and the isolated compounds (RA and L-7-G) demonstrated chemopreventive activity by protecting cells against oxidative DNA damage and stimulating DNA repair (SO, SF, and L-7-G).

  3. Polyphenolic compounds from Salvia species protect cellular DNA from oxidation and stimulate DNA repair in cultured human cells.

    PubMed

    Ramos, Alice A; Azqueta, Amaya; Pereira-Wilson, Cristina; Collins, Andrew R

    2010-06-23

    DNA damage can lead to carcinogenesis if replication proceeds without proper repair. This study evaluated the effects of the water extracts of three Salvia sp., Salvia officinalis (SO), Salvia fruticosa (SF), and Salvia lavandulifolia (SL), and of the major phenolic constituents, rosmarinic acid (RA) and luteolin-7-glucoside (L-7-G), on DNA protection in Caco-2 and HeLa cells exposed to oxidative agents and on DNA repair in Caco-2 cells. The comet assay was used to measure DNA damage and repair capacity. The final concentration of each sage extract was 50 microg/mL, and concentrations of RA and L-7-G were 50 and 20 microM, respectively. After a short incubation (2 h), L-7-G protected DNA in Caco-2 cells from damage induced by H(2)O(2) (75 microM); also, after a long incubation (24 h), SF, RA, and L-7-G had protective effects in Caco-2 cells. In HeLa cells, SO, SF, and RA protected against damage induced by H(2)O(2) after 24 h of incubation. Assays of DNA repair show that SO, SF, and L-7-G increased the rate of DNA repair (rejoining of strand breaks) in Caco-2 cells treated with H(2)O(2). The incision activity of a Caco-2 cell extract on a DNA substrate containing specific damage (8-oxoGua) was also measured to evaluate effects on base excision repair (BER) activity. Preincubation for 24 h with SO and L-7-G had a BER inductive effect, increasing incision activity in Caco-2 cells. In conclusion, SO, SF, and the isolated compounds (RA and L-7-G) demonstrated chemopreventive activity by protecting cells against oxidative DNA damage and stimulating DNA repair (SO, SF, and L-7-G). PMID:20486687

  4. Protection of cellular DNA from gamma-radiation-induced damages and enhancement in DNA repair by troxerutin.

    PubMed

    Maurya, Dharmendra Kumar; Balakrishnan, Sreedevi; Salvi, Veena Prakash; Nair, Cherupally Krishnan Krishnan

    2005-12-01

    The effect of troxerutin on gamma-radiation-induced DNA strand breaks in different tissues of mice in vivo and formations of the micronuclei were studied in human peripheral blood lymphocytes ex vivo and mice blood reticulocytes in vivo. Treatments with 1 mM troxerutin significantly inhibited the micronuclei induction in the human lymphocytes. Troxerutin protected the human peripheral blood leucocytes from radiation-induced DNA strand breaks in a concentration dependent manner under ex vivo condition of irradiation (2 Gy). Intraperitoneal administration of troxerutin (175 mg/kg body weight) to mice before and after whole body radiation exposure inhibited micronuclei formation in blood reticulocytes significantly. The administration of different doses (75, 125 and 175 mg/kg body weight) of troxerutin 1 h prior to 4 Gy gamma-radiation exposure showed dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and bone marrow cells. The dose-dependent protection was more pronounced in bone marrow cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1 h prior or immediately after whole body irradiation of mice showed that the decrease in strand breaks depended on the post-irradiation interval at which the analysis was done. The observed time-dependent decrease in the DNA strand breaks could be attributed to enhanced DNA repair in troxerutin administered animals. Thus in addition to anti-erythrocytic, anti-thrombic, fibrinolytic and oedema-protective rheological activity, troxerutin offers protection against gamma-radiation-induced micronuclei formation and DNA strand breaks and enhances repair of radiation-induced DNA strand breaks.

  5. Intradermal DNA Electroporation Induces Cellular and Humoral Immune Response and Confers Protection against HER2/neu Tumor.

    PubMed

    Lamolinara, Alessia; Stramucci, Lorenzo; Hysi, Albana; Iezzi, Manuela; Marchini, Cristina; Mariotti, Marianna; Amici, Augusto; Curcio, Claudia

    2015-01-01

    Skin represents an attractive target for DNA vaccine delivery because of its natural richness in APCs, whose targeting may potentiate the effect of vaccination. Nevertheless, intramuscular electroporation is the most common delivery method for ECTM vaccination. In this study we assessed whether intradermal administration could deliver the vaccine into different cell types and we analyzed the evolution of tissue infiltrate elicited by the vaccination protocol. Intradermal electroporation (EP) vaccination resulted in transfection of different skin layers, as well as mononuclear cells. Additionally, we observed a marked recruitment of reactive infiltrates mainly 6-24 hours after treatment and inflammatory cells included CD11c(+). Moreover, we tested the efficacy of intradermal vaccination against Her2/neu antigen in cellular and humoral response induction and consequent protection from a Her2/neu tumor challenge in Her2/neu nontolerant and tolerant mice. A significant delay in transplantable tumor onset was observed in both BALB/c (p ≤ 0,0003) and BALB-neuT mice (p = 0,003). Moreover, BALB-neuT mice displayed slow tumor growth as compared to control group (p < 0,0016). In addition, while in vivo cytotoxic response was observed only in BALB/c mice, a significant antibody response was achieved in both mouse models. Our results identify intradermal EP vaccination as a promising method for delivering Her2/neu DNA vaccine. PMID:26247038

  6. Reciprocal Paracrine Interactions Between Normal Human Epithelial and Mesenchymal Cells Protect Cellular DNA from Radiation-Induced Damage

    SciTech Connect

    Nakazawa, Yuka; Saenko, Vladimir Rogounovitch, Tatiana; Suzuki, Keiji; Mitsutake, Norisato; Matsuse, Michiko; Yamashita, Shunichi

    2008-06-01

    Purpose: To explore whether interactions between normal epithelial and mesenchymal cells can modulate the extent of radiation-induced DNA damage in one or both types of cells. Methods and Materials: Human primary thyrocytes (PT), diploid fibroblasts BJ, MRC-5, and WI-38, normal human mammary epithelial cells (HMEC), and endothelial human umbilical cord vein endothelial cells (HUV-EC-C), cultured either individually or in co-cultures or after conditioned medium transfer, were irradiated with 0.25 to 5 Gy of {gamma}-rays and assayed for the extent of DNA damage. Results: The number of {gamma}-H2AX foci in co-cultures of PT and BJ fibroblasts was approximately 25% lower than in individual cultures at 1 Gy in both types of cells. Reciprocal conditioned medium transfer to individual cultures before irradiation resulted in approximately a 35% reduction of the number {gamma}-H2AX foci at 1 Gy in both types of cells, demonstrating the role of paracrine soluble factors. The DNA-protected state of cells was achieved within 15 min after conditioned medium transfer; it was reproducible and reciprocal in several lines of epithelial cells and fibroblasts, fibroblasts, and endothelial cells but not in epithelial and endothelial cells. Unlike normal cells, human epithelial cancer cells failed to establish DNA-protected states in fibroblasts and vice versa. Conclusions: The results imply the existence of a network of reciprocal interactions between normal epithelial and some types of mesenchymal cells mediated by soluble factors that act in a paracrine manner to protect DNA from genotoxic stress.

  7. Protective effect of ferulic acid on nicotine-induced DNA damage and cellular changes in cultured rat peripheral blood lymphocytes: a comparison with N-acetylcysteine.

    PubMed

    Sudheer, Adluri Ram; Muthukumaran, Shanmugavelu; Kalpana, Chandran; Srinivasan, Marimuthu; Menon, Venugopal Padmanabhan

    2007-06-01

    Nicotine is the major pharmacologically active substance in cigarette smoke and plays an important etiological role in the development of lung cancer. Incidence of cancer may be related to oxidative damage to host genome by nicotine. These oxidative actions may be modified by the phytochemicals present in food. The present study describes the protective effect of ferulic acid (FA), a naturally occurring nutritional compound on nicotine-induced DNA damage and cellular changes in cultured rat peripheral blood lymphocytes in comparison with N-acetylcysteine (NAC), a well-known antioxidant. One-hour exposure of lymphocytes to nicotine at the doses of 0.125, 0.25, 0.5, 1, 2, 3 and 4 mM induced a statistically significant dose-dependent increase in the levels of thiobarbituric acid reactive substances (TBARS), a lipid peroxidative marker and decrease in the levels of reduced glutathione (GSH), an important endogenous antioxidant. The lowest concentration eliciting significant damage was 1 mM nicotine and maximum damage was observed with 3 mM concentration. Hence, the test concentration was fixed at 3 mM nicotine. We have used 5 different doses of FA (10, 50, 100, 150 and 300 microM) and NAC (0.25, 0.5, 1, 2 and 4 mM) to test their protective effects. In all the groups, FA and NAC showed a dose-dependent inhibitory effect. Maximum protection was observed at the dose of 150 microM FA and 1mM NAC. So, 150 microM FA and 1mM NAC were used for further studies. There was a significant increase in the levels of lipid peroxidative index (TBARS and hydroperoxides (HP)), severity of DNA damage (evaluated by comet assay) in nicotine-treated group, which were significantly decreased in FA and NAC-treated groups. Nicotine treatment significantly decreased the endogenous antioxidant status viz., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), GSH, vitamin A, E and C. Co-administration of FA and NAC to nicotine-treated lymphocytes showed a significant increase

  8. Cellular responses to environmental DNA damage

    SciTech Connect

    Not Available

    1994-08-01

    This volume contains the proceedings of the conference entitled Cellular Responses to Environmental DNA Damage held in Banff,Alberta December 1--6, 1991. The conference addresses various aspects of DNA repair in sessions titled DNA repair; Basic Mechanisms; Lesions; Systems; Inducible Responses; Mutagenesis; Human Population Response Heterogeneity; Intragenomic DNA Repair Heterogeneity; DNA Repair Gene Cloning; Aging; Human Genetic Disease; and Carcinogenesis. Individual papers are represented as abstracts of about one page in length.

  9. [Pulse-modulated Electromagnetic Radiation of Extremely High Frequencies Protects Cellular DNA against Damaging Effect of Physico-Chemical Factors in vitro].

    PubMed

    Gapeyev, A B; Lukyanova, N A

    2015-01-01

    Using a comet assay technique, we investigated protective effects of. extremely high frequency electromagnetic radiation in combination with the damaging effect of X-ray irradiation, the effect of damaging agents hydrogen peroxide and methyl methanesulfonate on DNA in mouse whole blood leukocytes. It was shown that the preliminary exposure of the cells to low intensity pulse-modulated electromagnetic radiation (42.2 GHz, 0.1 mW/cm2, 20-min exposure, modulation frequencies of 1 and 16 Hz) caused protective effects decreasing the DNA damage by 20-45%. The efficacy of pulse-modulated electromagnetic radiation depended on the type of genotoxic agent and increased in a row methyl methanesulfonate--X-rays--hydrogen peroxide. Continuous electromagnetic radiation was ineffective. The mechanisms of protective effects may be connected with an induction of the adaptive response by nanomolar concentrations of reactive oxygen species formed by pulse-modulated electromagnetic radiation.

  10. [Pulse-modulated Electromagnetic Radiation of Extremely High Frequencies Protects Cellular DNA against Damaging Effect of Physico-Chemical Factors in vitro].

    PubMed

    Gapeyev, A B; Lukyanova, N A

    2015-01-01

    Using a comet assay technique, we investigated protective effects of. extremely high frequency electromagnetic radiation in combination with the damaging effect of X-ray irradiation, the effect of damaging agents hydrogen peroxide and methyl methanesulfonate on DNA in mouse whole blood leukocytes. It was shown that the preliminary exposure of the cells to low intensity pulse-modulated electromagnetic radiation (42.2 GHz, 0.1 mW/cm2, 20-min exposure, modulation frequencies of 1 and 16 Hz) caused protective effects decreasing the DNA damage by 20-45%. The efficacy of pulse-modulated electromagnetic radiation depended on the type of genotoxic agent and increased in a row methyl methanesulfonate--X-rays--hydrogen peroxide. Continuous electromagnetic radiation was ineffective. The mechanisms of protective effects may be connected with an induction of the adaptive response by nanomolar concentrations of reactive oxygen species formed by pulse-modulated electromagnetic radiation. PMID:26591599

  11. Ellagic acid, a natural polyphenol protects rat peripheral blood lymphocytes against nicotine-induced cellular and DNA damage in vitro: with the comparison of N-acetylcysteine.

    PubMed

    Sudheer, Adluri Ram; Muthukumaran, Shanmugavelu; Devipriya, Nagarajan; Menon, Venugopal Padmanabhan

    2007-01-25

    The present work is aimed at evaluating the protective effect of ellagic acid (EA), a natural polyphenolic compound that is widely distributed in fruits and nuts against nicotine-induced toxicity in rat peripheral blood lymphocytes. The effect of EA against nicotine toxicity was compared with N-acetylcysteine (NAC), a well-known antioxidant. Lymphocytes were exposed to nicotine at the doses of 0.125, 0.25, 0.5, 1, 2, 3 and 4 mM for 1h in culture media. Thiobarbituric acid reactive substances (TBARS), a lipid peroxidative marker and reduced glutathione (GSH), as indicative of endogenous antioxidant status were analyzed to fix the optimum dose. The lowest concentration eliciting significant damage was 1 mM nicotine and maximum damage was observed with 3 mM concentration, as evidenced by increased levels of TBARS and decreased levels of GSH. Hence, the test concentration was fixed at 3 mM nicotine. To establish most effective protective support we used five different concentrations of EA (10, 50, 100, 150 and 300 microM) against 3 mM nicotine. A dose-dependent inhibitory effect was observed with all doses of EA. Maximum protection was observed at the dose of 100 microM EA. So, 100 microM dose was used for further studies. We have tested five different concentrations of NAC-0.25, 0.5, 1, 2 and 4 mM to elucidate the optimum protective dose against nicotine toxicity. One millimolar NAC showed a significant protection against nicotine toxicity. Protective effect of EA against nicotine toxicity was elucidated by analyzing the lipid peroxidative index, viz., TBARS, hydroperoxides (HP) and endogenous antioxidant status, viz., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), Vitamins A, E and C. DNA damage and repair were assessed by using alkaline single-cell microgel electrophoresis (Comet assay) and micronucleus assay. There was a significant increase in the levels of lipid peroxidative index, severity in DNA damage and

  12. The mitochondrial nucleoid: integrating mitochondrial DNA into cellular homeostasis.

    PubMed

    Gilkerson, Robert; Bravo, Liliana; Garcia, Iraselia; Gaytan, Norma; Herrera, Alan; Maldonado, Alicia; Quintanilla, Brandi

    2013-05-01

    The packaging of mitochondrial DNA (mtDNA) into DNA-protein assemblies called nucleoids provides an efficient segregating unit of mtDNA, coordinating mtDNA's involvement in cellular metabolism. From the early discovery of mtDNA as "extranuclear" genetic material, its organization into nucleoids and integration into both the mitochondrial organellar network and the cell at large via a variety of signal transduction pathways, mtDNA is a crucial component of the cell's homeostatic network. The mitochondrial nucleoid is composed of a set of DNA-binding core proteins involved in mtDNA maintenance and transcription, and a range of peripheral factors, which are components of signaling pathways controlling mitochondrial biogenesis, metabolism, apoptosis, and retrograde mitochondria-to-nucleus signaling. The molecular interactions of nucleoid components with the organellar network and cellular signaling pathways provide exciting clues to the dynamic integration of mtDNA into cellular metabolic homeostasis.

  13. p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus

    NASA Astrophysics Data System (ADS)

    Puisieux, Alain; Ji, Jingwei; Guillot, Celine; Legros, Yann; Soussi, Thierry; Isselbacher, Kurt; Ozturk, Mehmet

    1995-02-01

    Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G_1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

  14. REC-2006-A Fractionated Extract of Podophyllum hexandrum Protects Cellular DNA from Radiation-Induced Damage by Reducing the Initial Damage and Enhancing Its Repair In Vivo.

    PubMed

    Chaudhary, Pankaj; Shukla, Sandeep Kumar; Sharma, Rakesh Kumar

    2011-01-01

    Podophyllum hexandrum, a perennial herb commonly known as the Himalayan May Apple, is well known in Indian and Chinese traditional systems of medicine. P. hexandrum has been widely used for the treatment of venereal warts, skin infections, bacterial and viral infections, and different cancers of the brain, lung and bladder. This study aimed at elucidating the effect of REC-2006, a bioactive fractionated extract from the rhizome of P. hexandrum, on the kinetics of induction and repair of radiation-induced DNA damage in murine thymocytes in vivo. We evaluated its effect on non-specific radiation-induced DNA damage by the alkaline halo assay in terms of relative nuclear spreading factor (RNSF) and gene-specific radiation-induced DNA damage via semi-quantitative polymerase chain reaction. Whole body exposure of animals with gamma rays (10 Gy) caused a significant amount of DNA damage in thymocytes (RNSF values 17.7 ± 0.47, 12.96 ± 1.64 and 3.3 ± 0.014) and a reduction in the amplification of β-globin gene to 0, 28 and 43% at 0, 15 and 60 min, respectively. Administrating REC-2006 at a radioprotective concentration (15 mg kg(-1) body weight) 1 h before irradiation resulted in time-dependent reduction of DNA damage evident as a decrease in RNSF values 6.156 ± 0.576, 1.647 ± 0.534 and 0.496 ± 0.012, and an increase in β-globin gene amplification 36, 95 and 99%, at 0, 15 and 60 min, respectively. REC-2006 scavenged radiation-induced hydroxyl radicals in a dose-dependent manner stabilized DPPH free radicals and also inhibited superoxide anions. Various polyphenols and flavonoides present in REC-2006 might contribute to scavenging of radiation-induced free radicals, thereby preventing DNA damage and stimulating its repair.

  15. Ejecting Phage DNA against Cellular Turgor Pressure

    PubMed Central

    Marion, Sanjin; Šiber, Antonio

    2014-01-01

    We examine in vivo ejection of noncondensed DNA from tailed bacteriophages into bacteria. The ejection is dominantly governed by the physical conditions in the bacteria. The confinement of the DNA in the virus capsid only slightly helps the ejection, becoming completely irrelevant during its last stages. A simple calculation based on the premise of condensed DNA in the cell enables us to estimate the maximal bacterial turgor pressure against which the ejection can still be fully realized. The calculated pressure (∼5 atm) shows that the ejection of DNA into Gram-negative bacteria could proceed spontaneously, i.e., without the need to invoke active mechanisms. PMID:25418173

  16. Ejecting phage DNA against cellular turgor pressure.

    PubMed

    Marion, Sanjin; Siber, Antonio

    2014-10-21

    We examine in vivo ejection of noncondensed DNA from tailed bacteriophages into bacteria. The ejection is dominantly governed by the physical conditions in the bacteria. The confinement of the DNA in the virus capsid only slightly helps the ejection, becoming completely irrelevant during its last stages. A simple calculation based on the premise of condensed DNA in the cell enables us to estimate the maximal bacterial turgor pressure against which the ejection can still be fully realized. The calculated pressure (~5 atm) shows that the ejection of DNA into Gram-negative bacteria could proceed spontaneously, i.e., without the need to invoke active mechanisms.

  17. DNA origami: Nanorobots grab cellular control

    NASA Astrophysics Data System (ADS)

    Elbaz, Johann; Willner, Itamar

    2012-04-01

    Self-assembled barrel-like DNA nanostructures carrying active payloads and pre-programmed with logic operations to reconfigure in response to cell-surface cues can trigger a variety of intracellular functions.

  18. Characterization of cellular and extracellular DNA in saliva.

    PubMed

    Taki, Takashi; Kibayashi, Kazuhiko

    2015-11-01

    Although the presence of extracellular DNA in various body fluids was discovered long ago, only recently has it begun to attract attention for examining the genetic profiles of individuals in forensics studies. However, information on extracellular DNA is scarce. Among human body fluids, saliva is known to be rich in extracellular DNA. In this study, to analyze the possibility of identifying individuals and body fluids by using only extracellular DNA of saliva, we investigated the amount, size distribution, short tandem repeat (STR) profile, and methylation pattern of extracellular DNA from saliva and compared these with those of cellular DNA. The amount and size distribution of extracellular DNA was different from that of cellular DNA. However, their respective STR profiles were the same. The methylation patterns of the BCAS4 gene were different among donors, but no significant difference was observed between cellular and extracellular DNA. The results of our study suggest that identification of individuals and body fluids from saliva may be possible without the need for cells.

  19. Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity.

    PubMed

    Habermeyer, Michael; Fritz, Jessica; Barthelmes, Hans U; Christensen, Morten O; Larsen, Morten K; Boege, Fritz; Marko, Doris

    2005-09-01

    In the present study, we investigated the effect of anthocyanidins on human topoisomerases I and II and its relevance for DNA integrity within human cells. Anthocyanidins bearing vicinal hydroxy groups at the B-ring (delphinidin, DEL; cyanidin, CY) were found to potently inhibit the catalytic activity of human topoisomerases I and II, without discriminating between the IIalpha and the IIbeta isoforms. However, in contrast to topoisomerase poisons, DEL and CY did not stabilize the covalent DNA-topoisomerase intermediates (cleavable complex) of topoisomerase I or II. Using recombinant topoisomerase I, the presence of CY or DEL (> or = 1 microM) effectively prohibited the stabilization of the cleavable complex by the topoisomerase I poison camptothecin. We furthermore investigated whether the potential protective effect vs topoisomerase I poisons is reflected also on the cellular level, affecting the DNA damaging properties of camptothecin. Indeed, in HT29 cells, low micromolar concentrations of DEL (1-10 microM) significantly diminished the DNA strand breaking effect of camptothecin (100 microM). However, at concentrations > or = 50 microM, all anthocyanidins tested (delphinidin, cyanidin, malvidin, pelargonidin, and paeonidin), including those not interfering with topoisomerases, were found to induce DNA strand breaks in the comet assay. All of these analogues were able to compete with ethidium bromide for the intercalation into calf thymus DNA and to replace the minor groove binder Hoechst 33258. These data indicate substantial affinity to double-stranded DNA, which might contribute at least to the DNA strand breaking effect of anthocyanidins at higher concentrations (> or = 50 microM).

  20. Analysis of cellular and extracellular DNA in fingerprints

    SciTech Connect

    Button, Julie M.

    2014-09-09

    It has been previously shown that DNA can be recovered from latent fingerprints left on various surfaces [R. A. H. van Oorschot and M. K. Jones, Nature 387, 767 (1997)]. However, the source of the DNA, extracellular versus cellular origin, is difficult to determine. If the DNA is cellular, it is believed to belong to skin cells while extracellular DNA is believed to originate from body fluids such as sweat [D. J. Daly et. al, Forensic Sci. Int. Genet. 6, 41-46 (2012); V. V. Vlassov et. al, BioEssays 29, 654-667 (2007)]. The origin of the DNA in fingerprints has implications for processing and interpretation of forensic evidence. The determination of the origin of DNA in fingerprints is further complicated by the fact that the DNA in fingerprints tends to be at a very low quantity [R. A. H. van Oorschot and M. K. Jones, Nature 387, 767 (1997)]. This study examined fingerprints from five volunteers left on sterilized glass slides and plastic pens. Three fingerprints were left on each glass slide (thumb, index, and middle fingers) while the pens were held as if one was writing with them. The DNA was collected from the objects using the wet swabbing technique (TE buffer). Following collection, the cellular and extracellular components of each sample were separated using centrifugation and an acoustofluidics system. Centrifugation is still the primary separation technique utilized in forensics laboratories, while acoustic focusing uses sound waves to focus large particles (cells) into low pressure nodes, separating them from the rest of the sample matrix. After separation, all samples were quantified using real-time quantitative PCR (qPCR). The overall trend is that there is more DNA in the extracellular fractions than cellular fractions for both centrifugation and acoustofluidic processing. Additionally, more DNA was generally collected from the pen samples than the samples left on glass slides.

  1. Does a DNA-less cellular organism exist on Earth?

    PubMed

    Hiyoshi, Akira; Miyahara, Kohji; Kato, Chiaki; Ohshima, Yasumi

    2011-12-01

    All the self-reproducing cellular organisms so far examined have DNA as the genome. However, a DNA-less organism carrying an RNA genome is suggested by the fact that many RNA viruses exist and the widespread view that an RNA world existed before the present DNA world. Such a possibility is most plausible in the microbial world where biological diversity is enormous and most organisms have not been identified. We have developed experimental methodology to search DNA-less microorganisms, which is based on cultivation with drugs that inhibit replication or expression of DNA, detection of DNA in colonies with a fluorescent dye and double staining for DNA and RNA at a cellular level. These methods have been applied for about 100 microbial samples from various waters including hot springs, soils including deep sea sediments, and organisms. We found many colonies and cells which apparently looked DNA-less and examined them further. So far, all such colonies that reformed colonies on isolation were identified to be DNA-positive. However, considering the difficulty in cultivation, we think it possible for DNA-less microorganisms to live around us. We believe that our ideas and results will be of interest and useful to discover one in the future.

  2. Protein ADP-ribosylation and the cellular response to DNA strand breaks.

    PubMed

    Caldecott, K W

    2014-07-01

    DNA strand breaks arise continuously in cells and can lead to chromosome rearrangements and genome instability or cell death. The commonest DNA breaks are DNA single-strand breaks, which arise at a frequency of tens-of-thousands per cell each day and which can block the progression of RNA/DNA polymerases and disrupt gene transcription and genome duplication. If not rapidly repaired, SSBs can be converted into DNA double-strand breaks (DSBs) during genome duplication, eliciting a complex series of DNA damage responses that attempt to protect cells from irreversible replication fork collapse. DSBs are the most cytotoxic and clastogenic type of DNA breaks, and can also arise independently of DNA replication, albeit at a frequency several orders of magnitude lower than SSBs. Here, I discuss the evidence that DNA single- and double -strand break repair pathways, and cellular tolerance mechanisms for protecting replication forks during genome duplication, utilize signalling by protein ADP-ribosyltransferases to protect cells from the harmful impact of DNA strand breakage.

  3. Cellular sensing of viral DNA and viral evasion mechanisms.

    PubMed

    Orzalli, Megan H; Knipe, David M

    2014-01-01

    Mammalian cells detect foreign DNA introduced as free DNA or as a result of microbial infection, leading to the induction of innate immune responses that block microbial replication and the activation of mechanisms that epigenetically silence the genes encoded by the foreign DNA. A number of DNA sensors localized to a variety of sites within the cell have been identified, and this review focuses on the mechanisms that detect viral DNA and how the resulting responses affect viral infections. Viruses have evolved mechanisms that inhibit these host sensors and signaling pathways, and the study of these antagonistic viral strategies has provided insight into the mechanisms of these host responses. The field of cellular sensing of foreign DNA is in its infancy, but our currently limited knowledge has raised a number of important questions for study.

  4. DNA vaccines targeting heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge.

    PubMed

    Scott, Veronica L; Villarreal, Daniel O; Hutnick, Natalie A; Walters, Jewell N; Ragwan, Edwin; Bdeir, Khalil; Yan, Jian; Sardesai, Niranjan Y; Finnefrock, Adam C; Casimiro, Danilo R; Weiner, David B

    2015-01-01

    Botulinum neurotoxins (BoNTs) are deadly, toxic proteins produced by the bacterium Clostridium botulinum that can cause significant diseases in humans. The use of the toxic substances as potential bioweapons has raised concerns by the Centers for Disease Control and Prevention and the United States Military. Currently, there is no licensed vaccine to prevent botulinum intoxication. Here we present an immunogenicity study to evaluate the efficacy of novel monovalent vaccines and a trivalent cocktail DNA vaccine targeting the heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E. These synthetic DNA vaccines induced robust humoral and polyfunctional CD4(+) T-cell responses which fully protected animals against lethal challenge after just 2 immunizations. In addition, naïve animals administered immunized sera mixed with the lethal neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by a combinative synthetic DNA vaccine approach. This study has importance for the development of vaccines that provide protective immunity against C. botulinum neurotoxins and other toxins.

  5. DNA vaccines targeting heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge

    PubMed Central

    Scott, Veronica L; Villarreal, Daniel O; Hutnick, Natalie A; Walters, Jewell N; Ragwan, Edwin; Bdeir, Khalil; Yan, Jian; Sardesai, Niranjan Y; Finnefrock, Adam C; Casimiro, Danilo R; Weiner, David B

    2015-01-01

    Botulinum neurotoxins (BoNTs) are deadly, toxic proteins produced by the bacterium Clostridium botulinum that can cause significant diseases in humans. The use of the toxic substances as potential bioweapons has raised concerns by the Centers for Disease Control and Prevention and the United States Military. Currently, there is no licensed vaccine to prevent botulinum intoxication. Here we present an immunogenicity study to evaluate the efficacy of novel monovalent vaccines and a trivalent cocktail DNA vaccine targeting the heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E. These synthetic DNA vaccines induced robust humoral and polyfunctional CD4+ T-cell responses which fully protected animals against lethal challenge after just 2 immunizations. In addition, naïve animals administered immunized sera mixed with the lethal neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by a combinative synthetic DNA vaccine approach. This study has importance for the development of vaccines that provide protective immunity against C. botulinum neurotoxins and other toxins. PMID:26158319

  6. HSV-I and the cellular DNA damage response

    PubMed Central

    Smith, Samantha; Weller, Sandra K

    2015-01-01

    Peter Wildy first observed genetic recombination between strains of HSV in 1955. At the time, knowledge of DNA repair mechanisms was limited, and it has only been in the last decade that particular DNA damage response (DDR) pathways have been examined in the context of viral infections. One of the first reports addressing the interaction between a cellular DDR protein and HSV-1 was the observation by Lees-Miller et al. that DNA-dependent protein kinase catalytic subunit levels were depleted in an ICP0-dependent manner during Herpes simplex virus 1 infection. Since then, there have been numerous reports describing the interactions between HSV infection and cellular DDR pathways. Due to space limitations, this review will focus predominantly on the most recent observations regarding how HSV navigates a potentially hostile environment to replicate its genome. PMID:26213561

  7. Nitroxides block DNA scission and protect cells from oxidative damage

    SciTech Connect

    Samuni, A.; Godinger, D.; Aronovitch, J. ); Russo, A.; Mitchell, J. )

    1991-01-01

    The protective effect of cyclic stable nitroxide free radicals, having SOD-like activity, against oxidative damage was studied by using Escherichia coli xthA DNA repair-deficient mutant hypersensitive to H{sub 2}O{sub 2}. Oxidative damage induced by H{sub 2}O{sub 2} was assayed by monitoring cell survival. The metal chelator 1,10-phenanthroline (OP), which readily intercalates into DNA, potentiated with H{sub 2}O{sub 2}-induced damage. The extent of in vivo DNA scission and degradation was studied and compared with the loss of cell viability. The extent of DNA breakage correlated with cell killing, supporting previous suggestions that DNA is the crucial cellular target of H{sub 2}O{sub 2} cytotoxicity. The xthA cells were protected by catalase but not by superoxide dismutase (SOD). Both five- and six-membered ring nitroxides, having SOD-like activity, protected growing and resting cells from H{sub 2}O{sub 2} toxicity, without lowering H{sub 2}O{sub 2} concentration. To check whether nitroxides protect against O{sub 2}{sup {center dot}{minus}}-independent injury also, experiments were repeated under hypoxia. These nitroxides also protected hypoxic cells against H{sub 2}O{sub 2}, suggesting alternative modes of protection. Since nitroxides were found to reoxidize DNA-bound iron(II), the present results suggest that nitroxides protect by oxidizing reduced transition metals, thus interfering with the Fenton reaction.

  8. DNA damage emergency: cellular garbage disposal to the rescue?

    PubMed

    Stone, H R; Morris, J R

    2014-02-13

    The proteasome is a cellular machine found in the cytosol, nucleus and on chromatin that performs much of the proteolysis in eukaryotic cells. Recent reports show it is enriched at sites of double-stranded DNA breaks (DSBs) in mammalian cells. What is it doing there? This review will address three possibilities suggested by recent reports: in degrading proteins after their ubiquitination at and eviction from chromatin; as a deubiquitinase, specific to the antagonism of ubiquitin conjugates generated as part of the signalling of a DSB; and as a functional component of DNA repair mechanism itself. These findings add complexity to the proteasome as a potential therapeutic target in cancer treatment.

  9. DNA damage emergency: cellular garbage disposal to the rescue?

    PubMed

    Stone, H R; Morris, J R

    2014-02-13

    The proteasome is a cellular machine found in the cytosol, nucleus and on chromatin that performs much of the proteolysis in eukaryotic cells. Recent reports show it is enriched at sites of double-stranded DNA breaks (DSBs) in mammalian cells. What is it doing there? This review will address three possibilities suggested by recent reports: in degrading proteins after their ubiquitination at and eviction from chromatin; as a deubiquitinase, specific to the antagonism of ubiquitin conjugates generated as part of the signalling of a DSB; and as a functional component of DNA repair mechanism itself. These findings add complexity to the proteasome as a potential therapeutic target in cancer treatment. PMID:23503465

  10. Cellular processing and destinies of artificial DNA nanostructures.

    PubMed

    Lee, Di Sheng; Qian, Hang; Tay, Chor Yong; Leong, David Tai

    2016-08-01

    Since many bionanotechnologies are targeted at cells, understanding how and where their interactions occur and the subsequent results of these interactions is important. Changing the intrinsic properties of DNA nanostructures and linking them with interactions presents a holistic and powerful strategy for understanding dual nanostructure-biological systems. With the recent advances in DNA nanotechnology, DNA nanostructures present a great opportunity to understand the often convoluted mass of information pertaining to nanoparticle-biological interactions due to the more precise control over their chemistry, sizes, and shapes. Coupling just some of these designs with an understanding of biological processes is both a challenge and a source of opportunities. Despite continuous advances in the field of DNA nanotechnology, the intracellular fate of DNA nanostructures has remained unclear and controversial. Because understanding its cellular processing and destiny is a necessary prelude to any rational design of exciting and innovative bionanotechnology, in this review, we will discuss and provide a comprehensive picture relevant to the intracellular processing and the fate of various DNA nanostructures which have been remained elusive for some time. We will also link the unique capabilities of DNA to some novel ideas for developing next-generation bionanotechnologies. PMID:27119124

  11. Viral and Cellular Genomes Activate Distinct DNA Damage Responses

    PubMed Central

    Shah, Govind A.; O’Shea, Clodagh C.

    2015-01-01

    Summary In response to cellular genome breaks, MRE11/RAD50/NBS1 (MRN) activates a global ATM DNA damage response (DDR) that prevents cellular replication. Here we show that MRN-ATM also has critical functions in defending the cell against DNA viruses. We reveal temporally distinct responses to adenovirus genomes: a critical MRN-ATM DDR that must be inactivated by E1B-55K/E4-ORF3 viral oncoproteins and a global MRN independent ATM DDR to viral nuclear domains that does not impact viral replication. We show that MRN binds to adenovirus genomes and activates a localized ATM response that specifically prevents viral DNA replication. In contrast to chromosomal breaks, ATM activation is not amplified by H2AX across megabases of chromatin to induce global signaling and replicative arrest. Thus, γH2AX foci discriminate ‘self’ and ‘non-self’ genomes and determine if a localized anti-viral or global ATM response is appropriate. This provides an elegant mechanism to neutralize viral genomes without jeopardizing cellular viability. PMID:26317467

  12. Effect of mtDNA point mutations on cellular bioenergetics.

    PubMed

    Szczepanowska, Joanna; Malinska, Dominika; Wieckowski, Mariusz R; Duszynski, Jerzy

    2012-10-01

    This overview discusses the results of research on the effects of most frequent mtDNA point mutations on cellular bioenergetics. Thirteen proteins coded by mtDNA are crucial for oxidative phosphorylation, 11 of them constitute key components of the respiratory chain complexes I, III and IV and 2 of mitochondrial ATP synthase. Moreover, pathogenic point mutations in mitochondrial tRNAs and rRNAs generate abnormal synthesis of the mtDNA coded proteins. Thus, pathogenic point mutations in mtDNA usually disturb the level of key parameter of the oxidative phosphorylation, i.e. the electric potential on the inner mitochondrial membrane (Δψ), and in a consequence calcium signalling and mitochondrial dynamics in the cell. Mitochondrial generation of reactive oxygen species is also modified in the mutated cells. The results obtained with cultured cells and describing biochemical consequences of mtDNA point mutations are full of contradictions. Still they help elucidate the biochemical basis of pathologies and provide a valuable tool for finding remedies in the future. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). PMID:22406627

  13. Molecular targets in cellular response to ionizing radiation and implications in space radiation protection.

    PubMed

    Belli, Mauro; Sapora, Orazio; Tabocchini, Maria Antonella

    2002-12-01

    DNA repair systems and cell cycle checkpoints closely co-operate in the attempt of maintaining the genomic integrity of cells damaged by ionizing radiation. DNA double-strand breaks (DSB) are considered as the most biologically important radiation-induced damage. Their spatial distribution and association with other types of damage depend on radiation quality. It is believed these features affect damage reparability, thus explaining the higher efficiency for cellular effects of densely ionizing radiation with respect to gamma-rays. DSB repair systems identified in mammalian cells are homologous recombination (HR), single-strand annealing (SSA) and non-homologous end-joining (NHEJ). Some enzymes may participate in more than one of these repair systems. DNA damage also triggers biochemical signals activating checkpoints responsible for delay in cell cycle progression that allows more time for repair. Those at G1/S and S phases prevent replication of damaged DNA and those at G2/M phase prevent segregation of changed chromosomes. Individuals with lack or alterations of genes involved in DNA DSB repair and cell cycle checkpoints exhibit syndromes characterized by genome instability and predisposition to cancer. Information reviewed in this paper on the basic mechanisms of cellular response to ionizing radiation indicates their importance for a number of issues relevant to protection of astronauts from space radiation. PMID:12793724

  14. Degradation of DNA RNA Hybrids by Ribonuclease H and DNA Polymerases of Cellular and Viral Origin

    PubMed Central

    Keller, Walter; Crouch, Robert

    1972-01-01

    Ribonuclease H from human KB cells, chick embryos, calf thymus, avian myeloblastosis virus, and Rous associated virus specifically degrades the RNA of DNA·RNA hybrids, producing mono- and oligoribonucleotides terminated in 5′-phosphates. The cellular RNase H is an endonuclease, whereas the viral enzyme appears to be an exonuclease. Viral DNA polymerase and RNase H copurify through all separation steps. Therefore, RNase H activity is an intrinsic part of the viral DNA polymerase. DNA·RNA hybrids are also degraded by nucleases associated with cellular DNA polymerases and by exonuclease III. However, these nucleases differ from RNase H in their ability to degrade both strands of DNA·RNA hybrids. Images PMID:4343966

  15. PTEN regulates RPA1 and protects DNA replication forks.

    PubMed

    Wang, Guangxi; Li, Yang; Wang, Pan; Liang, Hui; Cui, Ming; Zhu, Minglu; Guo, Limei; Su, Qian; Sun, Yujie; McNutt, Michael A; Yin, Yuxin

    2015-11-01

    Tumor suppressor PTEN regulates cellular activities and controls genome stability through multiple mechanisms. In this study, we report that PTEN is necessary for the protection of DNA replication forks against replication stress. We show that deletion of PTEN leads to replication fork collapse and chromosomal instability upon fork stalling following nucleotide depletion induced by hydroxyurea. PTEN is physically associated with replication protein A 1 (RPA1) via the RPA1 C-terminal domain. STORM and iPOND reveal that PTEN is localized at replication sites and promotes RPA1 accumulation on replication forks. PTEN recruits the deubiquitinase OTUB1 to mediate RPA1 deubiquitination. RPA1 deletion confers a phenotype like that observed in PTEN knockout cells with stalling of replication forks. Expression of PTEN and RPA1 shows strong correlation in colorectal cancer. Heterozygous disruption of RPA1 promotes tumorigenesis in mice. These results demonstrate that PTEN is essential for DNA replication fork protection. We propose that RPA1 is a target of PTEN function in fork protection and that PTEN maintains genome stability through regulation of DNA replication.

  16. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2015-01-01

    Outside the protection of the geomagnetic field, astronauts and other living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, have effects on cellular responses to DNA damage induced by exposure to radiation or cytotoxic chemicals is still unknown, as is their impact on the radiation risks for astronauts and on the mutation rate in microorganisms. Although possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on cellular responses to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB) similar to the ionizing radiation. Damages in the DNA were measured by the phosphorylation of a histone protein H2AX (g-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ki-67 signals. Our results suggested that the difference in g-H2AX focus counts between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect initial transcriptional responses of the DNA damage response genes to

  17. Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors

    SciTech Connect

    Pasquinelli, C.; Garreau, F.; Bougueleret, L.; Cariani, E.; Thiers, V.; Croissant, O.; Hadchouel, M.; Tiollais, P.; Brechot, C. ); Grzeschik, K.H. )

    1988-02-01

    Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. The authors have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possible related to HBV integration.

  18. A Germline Polymorphism of DNA Polymerase Beta Induces Genomic Instability and Cellular Transformation

    PubMed Central

    Keh, Agnes; Sweasy, Joann B.

    2012-01-01

    Several germline single nucleotide polymorphisms (SNPs) have been identified in the POLB gene, but little is known about their cellular and biochemical impact. DNA Polymerase β (Pol β), encoded by the POLB gene, is the main gap-filling polymerase involved in base excision repair (BER), a pathway that protects the genome from the consequences of oxidative DNA damage. In this study we tested the hypothesis that expression of the POLB germline coding SNP (rs3136797) in mammalian cells could induce a cancerous phenotype. Expression of this SNP in both human and mouse cells induced double-strand breaks, chromosomal aberrations, and cellular transformation. Following treatment with an alkylating agent, cells expressing this coding SNP accumulated BER intermediate substrates, including single-strand and double-strand breaks. The rs3136797 SNP encodes the P242R variant Pol β protein and biochemical analysis showed that P242R protein had a slower catalytic rate than WT, although P242R binds DNA similarly to WT. Our results suggest that people who carry the rs3136797 germline SNP may be at an increased risk for cancer susceptibility. PMID:23144635

  19. Cosmid library of the turkey herpesvirus genome constructed from nanogram quantities of viral DNA associated with an excess of cellular DNA.

    PubMed

    Reilly, J D; Silva, R F

    1993-03-01

    A protocol was designed for the rapid and efficient construction of cosmid libraries from cell-associated viral genomes available in very low quantities. Purification of viral DNA from cellular DNA was unnecessary. The vast excess of cellular DNA compensated for the limited amount of available viral DNA, enabling titration of the restriction endonuclease partial digest. A cosmid library of the turkey herpesvirus DNA genome was constructed from 1.5 micrograms of cellular DNA containing approximately 6 nanograms of viral DNA.

  20. Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan

    PubMed Central

    Perić, Matea; Dib, Peter Bou; Dennerlein, Sven; Musa, Marina; Rudan, Marina; Lovrić, Anita; Nikolić, Andrea; Šarić, Ana; Sobočanec, Sandra; Mačak, Željka; Raimundo, Nuno; Kriško, Anita

    2016-01-01

    In cells living under optimal conditions, protein folding defects are usually prevented by the action of chaperones. Here, we investigate the cell-wide consequences of loss of chaperone function in cytosol, mitochondria or the endoplasmic reticulum (ER) in budding yeast. We find that the decline in chaperone activity in each compartment results in loss of respiration, demonstrating the dependence of mitochondrial activity on cell-wide proteostasis. Furthermore, each chaperone deficiency triggers a response, presumably via the communication among the folding environments of distinct cellular compartments, termed here the cross-organelle stress response (CORE). The proposed CORE pathway encompasses activation of protein conformational maintenance machineries, antioxidant enzymes, and metabolic changes simultaneously in the cytosol, mitochondria, and the ER. CORE induction extends replicative and chronological lifespan in budding yeast, highlighting its protective role against moderate proteotoxicity and its consequences such as the decline in respiration. Our findings accentuate that organelles do not function in isolation, but are integrated in a functional crosstalk, while also highlighting the importance of organelle communication in aging and age-related diseases. PMID:27346163

  1. Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan.

    PubMed

    Perić, Matea; Dib, Peter Bou; Dennerlein, Sven; Musa, Marina; Rudan, Marina; Lovrić, Anita; Nikolić, Andrea; Šarić, Ana; Sobočanec, Sandra; Mačak, Željka; Raimundo, Nuno; Kriško, Anita

    2016-01-01

    In cells living under optimal conditions, protein folding defects are usually prevented by the action of chaperones. Here, we investigate the cell-wide consequences of loss of chaperone function in cytosol, mitochondria or the endoplasmic reticulum (ER) in budding yeast. We find that the decline in chaperone activity in each compartment results in loss of respiration, demonstrating the dependence of mitochondrial activity on cell-wide proteostasis. Furthermore, each chaperone deficiency triggers a response, presumably via the communication among the folding environments of distinct cellular compartments, termed here the cross-organelle stress response (CORE). The proposed CORE pathway encompasses activation of protein conformational maintenance machineries, antioxidant enzymes, and metabolic changes simultaneously in the cytosol, mitochondria, and the ER. CORE induction extends replicative and chronological lifespan in budding yeast, highlighting its protective role against moderate proteotoxicity and its consequences such as the decline in respiration. Our findings accentuate that organelles do not function in isolation, but are integrated in a functional crosstalk, while also highlighting the importance of organelle communication in aging and age-related diseases. PMID:27346163

  2. Protection of DNA From Ionizing Radiation-Induced Lesions by Asiaticoside.

    PubMed

    Joy, Jisha; Alarifi, Saud; Alsuhaibani, Entissar; Nair, Cherupally K Krishnan

    2015-01-01

    This study aims to investigate whether asiaticoside, a triterpene glycoside, can afford protection to DNA from alterations induced by gamma radiation under in vitro, ex vivo, and in vivo conditions. In vitro studies were done on plasmid pBR322 DNA, ex vivo studies were done on cellular DNA of human peripheral blood leukocytes, and in vivo investigations were conducted on cellular DNA of spleen and bone marrow cells of mice exposed to whole-body gamma radiation. The supercoiled form of the plasmid pBR322 DNA upon exposure to the radiation was converted into relaxed open circular form due to induction of strand breaks. Presence of asiaticoside along with the DNA during irradiation prevented the relaxation of the supercoiled form to the open circular form. When human peripheral blood leukocytes were exposed to gamma radiation, the cellular DNA suffered strand breaks as evidenced by the increased comet parameters in an alkaline comet assay. Asiaticoside, when present along with blood during irradiation ex vivo, prevented the strand breaks and the comet parameters were closer to that of the controls. Whole-body exposure of mice to gamma radiation resulted in a significant increase in comet parameters of DNA of bone marrow and spleen cells of mice as a result of radiation-induced strand breaks in DNA. Administration of asiaticoside prior to whole-body radiation exposure of the mice prevented this increase in radiation-induced increase in comet parameters, which could be the result of protection to DNA under in vivo conditions of radiation exposure. Thus, it can be concluded from the results that asiaticoside can offer protection to DNA from radiation-induced alterations under in vitro, ex vivo, and in vivo conditions.

  3. Protection of DNA From Ionizing Radiation-Induced Lesions by Asiaticoside.

    PubMed

    Joy, Jisha; Alarifi, Saud; Alsuhaibani, Entissar; Nair, Cherupally K Krishnan

    2015-01-01

    This study aims to investigate whether asiaticoside, a triterpene glycoside, can afford protection to DNA from alterations induced by gamma radiation under in vitro, ex vivo, and in vivo conditions. In vitro studies were done on plasmid pBR322 DNA, ex vivo studies were done on cellular DNA of human peripheral blood leukocytes, and in vivo investigations were conducted on cellular DNA of spleen and bone marrow cells of mice exposed to whole-body gamma radiation. The supercoiled form of the plasmid pBR322 DNA upon exposure to the radiation was converted into relaxed open circular form due to induction of strand breaks. Presence of asiaticoside along with the DNA during irradiation prevented the relaxation of the supercoiled form to the open circular form. When human peripheral blood leukocytes were exposed to gamma radiation, the cellular DNA suffered strand breaks as evidenced by the increased comet parameters in an alkaline comet assay. Asiaticoside, when present along with blood during irradiation ex vivo, prevented the strand breaks and the comet parameters were closer to that of the controls. Whole-body exposure of mice to gamma radiation resulted in a significant increase in comet parameters of DNA of bone marrow and spleen cells of mice as a result of radiation-induced strand breaks in DNA. Administration of asiaticoside prior to whole-body radiation exposure of the mice prevented this increase in radiation-induced increase in comet parameters, which could be the result of protection to DNA under in vivo conditions of radiation exposure. Thus, it can be concluded from the results that asiaticoside can offer protection to DNA from radiation-induced alterations under in vitro, ex vivo, and in vivo conditions. PMID:26756427

  4. Protection after stroke: cellular effectors of neurovascular unit integrity

    PubMed Central

    Posada-Duque, Rafael Andres; Barreto, George E.; Cardona-Gomez, Gloria Patricia

    2014-01-01

    Neurological disorders are prevalent worldwide. Cerebrovascular diseases (CVDs), which account for 55% of all neurological diseases, are the leading cause of permanent disability, cognitive and motor disorders and dementia. Stroke affects the function and structure of blood-brain barrier, the loss of cerebral blood flow regulation, oxidative stress, inflammation and the loss of neural connections. Currently, no gold standard treatments are available outside the acute therapeutic window to improve outcome in stroke patients. Some promising candidate targets have been identified for the improvement of long-term recovery after stroke, such as Rho GTPases, cell adhesion proteins, kinases, and phosphatases. Previous studies by our lab indicated that Rho GTPases (Rac and RhoA) are involved in both tissue damage and survival, as these proteins are essential for the morphology and movement of neurons, astrocytes and endothelial cells, thus playing a critical role in the balance between cell survival and death. Treatment with a pharmacological inhibitor of RhoA/ROCK blocks the activation of the neurodegeneration cascade. In addition, Rac and synaptic adhesion proteins (p120 catenin and N-catenin) play critical roles in protection against cerebral infarction and in recovery by supporting the neurovascular unit and cytoskeletal remodeling activity to maintain the integrity of the brain parenchyma. Interestingly, neuroprotective agents, such as atorvastatin, and CDK5 silencing after cerebral ischemia and in a glutamate-induced excitotoxicity model may act on the same cellular effectors to recover neurovascular unit integrity. Therefore, future efforts must focus on individually targeting the structural and functional roles of each effector of neurovascular unit and the interactions in neural and non-neural cells in the post-ischemic brain and address how to promote the recovery or prevent the loss of homeostasis in the short, medium and long term. PMID:25177270

  5. Protection after stroke: cellular effectors of neurovascular unit integrity.

    PubMed

    Posada-Duque, Rafael Andres; Barreto, George E; Cardona-Gomez, Gloria Patricia

    2014-01-01

    Neurological disorders are prevalent worldwide. Cerebrovascular diseases (CVDs), which account for 55% of all neurological diseases, are the leading cause of permanent disability, cognitive and motor disorders and dementia. Stroke affects the function and structure of blood-brain barrier, the loss of cerebral blood flow regulation, oxidative stress, inflammation and the loss of neural connections. Currently, no gold standard treatments are available outside the acute therapeutic window to improve outcome in stroke patients. Some promising candidate targets have been identified for the improvement of long-term recovery after stroke, such as Rho GTPases, cell adhesion proteins, kinases, and phosphatases. Previous studies by our lab indicated that Rho GTPases (Rac and RhoA) are involved in both tissue damage and survival, as these proteins are essential for the morphology and movement of neurons, astrocytes and endothelial cells, thus playing a critical role in the balance between cell survival and death. Treatment with a pharmacological inhibitor of RhoA/ROCK blocks the activation of the neurodegeneration cascade. In addition, Rac and synaptic adhesion proteins (p120 catenin and N-catenin) play critical roles in protection against cerebral infarction and in recovery by supporting the neurovascular unit and cytoskeletal remodeling activity to maintain the integrity of the brain parenchyma. Interestingly, neuroprotective agents, such as atorvastatin, and CDK5 silencing after cerebral ischemia and in a glutamate-induced excitotoxicity model may act on the same cellular effectors to recover neurovascular unit integrity. Therefore, future efforts must focus on individually targeting the structural and functional roles of each effector of neurovascular unit and the interactions in neural and non-neural cells in the post-ischemic brain and address how to promote the recovery or prevent the loss of homeostasis in the short, medium and long term.

  6. Cellular prion protein and NMDA receptor modulation: protecting against excitotoxicity

    PubMed Central

    Black, Stefanie A. G.; Stys, Peter K.; Zamponi, Gerald W.; Tsutsui, Shigeki

    2014-01-01

    Although it is well established that misfolding of the cellular prion protein (PrPC) into the β-sheet-rich, aggregated scrapie conformation (PrPSc) causes a variety of transmissible spongiform encephalopathies (TSEs), the physiological roles of PrPC are still incompletely understood. There is accumulating evidence describing the roles of PrPC in neurodegeneration and neuroinflammation. Recently, we identified a functional regulation of NMDA receptors by PrPC that involves formation of a physical protein complex between these proteins. Excessive NMDA receptor activity during conditions such as ischemia mediates enhanced Ca2+ entry into cells and contributes to excitotoxic neuronal death. In addition, NMDA receptors and/or PrPC play critical roles in neuroinflammation and glial cell toxicity. Inhibition of NMDA receptor activity protects against PrPSc-induced neuronal death. Moreover, in mice lacking PrPC, infarct size is increased after focal cerebral ischemia, and absence of PrPC increases susceptibility of neurons to NMDA receptor-dependent death. Recently, PrPC was found to be a receptor for oligomeric beta-amyloid (Aβ) peptides, suggesting a role for PrPC in Alzheimer's disease (AD). Our recent findings suggest that Aβ peptides enhance NMDA receptor current by perturbing the normal copper- and PrPC-dependent regulation of these receptors. Here, we review evidence highlighting a role for PrPC in preventing NMDA receptor-mediated excitotoxicity and inflammation. There is a need for more detailed molecular characterization of PrPC-mediated regulation of NMDA receptors, such as determining which NMDA receptor subunits mediate pathogenic effects upon loss of PrPC-mediated regulation and identifying PrPC binding site(s) on the receptor. This knowledge will allow development of novel therapeutic interventions for not only TSEs, but also for AD and other neurodegenerative disorders involving dysfunction of PrPC. PMID:25364752

  7. Mechanistic Modelling of DNA Repair and Cellular Survival Following Radiation-Induced DNA Damage

    PubMed Central

    McMahon, Stephen J.; Schuemann, Jan; Paganetti, Harald; Prise, Kevin M.

    2016-01-01

    Characterising and predicting the effects of ionising radiation on cells remains challenging, with the lack of robust models of the underlying mechanism of radiation responses providing a significant limitation to the development of personalised radiotherapy. In this paper we present a mechanistic model of cellular response to radiation that incorporates the kinetics of different DNA repair processes, the spatial distribution of double strand breaks and the resulting probability and severity of misrepair. This model enables predictions to be made of a range of key biological endpoints (DNA repair kinetics, chromosome aberration and mutation formation, survival) across a range of cell types based on a set of 11 mechanistic fitting parameters that are common across all cells. Applying this model to cellular survival showed its capacity to stratify the radiosensitivity of cells based on aspects of their phenotype and experimental conditions such as cell cycle phase and plating delay (correlation between modelled and observed Mean Inactivation Doses R2 > 0.9). By explicitly incorporating underlying mechanistic factors, this model can integrate knowledge from a wide range of biological studies to provide robust predictions and may act as a foundation for future calculations of individualised radiosensitivity. PMID:27624453

  8. Mechanistic Modelling of DNA Repair and Cellular Survival Following Radiation-Induced DNA Damage.

    PubMed

    McMahon, Stephen J; Schuemann, Jan; Paganetti, Harald; Prise, Kevin M

    2016-01-01

    Characterising and predicting the effects of ionising radiation on cells remains challenging, with the lack of robust models of the underlying mechanism of radiation responses providing a significant limitation to the development of personalised radiotherapy. In this paper we present a mechanistic model of cellular response to radiation that incorporates the kinetics of different DNA repair processes, the spatial distribution of double strand breaks and the resulting probability and severity of misrepair. This model enables predictions to be made of a range of key biological endpoints (DNA repair kinetics, chromosome aberration and mutation formation, survival) across a range of cell types based on a set of 11 mechanistic fitting parameters that are common across all cells. Applying this model to cellular survival showed its capacity to stratify the radiosensitivity of cells based on aspects of their phenotype and experimental conditions such as cell cycle phase and plating delay (correlation between modelled and observed Mean Inactivation Doses R(2) > 0.9). By explicitly incorporating underlying mechanistic factors, this model can integrate knowledge from a wide range of biological studies to provide robust predictions and may act as a foundation for future calculations of individualised radiosensitivity. PMID:27624453

  9. DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair

    PubMed Central

    Abdou, Ismail; Poirier, Guy G.; Hendzel, Michael J.; Weinfeld, Michael

    2015-01-01

    In the current model of DNA SSBR, PARP1 is regarded as the sensor of single-strand breaks (SSBs). However, biochemical studies have implicated LIG3 as another possible SSB sensor. Using a laser micro-irradiation protocol that predominantly generates SSBs, we were able to demonstrate that PARP1 is dispensable for the accumulation of different single-strand break repair (SSBR) proteins at sites of DNA damage in live cells. Furthermore, we show in live cells for the first time that LIG3 plays a role in mediating the accumulation of the SSBR proteins XRCC1 and PNKP at sites of DNA damage. Importantly, the accumulation of LIG3 at sites of DNA damage did not require the BRCT domain-mediated interaction with XRCC1. We were able to show that the N-terminal ZnF domain of LIG3 plays a key role in the enzyme's SSB sensing function. Finally, we provide cellular evidence that LIG3 and not PARP1 acts as the sensor for DNA damage caused by the topoisomerase I inhibitor, irinotecan. Our results support the existence of a second damage-sensing mechanism in SSBR involving the detection of nicks in the genome by LIG3. PMID:25539916

  10. Crosslinking of cellular DNA by nitracrine and furocoumarin derivatives.

    PubMed

    Studzian, K; Tołwińska-Stańczyk, Z; Wilmańska, D; Palumbo, M; Gniazdowski, M

    1999-01-01

    The anticancer drug, nitracrine, a 1-nitro-9-aminoalkyl derivative of acridine exhibits potent cytotoxic effects which are due to its metabolic activation, followed by covalent binding to macromolecules--DNA being the target for the drug. The renaturable fraction of DNA from L-1210 cells pretreated with nitracrine is assayed by means of ethidium bromide fluorescence assay and chromatography on hydroxyapatite column. The effect of the drug was compared with furocoumarins of different DNA crosslinking potencies. The existence of crosslinks in DNA upon incubation of cells with nitracrine (1-4 microM) have been confirmed with two different methods under the conditions where 8-methoxypsoralen, a classic crosslinking agent induced the renaturation. The DNA preparation isolated from the drug pretreated cells exhibited decreased transcriptional template activity with E. coli DNA-dependent RNA polymerase. PMID:10355534

  11. DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus

    PubMed Central

    Qi, Yonghe; Gao, Zhenchao; Peng, Bo; Yan, Huan; Tang, Dingbin; Song, Zilin; He, Wenhui; Sun, Yinyan; Guo, Ju-Tao; Li, Wenhui

    2016-01-01

    Hepatitis B virus (HBV) infection of hepatocytes begins by binding to its cellular receptor sodium taurocholate cotransporting polypeptide (NTCP), followed by the internalization of viral nucleocapsid into the cytoplasm. The viral relaxed circular (rc) DNA genome in nucleocapsid is transported into the nucleus and converted into covalently closed circular (ccc) DNA to serve as a viral persistence reservoir that is refractory to current antiviral therapies. Host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA, however, the DNA polymerase(s) that fills the gap in the plus strand of rcDNA remains to be determined. Here we conducted targeted genetic screening in combination with chemical inhibition to identify the cellular DNA polymerase(s) responsible for cccDNA formation, and exploited recombinant HBV with capsid coding deficiency which infects HepG2-NTCP cells with similar efficiency of wild-type HBV to assure cccDNA synthesis is exclusively from de novo HBV infection. We found that DNA polymerase κ (POLK), a Y-family DNA polymerase with maximum activity in non-dividing cells, substantially contributes to cccDNA formation during de novo HBV infection. Depleting gene expression of POLK in HepG2-NTCP cells by either siRNA knockdown or CRISPR/Cas9 knockout inhibited the conversion of rcDNA into cccDNA, while the diminished cccDNA formation in, and hence the viral infection of, the knockout cells could be effectively rescued by ectopic expression of POLK. These studies revealed that POLK is a crucial host factor required for cccDNA formation during a de novo HBV infection and suggest that POLK may be a potential target for developing antivirals against HBV. PMID:27783675

  12. 3,5,7,3',4'-pentamethoxyflavone, a quercetin derivative protects DNA from oxidative challenges: potential mechanism of action.

    PubMed

    Jakhar, Rekha; Paul, Souren; Park, Young Rong; Han, Jaehong; Kang, Sun Chul

    2014-02-01

    DNA protection is one of the most important strategies in cancer therapy. Since quercetin and its derivatives are found to be potent antioxidant agents, they are able to scavenge radicals significantly. Therefore, we focused on the DNA protection activity of 3,5,7,3',4'-pentamethoxyflavone (PMF), a quercetin derivative isolated from Kaemperia parviflora. Although, PMF was found to be a very poor antioxidant compound, still it could remarkably protect DNA from oxidative damage. DNA binding assay showed that PMF bound to the minor groove of DNA, which suggests a possible mechanism for its DNA protective effects. Cellular toxicity assay on RAW 264.7 macrophages showed this compound is very safe for therapeutic applications.

  13. Protective effect of persimmon (Diospyros kaki) peel proanthocyanidin against oxidative damage under H2O2-induced cellular senescence.

    PubMed

    Lee, Young A; Cho, Eun Ju; Yokozawa, Takako

    2008-06-01

    8-Hydroxy-2'-deoxyguanosine (8-OHdG), one of the most abundant oxidative DNA adducts, is used as an indicator of oxidative DNA damage associated with aging. Among homologs of the silent information regulator (Sir), sirtuin 1 (SIRT1) is suggested as a regulator of the apoptotic response to DNA damage. Since it has been suggested that the aging process can be delayed by the attenuation of oxidative damage such as DNA damage or SIRT1 modulation, we focused on the protective effect against cellular oxidative damage of persimmon peel, a proanthocyanidin-rich food, in relation to its level of polymerization. We confirmed that 8-OHdG expression in TIG-1 human fibroblasts was increased by treatment with 300 microM H2O2 for 2 h. On the other hand, the nuclear SIRT1 level was decreased in H2O2-treated as compared with non-pretreated cells. However, pretreatments with polymers and oligomers led to a decrease in 8-OHdG and elevation in nuclear SIRT1 expression in a concentration-dependent manner. In particular, oligomers exerted a stronger effect. The present study supports the protective potential of proanthocyanidin from persimmon peel against oxidative damage under the aging process, and suggests that the polymerization of proanthocyanidin plays an important role in retarding aging in a cellular senescence model.

  14. Cobalamin inhibition of HIV-1 integrase and integration of HIV-1 DNA into cellular DNA.

    PubMed

    Weinberg, J B; Shugars, D C; Sherman, P A; Sauls, D L; Fyfe, J A

    1998-05-19

    Our prior studies showed that certain cobalamins inhibit productive HIV-1 infection of primary cultures of blood lymphocytes and monocytes. We demonstrate here that this antiviral activity may be mediated by an inhibition of HIV-1 integrase, an enzyme required for productive infection. Purified recombinant HIV-1 integrase activity was inhibited in vitro by hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl), adenosylcobalamin (Ado-Cbl), and dicyanocobinamide (CN2-Cbi) with IC50 values of approximately 17, 17, 17, and 4 microM, respectively. The agents inhibited HIV-1 infection of cultured monocytes (IC50 values for OH-Cbl, Me-Cbl, Ado-Cbl, and CN2-Cbi of 6, 7, 4, and 1 microM, respectively) and of cultured lymphocytes (IC50 values of 60, 50, 60, and 11 microM, respectively). Experiments using cultured monocytes or lymphocytes demonstrated that OH-Cbl inhibited integration of HIV-1 DNA into cellular DNA. Thus, cobalamins and cobinamides represent novel inhibitors of HIV-1 integrase. These or related agents may be useful as anti-viral treatments that target HIV-1 integrase. PMID:9610370

  15. Both Complexity and Location of DNA Damage Contribute to Cellular Senescence Induced by Ionizing Radiation

    PubMed Central

    Zhang, Xurui; Ye, Caiyong; Sun, Fang; Wei, Wenjun; Hu, Burong; Wang, Jufang

    2016-01-01

    Persistent DNA damage is considered as a main cause of cellular senescence induced by ionizing radiation. However, the molecular bases of the DNA damage and their contribution to cellular senescence are not completely clear. In this study, we found that both heavy ions and X-rays induced senescence in human uveal melanoma 92–1 cells. By measuring senescence associated-β-galactosidase and cell proliferation, we identified that heavy ions were more effective at inducing senescence than X-rays. We observed less efficient repair when DNA damage was induced by heavy ions compared with X-rays and most of the irreparable damage was complex of single strand breaks and double strand breaks, while DNA damage induced by X-rays was mostly repaired in 24 hours and the remained damage was preferentially associated with telomeric DNA. Our results suggest that DNA damage induced by heavy ion is often complex and difficult to repair, thus presents as persistent DNA damage and pushes the cell into senescence. In contrast, persistent DNA damage induced by X-rays is preferentially associated with telomeric DNA and the telomere-favored persistent DNA damage contributes to X-rays induced cellular senescence. These findings provide new insight into the understanding of high relative biological effectiveness of heavy ions relevant to cancer therapy and space radiation research. PMID:27187621

  16. Stapling monomeric GCN4 peptides allows for DNA binding and enhanced cellular uptake.

    PubMed

    Iyer, Abhishek; Van Lysebetten, Dorien; Ruiz García, Yara; Louage, Benoit; De Geest, Bruno G; Madder, Annemieke

    2015-04-01

    The basic DNA recognition region of the GCN4 protein comprising 23 amino acids has been modified to contain two optimally positioned cysteines which have been linked and stapled using cross-linkers of suitable lengths. This results in stapled peptides with a stabilized α-helical conformation which allows for DNA binding and concurrent enhancement of cellular uptake.

  17. Assessing cellular DNA damage from a helium plasma needle.

    PubMed

    Morales-Ramírez, P; Cruz-Vallejo, V; Peña-Eguiluz, R; López-Callejas, R; Rodríguez-Méndez, B G; Valencia-Alvarado, R; Mercado-Cabrera, A; Muñoz-Castro, A E

    2013-06-01

    The aim of the present study is to determine the deoxyribonucleic acid (DNA) damage by cells exposed to atmospheric pressure non-thermal plasma (APNTP). Mouse leukocytes embedded in agarose were exposed to the plasma at two different distances from a helium plasma needle outlet and during three different exposure periods. Damage was assessed by the single cell gel electrophoresis assay. The results indicate that, at 0.1 cm from the plasma needle, the exposure caused complete DNA fragmentation determined by the presence of so called "clouds". Samples exposed at 0.5 cm from the slide sample surface presented damage proportional to the exposure periods in terms of tail intensity, tail moment and "clouds" frequency. Studies performed with alkaline single cell gel electrophoresis assay to determine DNA breaks and alkali-labile sites, indicates that DNA damage produced by exposure to APNTP was caused mainly by oxidative radicals, rather than by UV light which causes cyclobutane pyrimidine dimers. These results allow us to conclude that plasma needle induced DNA breaks in mice leukocytes proportionally to exposure time.

  18. [Applications of DNA identification technology in protection of wild animals].

    PubMed

    Ni, Ping-Ya; Pei, Li; Ge, Wen-Dong; Zhang, Ying; Yang, Xue-Ying; Xu, Xiao-Yu; Tu, Zheng

    2011-12-01

    With the development of biotechnology, forensic DNA identification technology in protection of wild animals has been used more and more widely. This review introduces the global status of wildlife crime and the relevant protection to wildlife, outlines the practical applications of forensic DNA identification technology with regard to species identification, determination of geographic origin, individual identification and paternity identification. It focus on the techniques commonly used in DNA typing and their merits and demerits, as well as the problems and prospects of forensic DNA technology for wildlife conservation.

  19. Podophyllum hexandrum-Mediated Survival Protection and Restoration of Other Cellular Injuries in Lethally Irradiated Mice.

    PubMed

    Sankhwar, Sanghmitra; Gupta, Manju Lata; Gupta, Vanita; Verma, Savita; Suri, Krishna Avtar; Devi, Memita; Sharma, Punita; Khan, Ehsan Ahmed; Alam, M Sarwar

    2011-01-01

    This study aims at the development of a safe and effective formulation to counter the effects of lethal irradiation. The sub-fraction (G-001M), prepared from Podophyllum hexandrum has rendered high degree of survival (>90%) at a dose of 6 mg kg(-1) body weight (intramuscular) in lethally irradiated mice. Therapeutic dose of G-001M, at about 20 times lower concentration than its LD(100), has revealed a DRF of 1.62. Comet assay studies in peripheral blood leukocytes have reflected that, treatment of G-001M before irradiation has significantly reduced DNA tail length (P < .001) and DNA damage score (P < .001), as compared to radiation-only group. Spleen cell counts in irradiated animals had declined drastically at the very first day of exposure, and the fall continued till the 5th day (P < .001). In the treated irradiated groups, there was a steep reduction in the counts initially, but this phase did not prolong. More than 60% decline in thymocytes of irradiated group animals was registered at 5 h of irradiation when compared with controls, and the fall progressed further downwards with the similar pace till 5th day of exposure (P < .001). At later intervals, thymus was found fully regressed. In G-001M pre-treated irradiated groups also, thymocytes decreased till the 5th day but thereafter rejuvenated and within 30 days of treatment the values were close to normal. Current studies have explicitly indicated that, G-001M in very small doses has not only rendered high survivability in lethally irradiated mice, but also protected their cellular DNA, besides supporting fast replenishment of the immune system.

  20. DNA-PKcs-Dependent Modulation of Cellular Radiosensitivity by a Selective Cyclooxygenase-2 Inhibitor

    SciTech Connect

    Kodym, Elisabeth; Kodym, Reinhard; Chen, Benjamin P.; Chen, David J.; Morotomi-Yano, Keiko; Choy, Hak; Saha, Debabrata

    2007-09-01

    Purpose: Inhibition of cyclooxygenase-2 has been shown to increase radiosensitivity. Recently, the suppression of radiation-induced DNA-dependant protein kinase (DNA-PK) activity by the selective cyclooxygenase-2 inhibitor celecoxib was reported. Given the importance of DNA-PK for repair of radiation-induced DNA double-strand breaks by nonhomologous end-joining and the clinical use of the substance, we investigated the relevance of the DNA-PK catalytic subunit (DNA-PKcs) for the modulation of cellular radiosensitivity by celecoxib. Methods and Materials: We used a syngeneic model of Chinese hamster ovarian cell lines: AA8, possessing a wild-type DNK-PKcs; V3, lacking a functional DNA-PKcs; and V3/WT11, V3 stably transfected with the DNA-PKcs. The cells were treated with celecoxib (50 {mu}M) for 24 h before irradiation. The modulation of radiosensitivity was determined using the colony formation assay. Results: Treatment with celecoxib increased the cellular radiosensitivity in the DNA-PKcs-deficient cell line V3 with a dose-enhancement ratio of 1.3 for a surviving fraction of 0.5. In contrast, clonogenic survival was increased in DNA-PKcs wild-type-expressing AA8 cells and in V3 cells transfected with DNA-PKcs (V3/WT11). The decrease in radiosensitivity was comparable to the radiosensitization in V3 cells, with a dose-enhancement ratio of 0.76 (AA8) and 0.80 (V3/WT11) for a survival of 0.5. Conclusions: We have demonstrated a DNA-PKcs-dependent differential modulation of cellular radiosensitivity by celecoxib. These effects might be attributed to alterations in signaling cascades downstream of DNA-PK toward cell survival. These findings offer an explanation for the poor outcomes in some recently published clinical trials.

  1. Reducing the in-vitro electromagnetic field effect of cellular phones on human DNA and the intensity of their emitted radiation.

    PubMed

    Syldona, Maria

    2007-01-01

    Several studies have demonstrated detrimental effects of cellular phone radiation on in-vitro biological systems. This article introduces a novel in-vitro method for demonstrating conformational changes in human DNA induced by a 5 minute exposure to cellular phone radiation emitted by an actual contemporary cellular phone. Dynamic changes in DNA conformation was determined in real-time by measuring the rate of DNA rewinding (in a spectrophotometer) following exposure to heat which causes the unwinding of the two strands of the helix. Cellular phone radiation produced a 40% increase in the rate of DNA rewinding. This effect was 95% attenuated when the experiment was repeated with the same cellular phone to which was attached a commercially available shielding disk shaped sheet containing a paramagnetic mineral. In a separate series of experiments the intensity of the cellular phone radiation was measured using an electromagnetic frequency spectrum analyzer. The intensity was reduced by approximately 50% in the presence of the shielding disk. Taken together these studies indicate the efficacy of a shielding disk to protect the body from cellular phone radiation.

  2. Methylene blue protects astrocytes against glucose oxygen deprivation by improving cellular respiration.

    PubMed

    Roy Choudhury, Gourav; Winters, Ali; Rich, Ryan M; Ryou, Myoung-Gwi; Gryczynski, Zygmunt; Yuan, Fang; Yang, Shao-Hua; Liu, Ran

    2015-01-01

    Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration.

  3. Probabilistically determining the cellular source of DNA derived from differential extractions in sexual assault scenarios.

    PubMed

    Taylor, Duncan

    2016-09-01

    Sexual assault cases are the type of case that often produces questions about the cellular source of DNA. In these cases multiple findings of microscopy, DNA profiling and presumptive testing need to be considered when addressing source level propositions. In this work, I consider a line of questioning that has been raised a number of times in the recent past, where in court it was disputed that low levels of sperm seen on a microscope slide were the cellular source of the male DNA profile component generated from the sperm fraction of a differential DNA extraction. I demonstrate how the cell scoring results and DNA profiling results can be considered together, in helping address this source level question through the use of Bayesian Networks.

  4. Probabilistically determining the cellular source of DNA derived from differential extractions in sexual assault scenarios.

    PubMed

    Taylor, Duncan

    2016-09-01

    Sexual assault cases are the type of case that often produces questions about the cellular source of DNA. In these cases multiple findings of microscopy, DNA profiling and presumptive testing need to be considered when addressing source level propositions. In this work, I consider a line of questioning that has been raised a number of times in the recent past, where in court it was disputed that low levels of sperm seen on a microscope slide were the cellular source of the male DNA profile component generated from the sperm fraction of a differential DNA extraction. I demonstrate how the cell scoring results and DNA profiling results can be considered together, in helping address this source level question through the use of Bayesian Networks. PMID:27388428

  5. Cellular strategies for regulating DNA supercoiling: A single-molecule perspective

    PubMed Central

    Koster, Daniel A.; Crut, Aurélien; Shuman, Stewart; Bjornsti, Mary-Ann; Dekker, Nynke H.

    2010-01-01

    Summary Excess entangling and twisting of cellular DNA (i.e., DNA supercoiling) are problems inherent to the helical structure of double-stranded DNA. Supercoiling affects transcription, DNA replication, and chromosomal segregation. Consequently the cell must fine-tune supercoiling to optimize these key processes. Here, we summarize how supercoiling is generated and review experimental and theoretical insights into supercoil relaxation. We distinguish between the passive dissipation of supercoils by diffusion and the active removal of supercoils by topoisomerase enzymes. We also review single-molecule studies that elucidate the timescales and mechanisms of supercoil removal. PMID:20723754

  6. Aphidicolin-resistant polyomavirus and subgenomic cellular DNA synthesis occur early in the differentiation of cultured myoblasts to myotubes.

    PubMed Central

    DePolo, N J; Villarreal, L P

    1993-01-01

    Small DNA viruses have been historically used as probes of cellular control mechanisms of DNA replication, gene expression, and differentiation. Polyomavirus (Py) DNA replication is known to be linked to differentiation of may cells, including myoblasts. In this report, we use this linkage in myoblasts to simultaneously examine (i) cellular differentiation control of Py DNA replication and (ii) an unusual type of cellular and Py DNA synthesis during differentiation. Early proposals that DNA synthesis was involved in the induced differentiation of myoblasts to myotubes were apparently disproved by reliance on inhibitors of DNA synthesis (cytosine arabinoside and aphidicolin), which indicated that mitosis and DNA replication are not necessary for differentiation. Theoretical problems with the accessibility of inactive chromatin to trans-acting factors led us to reexamine possible involvement of DNA replication in myoblast differentiation. We show here that Py undergoes novel aphidicolin-resistant net DNA synthesis under specific conditions early in induced differentiation of myoblasts (following delayed aphidicolin addition). Under similar conditions, we also examined uninfected myoblast DNA synthesis, and we show that soon after differentiation induction, a period of aphidicolin-resistant cellular DNA synthesis can also be observed. This drug-resistant DNA synthesis appears to be subgenomic, not contributing to mitosis, and more representative of polyadenylated than of nonpolyadenylated RNA. These results renew the possibility that DNA synthesis plays a role in myoblast differentiation and suggest that the linkage of Py DNA synthesis to differentiation may involve a qualitative cellular alteration in Py DNA replication. Images PMID:8389922

  7. DNA phosphorothioate modifications influence the global transcriptional response and protect DNA from double-stranded breaks

    PubMed Central

    Gan, Rui; Wu, Xiaolin; He, Wei; Liu, Zhenhua; Wu, Shuangju; Chen, Chao; Chen, Si; Xiang, Qianrong; Deng, Zixin; Liang, Dequan; Chen, Shi; Wang, Lianrong

    2014-01-01

    The modification of DNA by phosphorothioate (PT) occurs when the non-bridging oxygen in the sugar-phosphate backbone of DNA is replaced with sulfur. This DNA backbone modification was recently discovered and is governed by the dndABCDE genes in a diverse group of bacteria and archaea. However, the biological function of DNA PT modifications is poorly understood. In this study, we employed the RNA-seq analysis to characterize the global transcriptional changes in response to PT modifications. Our results show that DNA without PT protection is susceptible to DNA damage caused by the dndFGHI gene products. The DNA double-stranded breaks then trigger the SOS response, cell filamentation and prophage induction. Heterologous expression of dndBCDE conferring DNA PT modifications at GPSA and GPST prevented the damage in Salmonella enterica. Our data provide insights into the physiological role of the DNA PT system. PMID:25319634

  8. DNA Mismatch Repair System: Repercussions in Cellular Homeostasis and Relationship with Aging

    PubMed Central

    Conde-Pérezprina, Juan Cristóbal; León-Galván, Miguel Ángel; Konigsberg, Mina

    2012-01-01

    The mechanisms that concern DNA repair have been studied in the last years due to their consequences in cellular homeostasis. The diverse and damaging stimuli that affect DNA integrity, such as changes in the genetic sequence and modifications in gene expression, can disrupt the steady state of the cell and have serious repercussions to pathways that regulate apoptosis, senescence, and cancer. These altered pathways not only modify cellular and organism longevity, but quality of life (“health-span”). The DNA mismatch repair system (MMR) is highly conserved between species; its role is paramount in the preservation of DNA integrity, placing it as a necessary focal point in the study of pathways that prolong lifespan, aging, and disease. Here, we review different insights concerning the malfunction or absence of the DNA-MMR and its impact on cellular homeostasis. In particular, we will focus on DNA-MMR mechanisms regulated by known repair proteins MSH2, MSH6, PMS2, and MHL1, among others. PMID:23213348

  9. Molecular and cellular functions of the FANCJ DNA helicase defective in cancer and in Fanconi anemia

    PubMed Central

    Brosh, Robert M.; Cantor, Sharon B.

    2014-01-01

    The FANCJ DNA helicase is mutated in hereditary breast and ovarian cancer as well as the progressive bone marrow failure disorder Fanconi anemia (FA). FANCJ is linked to cancer suppression and DNA double strand break repair through its direct interaction with the hereditary breast cancer associated gene product, BRCA1. FANCJ also operates in the FA pathway of interstrand cross-link repair and contributes to homologous recombination. FANCJ collaborates with a number of DNA metabolizing proteins implicated in DNA damage detection and repair, and plays an important role in cell cycle checkpoint control. In addition to its role in the classical FA pathway, FANCJ is believed to have other functions that are centered on alleviating replication stress. FANCJ resolves G-quadruplex (G4) DNA structures that are known to affect cellular replication and transcription, and potentially play a role in the preservation and functionality of chromosomal structures such as telomeres. Recent studies suggest that FANCJ helps to maintain chromatin structure and preserve epigenetic stability by facilitating smooth progression of the replication fork when it encounters DNA damage or an alternate DNA structure such as a G4. Ongoing studies suggest a prominent but still not well-understood role of FANCJ in transcriptional regulation, chromosomal structure and function, and DNA damage repair to maintain genomic stability. This review will synthesize our current understanding of the molecular and cellular functions of FANCJ that are critical for chromosomal integrity. PMID:25374583

  10. A threshold of endogenous stress is required to engage cellular response to protect against mutagenesis.

    PubMed

    Saintigny, Yannick; Chevalier, François; Bravard, Anne; Dardillac, Elodie; Laurent, David; Hem, Sonia; Dépagne, Jordane; Radicella, J Pablo; Lopez, Bernard S

    2016-07-11

    Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [(3)H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [(3)H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses.

  11. A threshold of endogenous stress is required to engage cellular response to protect against mutagenesis

    PubMed Central

    Saintigny, Yannick; Chevalier, François; Bravard, Anne; Dardillac, Elodie; Laurent, David; Hem, Sonia; Dépagne, Jordane; Radicella, J. Pablo; Lopez, Bernard S.

    2016-01-01

    Endogenous stress represents a major source of genome instability, but is in essence difficult to apprehend. Incorporation of labeled radionuclides into DNA constitutes a tractable model to analyze cellular responses to endogenous attacks. Here we show that incorporation of [3H]thymidine into CHO cells generates oxidative-induced mutagenesis, but, with a peak at low doses. Proteomic analysis showed that the cellular response differs between low and high levels of endogenous stress. In particular, these results confirmed the involvement of proteins implicated in redox homeostasis and DNA damage signaling pathways. Induced-mutagenesis was abolished by the anti-oxidant N-acetyl cysteine and plateaued, at high doses, upon exposure to L-buthionine sulfoximine, which represses cellular detoxification. The [3H]thymidine-induced mutation spectrum revealed mostly base substitutions, exhibiting a signature specific for low doses (GC > CG and AT > CG). Consistently, the enzymatic activity of the base excision repair protein APE-1 is induced at only medium or high doses. Collectively, the data reveal that a threshold of endogenous stress must be reached to trigger cellular detoxification and DNA repair programs; below this threshold, the consequences of endogenous stress escape cellular surveillance, leading to high levels of mutagenesis. Therefore, low doses of endogenous local stress can jeopardize genome integrity more efficiently than higher doses. PMID:27406380

  12. Extracellular ATP protects endothelial cells against DNA damage.

    PubMed

    Aho, Joonas; Helenius, Mikko; Vattulainen-Collanus, Sanna; Alastalo, Tero-Pekka; Koskenvuo, Juha

    2016-09-01

    Cell damage can lead to rapid release of ATP to extracellular space resulting in dramatic change in local ATP concentration. Evolutionary, this has been considered as a danger signal leading to adaptive responses in adjacent cells. Our aim was to demonstrate that elevated extracellular ATP or inhibition of ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39) activity could be used to increase tolerance against DNA-damaging conditions. Human endothelial cells, with increased extracellular ATP concentration in cell proximity, were more resistant to irradiation or chemically induced DNA damage evaluated with the DNA damage markers γH2AX and phosphorylated p53. In our rat models of DNA damage, inhibiting CD39-driven ATP hydrolysis with POM-1 protected the heart and lung tissues against chemically induced DNA damage. Interestingly, the phenomenon could not be replicated in cancer cells. Our results show that transient increase in extracellular ATP can promote resistance to DNA damage.

  13. Cellular Heterogeneity in the Level of mtDNA Heteroplasmy in Mouse Embryonic Stem Cells.

    PubMed

    Neupane, Jitesh; Ghimire, Sabitri; Vandewoestyne, Mado; Lu, Yuechao; Gerris, Jan; Van Coster, Rudy; Deroo, Tom; Deforce, Dieter; Vansteelandt, Stijn; De Sutter, Petra; Heindryckx, Björn

    2015-11-17

    Variation in the level of mtDNA heteroplasmy in adult tissues is commonly seen in patients with a mixture of wild-type and mutant mtDNA. A mixture of different mtDNA variants may influence such variation and cause mtDNA segregation bias. We analyzed cellular heterogeneity in embryonic stem cells (ESCs) derived from a polymorphic mouse model containing NZB and BALB mtDNA genotypes. In ESCs, inter-colony heterogeneity varied up to 61%, whereas intra-colony heterogeneity varied up to 100%. Three out of five cell lines displayed nearly homoplasmic BALB and NZB mtDNA haplotypes in differentiated single cells. The proportion of NZB mtDNA genotype increased with progressive passaging (0.39%; p = 0.002). These results demonstrate the bimodal segregation of mtDNA haplotypes, indicating the occurrence of tissues with variable levels of heteroplasmies in individuals with mtDNA mutations. Furthermore, proliferation of one mtDNA genotype over another may pose the risk of accumulating mutant mtDNAs during subsequent cell divisions.

  14. Protective Effects of Extracts from Fructus rhodomyrti against Oxidative DNA Damage In Vitro and In Vivo

    PubMed Central

    Ke, Yuebin; Xu, Xinyun; Wu, Shuang; Huang, Juan; Misra, Hara; Li, Yunbo

    2013-01-01

    Objective. To evaluate the potential protective effects of extracts from Fructus rhodomyrti (FR) against oxidative DNA damage using a cellular system and the antioxidant ability on potassium bromate- (KBrO3-) mediated oxidative stress in rats. Methods. The effects of FR on DNA damage induced by hydrogen peroxide (H2O2) were evaluated by comet assay in primary spleen lymphocytes cultures. The effects of FR on the activities of SOD, CAT, and GPx and the levels of GSH, hydroperoxides, and 8-OHdG were determined in the plasma and tissues of rats treated with KBrO3. Results. FR was shown to effectively protect against DNA damage induced by H2O2  in vitro, and the maximum protective effect was observed when FR was diluted 20 times. Endogenous antioxidant status, namely, the activities of SOD, CAT, and GPx and the levels of GSH were significantly decreased in the plasma, the liver, and the kidney of the KBrO3-treated rats, while the pretreatment of FR prevented the decreases of these parameters. In addition, the pretreatment of FR was also able to prevent KBrO3-induced increases in the levels of hydroperoxides and 8-OHdG in the plasma, the liver, and the kidney in rats. Conclusions. Our findings suggested that FR might act as a chemopreventive agent with antioxidant properties offering effective protection against oxidative DNA damage in a concentration-dependent manner in vitro and in vivo. PMID:24089629

  15. Microinjected pBR322 stimulates cellular DNA synthesis in Swiss 3T3 cells.

    PubMed Central

    Hyland, J K; Hirschhorn, R R; Avignolo, C; Mercer, W E; Ohta, M; Galanti, N; Jonak, G J; Baserga, R

    1984-01-01

    When pBR322 is manually microinjected into the nuclei of quiescent Swiss 3T3 cells it stimulates the incorporation of [3H]thymidine into DNA. The evidence clearly shows that this increased incorporation that is detected by in situ autoradiography in microinjected cells represents cellular DNA synthesis and not DNA repair or plasmid replication. The effect is due to pBR322 and not due to impurities, mechanical perturbances due to the microinjection technique, or aspecific effects. This stimulation is striking in Swiss 3T3 cells. Some NIH 3T3 cells show a slight stimulation, but hamster cells, derived from baby hamster kidney (BHK) cells, are not stimulated when microinjected with pBR322. The preliminary evidence seems to indicate that the integrity of the pBR322 genome is important for the stimulation of cellular DNA synthesis in quiescent Swiss 3T3 cells. These results, although of a preliminary nature, are of interest because they indicate that a prokaryotic genome may alter the cell cycle of mammalian cells. From a practical point of view the stimulatory effect of microinjected pBR322 on cellular DNA synthesis has a more immediate interest, because pBR322 is the vector most commonly used for molecular cloning and 3T3 cells are very frequently used for gene transfer experiments. Images PMID:6582497

  16. Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications

    PubMed Central

    Róna, Gergely; Scheer, Ildikó; Nagy, Kinga; Pálinkás, Hajnalka L.; Tihanyi, Gergely; Borsos, Máté; Békési, Angéla; Vértessy, Beáta G.

    2016-01-01

    The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell. PMID:26429970

  17. Effects of a Protecting Osmolyte on The Ion Atmosphere Surrounding DNA Duplexes

    PubMed Central

    Blose, Joshua M.; Pabit, Suzette A.; Meisburger, Steve P.; Li, Li; Jones, Christopher D.; Pollack, Lois

    2012-01-01

    Osmolytes are small, chemically diverse, organic solutes that function as an essential component of cellular stress response. Protecting osmolytes enhance protein stability via preferential exclusion, and non-protecting osmolytes, such as urea, destabilize protein structures. Although much is known about osmolyte effects on proteins, less is understood about osmolyte effects on nucleic acids and their counterion atmospheres. Non-protecting osmolytes destabilize nucleic acid structures, but effects of protecting osmolytes depend on numerous factors including the type of nucleic acid and the complexity of the functional fold. To begin quantifying protecting osmolyte effects on nucleic acid interactions we used small angle x-ray scattering (SAXS) techniques to monitor DNA duplexes in the presence of sucrose. This protecting osmolyte is a commonly used contrast matching agent in SAXS studies of protein-nucleic acid complexes, thus it is important to characterize interaction changes induced by sucrose. Measurements of interactions between duplexes showed no dependence on the presence of up to 30% sucrose except under high Mg2+ conditions where stacking interactions were disfavored. The number of excess ions associated with DNA duplexes, reported by anomalous small angle x-ray scattering (ASAXS) experiments, was sucrose independent. Although protecting osmolytes can destabilize secondary structures, our results suggest that ion atmospheres of individual duplexes remain unperturbed by sucrose. PMID:21882885

  18. Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation

    PubMed Central

    Shrivastav, Nidhi; Li, Deyu; Essigmann, John M.

    2010-01-01

    The reaction of DNA-damaging agents with the genome results in a plethora of lesions, commonly referred to as adducts. Adducts may cause DNA to mutate, they may represent the chemical precursors of lethal events and they can disrupt expression of genes. Determination of which adduct is responsible for each of these biological endpoints is difficult, but this task has been accomplished for some carcinogenic DNA-damaging agents. Here, we describe the respective contributions of specific DNA lesions to the biological effects of low molecular weight alkylating agents. PMID:19875697

  19. Assessment of the ability of seaweed extracts to protect against hydrogen peroxide and tert-butyl hydroperoxide induced cellular damage in Caco-2 cells.

    PubMed

    O'Sullivan, A M; O'Callaghan, Y C; O'Grady, M N; Queguineur, B; Hanniffy, D; Troy, D J; Kerry, J P; O'Brien, N M

    2012-09-15

    The ability of brown seaweed extracts, Ascophyllum nodosum, Laminaria hyperborea, Pelvetia canaliculata, Fucus vesiculosus and Fucus serratus to protect against tert-butyl hydroperoxide (tert-BOOH) induced stress in Caco-2 cells was investigated. Oxidative stress was determined by measuring alteration in the enzymatic activity of catalase (CAT) and superoxide dismutases (SOD) and cellular levels of glutathione (GSH). L. hyperborea, P. canaliculata and F. serratus significantly protected against tert-BOOH induced SOD reduction but did not protect against the reduction in CAT activity or the increased cellular levels of GSH. The ability of F. serratus and F. vesiculosus to protect against H(2)O(2) and tert-BOOH induced DNA damage was also assessed. The DNA protective effects of the two seaweed extracts was compared to those of three metal chelators; deferoxamine mesylate (DFO), 1,10-phenanthroline (o-phen) and 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA-AM). F. serratus and F. vesiculosus significantly protected (P<0.05) against H(2)O(2) (50 μM) induced DNA damage but not tert-BOOH induced damage. PMID:23107739

  20. Viral and cellular SOS-regulated motor proteins: dsDNA translocation mechanisms with divergent functions.

    PubMed

    Wolfe, Annie; Phipps, Kara; Weitao, Tao

    2014-01-01

    DNA damage attacks on bacterial cells have been known to activate the SOS response, a transcriptional response affecting chromosome replication, DNA recombination and repair, cell division and prophage induction. All these functions require double-stranded (ds) DNA translocation by ASCE hexameric motors. This review seeks to delineate the structural and functional characteristics of the SOS response and the SOS-regulated DNA translocases FtsK and RuvB with the phi29 bacteriophage packaging motor gp16 ATPase as a prototype to study bacterial motors. While gp16 ATPase, cellular FtsK and RuvB are similarly comprised of hexameric rings encircling dsDNA and functioning as ATP-driven DNA translocases, they utilize different mechanisms to accomplish separate functions, suggesting a convergent evolution of these motors. The gp16 ATPase and FtsK use a novel revolution mechanism, generating a power stroke between subunits through an entropy-DNA affinity switch and pushing dsDNA inward without rotation of DNA and the motor, whereas RuvB seems to employ a rotation mechanism that remains to be further characterized. While FtsK and RuvB perform essential tasks during the SOS response, their roles may be far more significant as SOS response is involved in antibiotic-inducible bacterial vesiculation and biofilm formation as well as the perspective of the bacteria-cancer evolutionary interaction.

  1. Temporal order of evolution of DNA replication systems inferred by comparison of cellular and viral DNA polymerases

    PubMed Central

    Koonin, Eugene V

    2006-01-01

    Background The core enzymes of the DNA replication systems show striking diversity among cellular life forms and more so among viruses. In particular, and counter-intuitively, given the central role of DNA in all cells and the mechanistic uniformity of replication, the core enzymes of the replication systems of bacteria and archaea (as well as eukaryotes) are unrelated or extremely distantly related. Viruses and plasmids, in addition, possess at least two unique DNA replication systems, namely, the protein-primed and rolling circle modalities of replication. This unexpected diversity makes the origin and evolution of DNA replication systems a particularly challenging and intriguing problem in evolutionary biology. Results I propose a specific succession for the emergence of different DNA replication systems, drawing argument from the differences in their representation among viruses and other selfish replicating elements. In a striking pattern, the DNA replication systems of viruses infecting bacteria and eukaryotes are dominated by the archaeal-type B-family DNA polymerase (PolB) whereas the bacterial replicative DNA polymerase (PolC) is present only in a handful of bacteriophage genomes. There is no apparent mechanistic impediment to the involvement of the bacterial-type replication machinery in viral DNA replication. Therefore, I hypothesize that the observed, markedly unequal distribution of the replicative DNA polymerases among the known cellular and viral replication systems has a historical explanation. I propose that, among the two types of DNA replication machineries that are found in extant life forms, the archaeal-type, PolB-based system evolved first and had already given rise to a variety of diverse viruses and other selfish elements before the advent of the bacterial, PolC-based machinery. Conceivably, at that stage of evolution, the niches for DNA-viral reproduction have been already filled with viruses replicating with the help of the archaeal

  2. Single mammalian cells compensate for differences in cellular volume and DNA copy number through independent global transcriptional mechanisms

    PubMed Central

    Padovan-Merhar, Olivia; Nair, Gautham P.; Biaesch, Andrew; Mayer, Andreas; Scarfone, Steven; Foley, Shawn W.; Wu, Angela R.; Churchman, L. Stirling; Singh, Abhyudai; Raj, Arjun

    2015-01-01

    Summary Individual mammalian cells exhibit large variability in cellular volume even with the same absolute DNA content and so must compensate for differences in DNA concentration in order to maintain constant concentration of gene expression products. Using single molecule counting and computational image analysis, we show that transcript abundance correlates with cellular volume at the single cell level due to increased global transcription in larger cells. Cell fusion experiments establish that increased cellular content itself can directly increase transcription. Quantitative analysis shows that this mechanism measures the ratio of cellular volume to DNA content, mostly likely through sequestration of a transcriptional factor to DNA. Analysis of transcriptional bursts reveals a separate mechanism for gene dosage compensation after DNA replication that enables proper transcriptional output during early and late S-phase. Our results provide a framework for quantitatively understanding the relationships between DNA content, cell size and gene expression variability in single cells. PMID:25866248

  3. DNA binding, antioxidant activity, and DNA damage protection of chiral macrocyclic Mn(III) salen complexes.

    PubMed

    Pandya, Nirali; Khan, Noor-ul H; Prathap, K Jeya; Kureshy, Rukhsana I; Abdi, Sayed H R; Mishra, Sandhya; Bajaj, Hari C

    2012-12-01

    We are reporting the synthesis, characterization, and calf thymus DNA binding studies of novel chiral macrocyclic Mn(III) salen complexes S-1, R-1, S-2, and R-2. These chiral complexes showed ability to bind with DNA, where complex S-1 exhibits the highest DNA binding constant 1.20 × 10(6) M(-1). All the compounds were screened for superoxide and hydroxyl radical scavenging activities; among them, complex S-1 exhibited significant activity with IC(50) 1.36 and 2.37 μM, respectively. Further, comet assay was used to evaluate the DNA damage protection in white blood cells against the reactive oxygen species wherein complex S-1 was found effective in protecting the hydroxyl radicals mediated plasmid and white blood cells DNA damage.

  4. Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

    NASA Technical Reports Server (NTRS)

    Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

    2002-01-01

    Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

  5. The influence of antigen targeting to sub-cellular compartments on the anti-allergic potential of a DNA vaccine☆

    PubMed Central

    Weinberger, Esther E.; Isakovic, Almedina; Scheiblhofer, Sandra; Ramsauer, Christina; Reiter, Katrin; Hauser-Kronberger, Cornelia; Thalhamer, Josef; Weiss, Richard

    2013-01-01

    Background Gene vaccines offer attractive rationales for prophylactic as well as therapeutic treatments of type I allergies. DNA and mRNA vaccines have been shown to prevent from allergic sensitization and to counterbalance established allergic immune reactions. Recent advances in gene vaccine manipulation offer additional opportunities for modulation of T helper cell profiles by specific targeting of cellular compartments. Methods DNA vaccines encoding the major birch pollen allergen Bet v 1.0101 were equipped with different leader sequences to shuttle the antigen to lysosomes (LIMP-II), to trigger cellular secretion (hTPA), or to induce proteasomal degradation via forced ubiquitination (ubi). Mice were pre-vaccinated with these constructs and the protective efficacy was tested by subcutaneous Th2-promoting challenges, followed by allergen inhalation. IgG antibody subclass distribution and allergen-specific IgE as well as cytokine profiles from re-stimulated splenocytes and from BALFs were assessed. The cellular composition of BALFs, and lung resistance and compliance were determined. Results Immunization with all targeting variants protected from allergic sensitization, i.e. IgE induction, airway hyperresponsiveness, lung inflammation, and systemic and local Th2 cytokine expression. Surprisingly, protection did not clearly correlate with the induction of a systemic Th1 cytokine profile, but rather with proliferating CD4+ CD25+ FoxP3+ T regulatory cells in splenocyte cultures. Targeting the allergen to proteasomal or lysosomal degradation severely down-regulated antibody induction after vaccination, while T cell responses remained unaffected. Although secretion of antigen promoted the highest numbers of Th1 cells, this vaccine type was the least efficient in suppressing the establishment of an allergic immune response. Conclusion This comparative analysis highlights the modulatory effect of antigen targeting on the resulting immune response, with a special

  6. EBNA-LP Associates with Cellular Proteins Including DNA-PK and HA95

    PubMed Central

    Han, Innoc; Harada, Shizuko; Weaver, David; Xue, Yong; Lane, William; Orstavik, Sigurd; Skalhegg, Bjorn; Kieff, Elliott

    2001-01-01

    EBNA-LP-associated proteins were identified by sequencing proteins that immunoprecipitated with Flag epitope-tagged EBNA-LP (FLP) from lymphoblasts in which FLP was stably expressed. The association of EBNA-LP with Hsp70 (72/73) was confirmed, and sequences of DNA-PK catalytic subunit (DNA-PKcs), HA95, Hsp27, prolyl 4-hydroxylase α-1 subunit, α-tubulin, and β-tubulin were identified. The fraction of total cellular HA95 that associated with FLP was very high, while progressively lower fractions of the total DNA-PKcs, Hsp70, Hsp 27, α-tubulin, and β-tubulin specifically associated with EBNA-LP as determined by immunoblotting with antibodies to these proteins. EBNA-LP bound to two domains in the DNA-PKcs C terminus and DNA-PKcs associated with the EBNA-LP repeat domain. DNA-PKcs that was bound to EBNA-LP phosphorylated p53 or EBNA-LP in vitro, and the phosphorylation of EBNA-LP was inhibited by Wortmannin, a specific in vitro inhibitor of DNA-PKcs. PMID:11160753

  7. UV protective effects of DNA repair enzymes and RNA lotion.

    PubMed

    Ke, Malcolm S; Camouse, Melissa M; Swain, Freddie R; Oshtory, Shaheen; Matsui, Mary; Mammone, Thomas; Maes, Daniel; Cooper, Kevin D; Stevens, Seth R; Baron, Elma D

    2008-01-01

    Solar UV radiation is known to cause immune suppression, believed to be a critical factor in cutaneous carcinogenesis. Although the mechanism is not entirely understood, DNA damage is clearly involved. Sunscreens function by attenuating the UV radiation that reaches the epidermis. However, once DNA damage ensues, repair mechanisms become essential for prevention of malignant transformation. DNA repair enzymes have shown efficacy in reducing cutaneous neoplasms among xeroderma pigmentosum patients. In vitro studies suggest that RNA fragments increase the resistance of human keratinocytes to UVB damage and enhance DNA repair but in vivo data are lacking. This study aimed to determine the effect of topical formulations containing either DNA repair enzymes (Micrococcus luteus) or RNA fragments (UVC-irradiated rabbit globin mRNA) on UV-induced local contact hypersensitivity (CHS) suppression in humans as measured in vivo using the contact allergen dinitrochlorobenzene. Immunohistochemistry was also employed in skin biopsies to evaluate the level of thymine dimers after UV. Eighty volunteers completed the CHS portion. A single 0.75 minimum erythema dose (MED) simulated solar radiation exposure resulted in 64% CHS suppression in unprotected subjects compared with unirradiated sensitized controls. In contrast, UV-induced CHS suppression was reduced to 19% with DNA repair enzymes, and 7% with RNA fragments. Sun protection factor (SPF) testing revealed an SPF of 1 for both formulations, indicating that the observed immune protection cannot be attributed to sunscreen effects. Biopsies from an additional nine volunteers showed an 18% decrease in thymine dimers by both DNA repair enzymes and RNA fragments, relative to unprotected UV-irradiated skin. These results suggest that RNA fragments may be useful as a photoprotective agent with in vivo effects comparable to DNA repair enzymes.

  8. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2015-01-01

    Living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, affects on the cellular response to DNA damage induced by exposures to radiation or other toxic chemicals will have an impact on the radiation risks for the astronauts, as well as on the mutation rate in microorganisms, is still an open question. Although the possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate the effects of spaceflight on the cellular response to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induces DNA damages including the double strand breaks (DSB) similar to the ionizing radiation. Damage in the DNA was measured by the phosphorylation of a histone protein H2AX (-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in the DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ti-67 signals. Our results suggested that the difference in -H2AX between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect the response of the DNA damage response genes to bleomycin treatment.

  9. Chemical induction of cellular antioxidants affords marked protection against oxidative injury in vascular smooth muscle cells.

    PubMed

    Cao, Zhuoxiao; Li, Yunbo

    2002-03-22

    Extensive evidence suggests that reactive oxygen species are critically involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis and myocardial ischemia-reperfusion injury. Consistent with this concept, administration of exogenous antioxidants has been shown to be protective against oxidative cardiovascular injury. However, whether induction of endogenous antioxidants by chemical inducers in vasculature also affords protection against oxidative vascular cell injury has not been extensively investigated. In this study, using rat aortic smooth muscle A10 cells as an in vitro system, we have studied the induction of cellular antioxidants by the unique chemoprotector, 3H-1,2-dithiole-3-thione [corrected] (D3T) and the protective effects of the D3T-induced cellular antioxidants against oxidative cell injury. Incubation of A10 cells with micromolar concentrations of D3T for 24 h resulted in a significant induction of a battery of cellular antioxidants in a concentration-dependent manner. These included reduced glutathione (GSH), GSH peroxidase, GSSG reductase, GSH S-transferase, superoxide dismutase, and catalase. To further examine the protective effects of the induced endogenous antioxidants against oxidative cell injury, A10 cells were pretreated with D3T and then exposed to either xanthine oxidase (XO)/xanthine, 4-hydroxynonenal, or cadmium. We observed that D3T pretreatment of A10 cells led to significant protection against the cytotoxicity induced by XO/xanthine, 4-hydroxynonenal or cadmium, as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium reduction assay. Taken together, this study demonstrates for the first time that a number of endogenous antioxidants in vascular smooth muscle cells can be induced by exposure to D3T, and that this chemical induction of cellular antioxidants is accompanied by markedly increased resistance to oxidative vascular cell injury.

  10. The distinctive cellular responses to DNA strand breaks caused by a DNA topoisomerase I poison in conjunction with DNA replication and RNA transcription.

    PubMed

    Sakasai, Ryo; Iwabuchi, Kuniyoshi

    2016-01-01

    Camptothecin (CPT) inhibits DNA topoisomerase I (Top1) through a non-catalytic mechanism that stabilizes the Top1-DNA cleavage complex (Top1cc) and blocks the DNA re-ligation step, resulting in the accumulation in the genome of DNA single-strand breaks (SSBs), which are converted to secondary strand breaks when they collide with the DNA replication and RNA transcription machinery. DNA strand breaks mediated by replication, which have one DNA end, are distinct in repair from the DNA double-strand breaks (DSBs) that have two ends and are caused by ionizing radiation and other agents. In contrast to two-ended DSBs, such one-ended DSBs are preferentially repaired through the homologous recombination pathway. Conversely, the repair of one-ended DSBs by the non-homologous end-joining pathway is harmful for cells and leads to cell death. The choice of repair pathway has a crucial impact on cell fate and influences the efficacy of anticancer drugs such as CPT derivatives. In addition to replication-mediated one-ended DSBs, transcription also generates DNA strand breaks upon collision with the Top1cc. Some reports suggest that transcription-mediated DNA strand breaks correlate with neurodegenerative diseases. However, the details of the repair mechanisms of, and cellular responses to, transcription-mediated DNA strand breaks still remain unclear. In this review, combining our recent results and those of previous reports, we introduce and discuss the responses to CPT-induced DNA damage mediated by DNA replication and RNA transcription.

  11. Autophagy confers DNA damage repair pathways to protect the hematopoietic system from nuclear radiation injury

    PubMed Central

    Lin, Weiwei; Yuan, Na; Wang, Zhen; Cao, Yan; Fang, Yixuan; Li, Xin; Xu, Fei; Song, Lin; Wang, Jian; Zhang, Han; Yan, Lili; Xu, Li; Zhang, Xiaoying; Zhang, Suping; Wang, Jianrong

    2015-01-01

    Autophagy is essentially a metabolic process, but its in vivo role in nuclear radioprotection remains unexplored. We observed that ex vivo autophagy activation reversed the proliferation inhibition, apoptosis, and DNA damage in irradiated hematopoietic cells. In vivo autophagy activation improved bone marrow cellularity following nuclear radiation exposure. In contrast, defective autophagy in the hematopoietic conditional mouse model worsened the hematopoietic injury, reactive oxygen species (ROS) accumulation and DNA damage caused by nuclear radiation exposure. Strikingly, in vivo defective autophagy caused an absence or reduction in regulatory proteins critical to both homologous recombination (HR) and non-homologous end joining (NHEJ) DNA damage repair pathways, as well as a failure to induce these proteins in response to nuclear radiation. In contrast, in vivo autophagy activation increased most of these proteins in hematopoietic cells. DNA damage assays confirmed the role of in vivo autophagy in the resolution of double-stranded DNA breaks in total bone marrow cells as well as bone marrow stem and progenitor cells upon whole body irradiation. Hence, autophagy protects the hematopoietic system against nuclear radiation injury by conferring and intensifying the HR and NHEJ DNA damage repair pathways and by removing ROS and inhibiting apoptosis. PMID:26197097

  12. Fisetin attenuates hydrogen peroxide-induced cell damage by scavenging reactive oxygen species and activating protective functions of cellular glutathione system.

    PubMed

    Kang, Kyoung Ah; Piao, Mei Jing; Kim, Ki Cheon; Cha, Ji Won; Zheng, Jian; Yao, Cheng Wen; Chae, Sungwook; Hyun, Jin Won

    2014-01-01

    Hydrogen peroxide (H2O2) can induce cell damage by generating reactive oxygen species (ROS), resulting in DNA damage and cell death. The aim of this study is to elucidate the protective effects of fisetin (3,7,3',4',-tetrahydroxy flavone) against H2O2-induced cell damage. Fisetin reduced the level of superoxide anion, hydroxyl radical in cell free system, and intracellular ROS generated by H2O2. Moreover, fisetin protected against H2O2-induced membrane lipid peroxidation, cellular DNA damage, and protein carbonylation, which are the primary cellular outcomes of H2O2 treatment. Furthermore, fisetin increased the level of reduced glutathione (GSH) and expression of glutamate-cysteine ligase catalytic subunit, which is decreased by H2O2. Conversely, a GSH inhibitor abolished the cytoprotective effect of fisetin against H2O2-induced cells damage. Taken together, our results suggest that fisetin protects against H2O2-induced cell damage by inhibiting ROS generation, thereby maintaining the protective role of the cellular GSH system.

  13. Antioxidant and DNA damage protection potentials of selected phenolic acids.

    PubMed

    Sevgi, Kemal; Tepe, Bektas; Sarikurkcu, Cengiz

    2015-03-01

    In this study, ten different phenolic acids (caffeic, chlorogenic, cinnamic, ferulic, gallic, p-hydroxybenzoic, protocatechuic, rosmarinic, syringic, and vanillic acids) were evaluated for their antioxidant and DNA damage protection potentials. Antioxidant activity was evaluated by using four different test systems named as β-carotene bleaching, DPPH free radical scavenging, reducing power and chelating effect. In all test systems, rosmarinic acid showed the maximum activity potential, while protocatechuic acid was determined as the weakest antioxidant in β-carotene bleaching, DPPH free radical scavenging, and chelating effect assays. Phenolic acids were also screened for their protective effects on pBR322 plasmid DNA against the mutagenic and toxic effects of UV and H2O2. Ferulic acid was found as the most active phytochemical among the others. Even at the lowest concentration value (0.002 mg/ml), ferulic acid protected all of the bands in the presence of H2O2 and UV. It is followed by caffeic, rosmarinic, and vanillic acids. On the other hand, cinnamic acid (at 0.002 mg/ml), gallic acid (at 0.002 mg/ml), p-hydroxybenzoic acid (at 0.002 and 0.004 mg/ml), and protocatechuic acid (at 0.002 and 0.004 mg/ml) could not protect plasmid DNA. PMID:25542528

  14. Sequencing of chloroplast genome using whole cellular DNA and solexa sequencing technology.

    PubMed

    Wu, Jian; Liu, Bo; Cheng, Feng; Ramchiary, Nirala; Choi, Su Ryun; Lim, Yong Pyo; Wang, Xiao-Wu

    2012-01-01

    Sequencing of the chloroplast (cp) genome using traditional sequencing methods has been difficult because of its size (>120 kb) and the complicated procedures required to prepare templates. To explore the feasibility of sequencing the cp genome using DNA extracted from whole cells and Solexa sequencing technology, we sequenced whole cellular DNA isolated from leaves of three Brassicarapa accessions with one lane per accession. In total, 246, 362, and 361 Mb sequence data were generated for the three accessions Chiifu-401-42, Z16, and FT, respectively. Micro-reads were assembled by reference-guided assembly using the cpDNA sequences of B. rapa, Arabidopsis thaliana, and Nicotiana tabacum. We achieved coverage of more than 99.96% of the cp genome in the three tested accessions using the B. rapa sequence as the reference. When A. thaliana or N. tabacum sequences were used as references, 99.7-99.8 or 95.5-99.7% of the B. rapa cp genome was covered, respectively. These results demonstrated that sequencing of whole cellular DNA isolated from young leaves using the Illumina Genome Analyzer is an efficient method for high-throughput sequencing of cp genome.

  15. Mitochondrial DNA Replication Defects Disturb Cellular dNTP Pools and Remodel One-Carbon Metabolism.

    PubMed

    Nikkanen, Joni; Forsström, Saara; Euro, Liliya; Paetau, Ilse; Kohnz, Rebecca A; Wang, Liya; Chilov, Dmitri; Viinamäki, Jenni; Roivainen, Anne; Marjamäki, Päivi; Liljenbäck, Heidi; Ahola, Sofia; Buzkova, Jana; Terzioglu, Mügen; Khan, Nahid A; Pirnes-Karhu, Sini; Paetau, Anders; Lönnqvist, Tuula; Sajantila, Antti; Isohanni, Pirjo; Tyynismaa, Henna; Nomura, Daniel K; Battersby, Brendan J; Velagapudi, Vidya; Carroll, Christopher J; Suomalainen, Anu

    2016-04-12

    Mitochondrial dysfunction affects cellular energy metabolism, but less is known about the consequences for cytoplasmic biosynthetic reactions. We report that mtDNA replication disorders caused by TWINKLE mutations-mitochondrial myopathy (MM) and infantile onset spinocerebellar ataxia (IOSCA)-remodel cellular dNTP pools in mice. MM muscle shows tissue-specific induction of the mitochondrial folate cycle, purine metabolism, and imbalanced and increased dNTP pools, consistent with progressive mtDNA mutagenesis. IOSCA-TWINKLE is predicted to hydrolyze dNTPs, consistent with low dNTP pools and mtDNA depletion in the disease. MM muscle also modifies the cytoplasmic one-carbon cycle, transsulfuration, and methylation, as well as increases glucose uptake and its utilization for de novo serine and glutathione biosynthesis. Our evidence indicates that the mitochondrial replication machinery communicates with cytoplasmic dNTP pools and that upregulation of glutathione synthesis through glucose-driven de novo serine biosynthesis contributes to the metabolic stress response. These results are important for disorders with primary or secondary mtDNA instability and offer targets for metabolic therapy. PMID:26924217

  16. Low-energy-electron interactions with DNA: approaching cellular conditions with atmospheric experiments

    NASA Astrophysics Data System (ADS)

    Alizadeh, Elahe; Sanche, Léon

    2014-04-01

    A novel technique has been developed to investigate low energy electron (LEE)-DNA interactions in the presence of small biomolecules (e.g., N2, O2, H2O) found near DNA in the cell nucleus, in order to simulate cellular conditions. In this technique, LEEs are emitted from a metallic surface exposed by soft X-rays and interact with DNA thin films at standard ambient temperature and pressure (SATP). Whereas atmospheric N2 had little effect on the yields of LEE-induced single and double strand breaks, both O2 and H2O considerably modified and increased such damage. The highest yields were obtained when DNA is embedded in a combined O2 and H2O atmosphere. In this case, the amount of additional double strand breaks was supper-additive. The effect of modifying the chemical and physical stability of DNA by platinum-based chemotherapeutic agents (Pt-drugs) including cisplatin, carboplatin and oxaliplatin was also investigated with this technique. The results obtained provide information on the role played by subexcitation-energy electrons and dissociative electron attachment in the radiosensitization of DNA by Pt-drugs, which is an important step to unravel the mechanisms of radiosensitisation of these agents in chemoradiation cancer therapy.

  17. Antioxidative Activity of Platinum Nanocolloid and Its Protective Effect Against Chemical-Induced Hepatic Cellular Damage.

    PubMed

    Choi, Mi-Ran; Do, Le Thanh; Chung, Yong-Hoon; Yoo, Hoon; Yu, Rina

    2015-08-01

    Oxidative stress, a major cause of cellular injuries, is closely associated with a variety of chronic diseases such as cancer, liver diseases, degenerative brain disease and aging. In this study, we investigated antioxidant properties of platinum nanocolloid (PNC) against various oxidative stress conditions in vitro/in vivo by treating PNC on liver cell or tissue. Antioxidant activities of the PNC were determined by measuring quenching capacity on reactive oxygen species and its protective action against hydrogen peroxide or CCl4-induced oxidative cellular damage in HepG2 cell or liver tissue of mice. In vitro study, PNC markedly suppressed the production H2O2, ·OH, α,α-diphenyl-β-picrylhydrazyl radical and nitric oxide in a dose-dependent manner. PNC also inhibited hydrogen peroxide-induced oxidative cellular damage in HepG2 hepatocytes. In vivo study with mice, PNC reduced hepatic lipid peroxidation and CCl4 induced toxicity. Our results support that platinum nanocolloid has antioxidant activities and protects hepatic cellular oxidative damage. Thus platinum nanocolloid may have a potential to be used as an antioxidant supplement.

  18. DNA vaccine encoding Haemonchus contortus actin induces partial protection in goats.

    PubMed

    Yan, Ruofeng; Wang, Jingjing; Xu, Lixin; Song, Xiaokai; Li, Xiangrui

    2014-10-01

    Actin is a globular multi-functional protein that forms microfilaments, and participates in many important cellular processes. Previous study found that Haemonchus contortus actin could be recognized by the serum of goats infected with the homology parasite. This indicated that H. contortus actin could be a potential candidate for vaccine. In this study, DNA vaccine encoding H. contortus actin was tested for protection against experimental H. contortus infections in goats. Fifteen goats were allocated into three trial groups. The animals of Actin group were vaccinated with the DNA vaccine on day 0 and 14, and challenged with 5000 infective H. contortus third stage larval (L3) on day 28. An unvaccinated positive control group was challenged with L3 at the same time. An unvaccinated negative control group was not challenged with L3. The results showed that DNA vaccine were transcribed at local injection sites and expressed in vivo post immunizations respectively. For goats in Actin vaccinated group, higher levels of serum IgG, serum IgA and mucosal IgA were produced, the percentages of CD4(+) T lymphocytes, CD8(+) T lymphocytes and B lymphocytes and the concentrations of TGF-β were increased significantly (P<0.05). Following L3 challenge, the mean eggs per gram feces (EPG) and worm burdens of Actin group were reduced by 34.4% and 33.1%, respectively. This study suggest that recombinant H. contortus Actin DNA vaccine induced partial immune response and has protective potential against goat haemonchosis.

  19. Two Immunologically Distinct Human DNA Polymerase α-Primase Subpopulations Are Involved in Cellular DNA Replication

    PubMed Central

    Dehde, Silke; Rohaly, Gabor; Schub, Oliver; Nasheuer, Heinz-Peter; Bohn, Wolfgang; Chemnitz, Jan; Deppert, Wolfgang; Dornreiter, Irena

    2001-01-01

    Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase α-primase (Pol-Prim) populations that are distinguishable by monoclonal antibodies. Cell cycle studies showed that the hypophosphorylated form was found in a complex with PP2A and cyclin E-Cdk2 in G1, whereas the phosphorylated enzyme was associated with a cyclin A kinase in S and G2. Modification of Pol-Prim by PP2A and Cdks regulated the interaction with the simian virus 40 origin-binding protein large T antigen and thus initiation of DNA replication. Confocal microscopy demonstrated nuclear colocalization of hypophosphorylated Pol-Prim with MCM2 in S phase nuclei, but its presence preceded 5-bromo-2′-deoxyuridine (BrdU) incorporation. The phosphorylated replicase exclusively colocalized with the BrdU signal, but not with MCM2. Immunoprecipitation experiments proved that only hypophosphorylated Pol-Prim associated with MCM2. The data indicate that the hypophosphorylated enzyme initiates DNA replication at origins, and the phosphorylated form synthesizes the primers for the lagging strand of the replication fork. PMID:11259605

  20. The catalytic A1 domains of cholera toxin and heat-labile enterotoxin are potent DNA adjuvants that evoke mixed Th1/Th17 cellular immune responses

    PubMed Central

    Bagley, Kenneth; Xu, Rong; Ota-Setlik, Ayuko; Egan, Michael; Schwartz, Jennifer; Fouts, Timothy

    2015-01-01

    DNA encoded adjuvants are well known for increasing the magnitude of cellular and/or humoral immune responses directed against vaccine antigens. DNA adjuvants can also tune immune responses directed against vaccine antigens to better protect against infection of the target organism. Two potent DNA adjuvants that have unique abilities to tune immune responses are the catalytic A1 domains of Cholera Toxin (CTA1) and Heat-Labile Enterotoxin (LTA1). Here, we have characterized the adjuvant activities of CTA1 and LTA1 using HIV and SIV genes as model antigens. Both of these adjuvants enhanced the magnitude of antigen-specific cellular immune responses on par with those induced by the well-characterized cytokine adjuvants IL-12 and GM-CSF. CTA1 and LTA1 preferentially enhanced cellular responses to the intracellular antigen SIVmac239-gag over those for the secreted HIVBaL-gp120 antigen. IL-12, GM-CSF and electroporation did the opposite suggesting differences in the mechanisms of actions of these diverse adjuvants. Combinations of CTA1 or LTA1 with IL-12 or GM-CSF generated additive and better balanced cellular responses to both of these antigens. Consistent with observations made with the holotoxin and the CTA1-DD adjuvant, CTA1 and LTA1 evoked mixed Th1/Th17 cellular immune responses. Together, these results show that CTA1 and LTA1 are potent DNA vaccine adjuvants that favor the intracellular antigen gag over the secreted antigen gp120 and evoke mixed Th1/Th17 responses against both of these antigens. The results also indicate that achieving a balanced immune response to multiple intracellular and extracellular antigens delivered via DNA vaccination may require combining adjuvants that have different and complementary mechanisms of action. PMID:26042527

  1. Functional DNA-Containing Nanomaterials: Cellular Applications in Biosensing, Imaging, and Targeted Therapy

    PubMed Central

    2015-01-01

    Conspectus DNA performs a vital function as a carrier of genetic code, but in the field of nanotechnology, DNA molecules can catalyze chemical reactions in the cell, that is, DNAzymes, or bind with target-specific ligands, that is, aptamers. These functional DNAs with different modifications have been developed for sensing, imaging, and therapeutic systems. Thus, functional DNAs hold great promise for future applications in nanotechnology and bioanalysis. However, these functional DNAs face challenges, especially in the field of biomedicine. For example, functional DNAs typically require the use of cationic transfection reagents to realize cellular uptake. Such reagents enter the cells, increasing the difficulty of performing bioassays in vivo and potentially damaging the cell’s nucleus. To address this obstacle, nanomaterials, such as metallic, carbon, silica, or magnetic materials, have been utilized as DNA carriers or assistants. In this Account, we describe selected examples of functional DNA-containing nanomaterials and their applications from our recent research and those of others. As models, we have chosen to highlight DNA/nanomaterial complexes consisting of gold nanoparticles, graphene oxides, and aptamer–micelles, and we illustrate the potential of such complexes in biosensing, imaging, and medical diagnostics. Under proper conditions, multiple ligand–receptor interactions, decreased steric hindrance, and increased surface roughness can be achieved from a high density of DNA that is bound to the surface of nanomaterials, resulting in a higher affinity for complementary DNA and other targets. In addition, this high density of DNA causes a high local salt concentration and negative charge density, which can prevent DNA degradation. For example, DNAzymes assembled on gold nanoparticles can effectively catalyze chemical reactions even in living cells. And it has been confirmed that DNA–nanomaterial complexes can enter cells more easily than free

  2. p53-dependent SIRT6 expression protects Aβ42-induced DNA damage

    PubMed Central

    Jung, Eun Sun; Choi, Hyunjung; Song, Hyundong; Hwang, Yu Jin; Kim, Ahbin; Ryu, Hoon; Mook-Jung, Inhee

    2016-01-01

    Alzheimer’s disease (AD) is the most common type of dementia and age-related neurodegenerative disease. Elucidating the cellular changes that occur during ageing is an important step towards understanding the pathogenesis and progression of neurodegenerative disorders. SIRT6 is a member of the mammalian sirtuin family of anti-aging genes. However, the relationship between SIRT6 and AD has not yet been elucidated. Here, we report that SIRT6 protein expression levels are reduced in the brains of both the 5XFAD AD mouse model and AD patients. Aβ42, a major component of senile plaques, decreases SIRT6 expression, and Aβ42-induced DNA damage is prevented by the overexpression of SIRT6 in HT22 mouse hippocampal neurons. Also, there is a strong negative correlation between Aβ42-induced DNA damage and p53 levels, a protein involved in DNA repair and apoptosis. In addition, upregulation of p53 protein by Nutlin-3 prevents SIRT6 reduction and DNA damage induced by Aβ42. Taken together, this study reveals that p53-dependent SIRT6 expression protects cells from Aβ42-induced DNA damage, making SIRT6 a promising new therapeutic target for the treatment of AD. PMID:27156849

  3. Dandelion Extracts Protect Human Skin Fibroblasts from UVB Damage and Cellular Senescence.

    PubMed

    Yang, Yafan; Li, Shuangshuang

    2015-01-01

    Ultraviolet (UV) irradiation causes damage in skin by generating excessive reactive oxygen species (ROS) and induction of matrix metalloproteinases (MMPs), leading to skin photoageing. Dandelion extracts have long been used for traditional Chinese medicine and native American medicine to treat cancers, hepatitis, and digestive diseases; however, less is known on the effects of dandelion extracts in skin photoageing. Here we found that dandelion leaf and flower extracts significantly protect UVB irradiation-inhibited cell viability when added before UVB irradiation or promptly after irradiation. Dandelion leaf and flower extracts inhibited UVB irradiation-stimulated MMP activity and ROS generation. Dandelion root extracts showed less action on protecting HDFs from UVB irradiation-induced MMP activity, ROS generation, and cell death. Furthermore, dandelion leaf and flower but not root extracts stimulated glutathione generation and glutathione reductase mRNA expression in the presence or absence of UVB irradiation. We also found that dandelion leaf and flower extracts help absorb UVB irradiation. In addition, dandelion extracts significantly protected HDFs from H2O2-induced cellular senescence. In conclusion, dandelion extracts especially leaf and flower extracts are potent protective agents against UVB damage and H2O2-induced cellular senescence in HDFs by suppressing ROS generation and MMP activities and helping UVB absorption. PMID:26576225

  4. Dandelion Extracts Protect Human Skin Fibroblasts from UVB Damage and Cellular Senescence.

    PubMed

    Yang, Yafan; Li, Shuangshuang

    2015-01-01

    Ultraviolet (UV) irradiation causes damage in skin by generating excessive reactive oxygen species (ROS) and induction of matrix metalloproteinases (MMPs), leading to skin photoageing. Dandelion extracts have long been used for traditional Chinese medicine and native American medicine to treat cancers, hepatitis, and digestive diseases; however, less is known on the effects of dandelion extracts in skin photoageing. Here we found that dandelion leaf and flower extracts significantly protect UVB irradiation-inhibited cell viability when added before UVB irradiation or promptly after irradiation. Dandelion leaf and flower extracts inhibited UVB irradiation-stimulated MMP activity and ROS generation. Dandelion root extracts showed less action on protecting HDFs from UVB irradiation-induced MMP activity, ROS generation, and cell death. Furthermore, dandelion leaf and flower but not root extracts stimulated glutathione generation and glutathione reductase mRNA expression in the presence or absence of UVB irradiation. We also found that dandelion leaf and flower extracts help absorb UVB irradiation. In addition, dandelion extracts significantly protected HDFs from H2O2-induced cellular senescence. In conclusion, dandelion extracts especially leaf and flower extracts are potent protective agents against UVB damage and H2O2-induced cellular senescence in HDFs by suppressing ROS generation and MMP activities and helping UVB absorption.

  5. Dandelion Extracts Protect Human Skin Fibroblasts from UVB Damage and Cellular Senescence

    PubMed Central

    Yang, Yafan; Li, Shuangshuang

    2015-01-01

    Ultraviolet (UV) irradiation causes damage in skin by generating excessive reactive oxygen species (ROS) and induction of matrix metalloproteinases (MMPs), leading to skin photoageing. Dandelion extracts have long been used for traditional Chinese medicine and native American medicine to treat cancers, hepatitis, and digestive diseases; however, less is known on the effects of dandelion extracts in skin photoageing. Here we found that dandelion leaf and flower extracts significantly protect UVB irradiation-inhibited cell viability when added before UVB irradiation or promptly after irradiation. Dandelion leaf and flower extracts inhibited UVB irradiation-stimulated MMP activity and ROS generation. Dandelion root extracts showed less action on protecting HDFs from UVB irradiation-induced MMP activity, ROS generation, and cell death. Furthermore, dandelion leaf and flower but not root extracts stimulated glutathione generation and glutathione reductase mRNA expression in the presence or absence of UVB irradiation. We also found that dandelion leaf and flower extracts help absorb UVB irradiation. In addition, dandelion extracts significantly protected HDFs from H2O2-induced cellular senescence. In conclusion, dandelion extracts especially leaf and flower extracts are potent protective agents against UVB damage and H2O2-induced cellular senescence in HDFs by suppressing ROS generation and MMP activities and helping UVB absorption. PMID:26576225

  6. Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro

    PubMed Central

    Hossain, M. Zulfiquer; Patel, Kalpesh; Kern, Scott E.

    2014-01-01

    Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution. PMID:24842839

  7. Protection of cisplatin-induced spermatotoxicity, DNA damage and chromatin abnormality by selenium nano-particles

    SciTech Connect

    Rezvanfar, Mohammad Amin; Rezvanfar, Mohammad Ali; Shahverdi, Ahmad Reza; Ahmadi, Abbas; Baeeri, Maryam; Mohammadirad, Azadeh; Abdollahi, Mohammad

    2013-02-01

    Cisplatin (CIS), an anticancer alkylating agent, induces DNA adducts and effectively cross links the DNA strands and so affects spermatozoa as a male reproductive toxicant. The present study investigated the cellular/biochemical mechanisms underlying possible protective effect of selenium nano-particles (Nano-Se) as an established strong antioxidant with more bioavailability and less toxicity, on reproductive toxicity of CIS by assessment of sperm characteristics, sperm DNA integrity, chromatin quality and spermatogenic disorders. To determine the role of oxidative stress (OS) in the pathogenesis of CIS gonadotoxicity, the level of lipid peroxidation (LPO), antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and peroxynitrite (ONOO) as a marker of nitrosative stress (NS) and testosterone (T) concentration as a biomarker of testicular function were measured in the blood and testes. Thirty-two male Wistar rats were equally divided into four groups. A single IP dose of CIS (7 mg/kg) and protective dose of Nano-Se (2 mg/kg/day) were administered alone or in combination. The CIS-exposed rats showed a significant increase in testicular and serum LPO and ONOO level, along with a significant decrease in enzymatic antioxidants levels, diminished serum T concentration and abnormal histologic findings with impaired sperm quality associated with increased DNA damage and decreased chromatin quality. Coadministration of Nano-Se significantly improved the serum T, sperm quality, and spermatogenesis and reduced CIS-induced free radical toxic stress and spermatic DNA damage. In conclusion, the current study demonstrated that Nano-Se may be useful to prevent CIS-induced gonadotoxicity through its antioxidant potential. Highlights: ► Cisplatin (CIS) affects spermatozoa as a male reproductive toxicant. ► Effect of Nano-Se on CIS-induced spermatotoxicity was investigated. ► CIS-exposure induces oxidative sperm DNA damage

  8. A DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus induces protective immunity in BALB/c mice.

    PubMed

    Oñate, Angel A; Céspedes, Sandra; Cabrera, Alex; Rivers, Rodolfo; González, Andrés; Muñoz, Carola; Folch, Hugo; Andrews, Edilia

    2003-09-01

    This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). Intramuscular injection of plasmid DNA carrying the SOD gene (pcDNA-SOD) into BALB/c mice elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD-specific antibodies which exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the DNA vaccine elicited a T-cell-proliferative response and also induced the production of gamma interferon, but not interleukin-10 (IL-10) or IL-4, upon restimulation with either recombinant SOD or crude Brucella protein, suggesting the induction of a typical T-helper-1-dominated immune response in mice. The pcDNA-SOD (but not the control vector) induced a strong, significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308; the level of protection was similar to the one induced by B. abortus vaccine strain RB51. Altogether, these data suggest that pcDNA-SOD is a good candidate for use in future studies of vaccination against brucellosis.

  9. Fisetin Protects DNA Against Oxidative Damage and Its Possible Mechanism

    PubMed Central

    Wang, Tingting; Lin, Huajuan; Tu, Qian; Liu, Jingjing; Li, Xican

    2016-01-01

    Purpose: The paper tries to assess the protective effect of fisetin against •OH-induced DNA damage, then to investigate the possible mechanism. Methods: The protective effect was evaluated based on the content of malondialdehyde (MDA). The possible mechanism was analyzed using various antioxidant methods in vitro, including •OH scavenging (deoxyribose degradation), •O2- scavenging (pyrogallol autoxidation), DPPH• scavenging, ABTS•+ scavenging, and Cu2+-reducing power assays. Results: Fisetin increased dose-dependently its protective percentages against •OH-induced DNA damage (IC50 value =1535.00±29.60 µM). It also increased its radical-scavenging percentages in a dose-dependent manner in various antioxidants assays. Its IC50 values in •OH scavenging, •O2- scavenging, DPPH• scavenging, ABTS•+ scavenging, and Cu2+-reducing power assays, were 47.41±4.50 µM, 34.05±0.87 µM, 9.69±0.53 µM, 2.43±0.14 µM, and 1.49±0.16 µM, respectively. Conclusion: Fisetin can effectively protect DNA against •OH-induced oxidative damage possibly via reactive oxygen species (ROS) scavenging approach, which is assumed to be hydrogen atom (H•) and/or single electron (e) donation (HAT/SET) pathways. In the HAT pathway, the 3’,4’-dihydroxyl moiety in B ring of fisetin is thought to play an important role, because it can be ultimately oxidized to a stable ortho-benzoquinone form. PMID:27478791

  10. Protective cellular retroviral immunity requires both CD4+ and CD8+ immune T cells.

    PubMed Central

    Hom, R C; Finberg, R W; Mullaney, S; Ruprecht, R M

    1991-01-01

    We have found previously that postexposure chemoprophylaxis with 3'-azido-3'-deoxythymidine (also known as zidovudine or AZT) in combination with recombinant human alpha A/D interferon fully protected mice exposed to a lethal dose of Rauscher murine leukemia virus (RLV) against viremia and disease. After cessation of therapy, over 90% of these mice were able to resist rechallenge with live RLV, thus demonstrating an acquired immunity. Adoptive cell transfer of 4 x 10(7) cells from immunized mice fully protected naive recipients from viremia and splenomegaly after RLV challenge. However, when these immune T cells were fractionated into CD4+ and CD8+ subpopulations, only partial protection was found when 4 x 10(7) T cells of either subset were given. Full protection against RLV challenge was seen again when the T-cell subsets from immunized mice were recombined and transferred at the same number into naive mice. We conclude that cellular immunity alone is protective and that both CD4+ and CD8+ cell types are required for conferring full protection against live virus challenge. Images PMID:1898666

  11. DNA-controlled dynamic colloidal nanoparticle systems for mediating cellular interaction.

    PubMed

    Ohta, Seiichi; Glancy, Dylan; Chan, Warren C W

    2016-02-19

    Precise control of biosystems requires development of materials that can dynamically change physicochemical properties. Inspired by the ability of proteins to alter their conformation to mediate function, we explored the use of DNA as molecular keys to assemble and transform colloidal nanoparticle systems. The systems consist of a core nanoparticle surrounded by small satellites, the conformation of which can be transformed in response to DNA via a toe-hold displacement mechanism. The conformational changes can alter the optical properties and biological interactions of the assembled nanosystem. Photoluminescent signal is altered by changes in fluorophore-modified particle distance, whereas cellular targeting efficiency is increased 2.5 times by changing the surface display of targeting ligands. These concepts provide strategies for engineering dynamic nanotechnology systems for navigating complex biological environments. PMID:26912892

  12. DNA-controlled dynamic colloidal nanoparticle systems for mediating cellular interaction

    NASA Astrophysics Data System (ADS)

    Ohta, Seiichi; Glancy, Dylan; Chan, Warren C. W.

    2016-02-01

    Precise control of biosystems requires development of materials that can dynamically change physicochemical properties. Inspired by the ability of proteins to alter their conformation to mediate function, we explored the use of DNA as molecular keys to assemble and transform colloidal nanoparticle systems. The systems consist of a core nanoparticle surrounded by small satellites, the conformation of which can be transformed in response to DNA via a toe-hold displacement mechanism. The conformational changes can alter the optical properties and biological interactions of the assembled nanosystem. Photoluminescent signal is altered by changes in fluorophore-modified particle distance, whereas cellular targeting efficiency is increased 2.5 times by changing the surface display of targeting ligands. These concepts provide strategies for engineering dynamic nanotechnology systems for navigating complex biological environments.

  13. Intranasal DNA vaccination induces potent mucosal and systemic immune responses and cross-protective immunity against influenza viruses.

    PubMed

    Torrieri-Dramard, Lea; Lambrecht, Bénédicte; Ferreira, Helena Lage; Van den Berg, Thierry; Klatzmann, David; Bellier, Bertrand

    2011-03-01

    The induction of potent virus-specific immune responses at mucosal surfaces where virus transmission occurs is a major challenge for vaccination strategies. In the case of influenza vaccination, this has been achieved only by intranasal delivery of live-attenuated vaccines that otherwise pose safety problems. Here, we demonstrate that potent mucosal and systemic immune responses, both cellular and humoral, are induced by intranasal immunization using formulated DNA. We show that formulation with the DNA carrier polyethylenimine (PEI) improved by a 1,000-fold the efficiency of gene transfer in the respiratory track following intranasal administration of luciferase-coding DNA. Using PEI formulation, intranasal vaccination with DNA-encoding hemagglutinin (HA) from influenza A H5N1 or (H1N1)2009 viruses induced high levels of HA-specific immunoglobulin A (IgA) antibodies that were detected in bronchoalveolar lavages (BALs) and the serum. No mucosal responses could be detected after parenteral or intranasal immunization with naked-DNA. Furthermore, intranasal DNA vaccination with HA from a given H5N1 virus elicited full protection against the parental strain and partial cross-protection against a distinct highly pathogenic H5N1 strain that could be improved by adding neuraminidase (NA) DNA plasmids. Our observations warrant further investigation of intranasal DNA as an effective vaccination route.

  14. Antioxidant and DNA Damage Protecting Activity of Exopolysaccharides from the Endophytic Bacterium Bacillus cereus SZ1.

    PubMed

    Zheng, Li Ping; Zou, Tin; Ma, Yan Jun; Wang, Jian Wen; Zhang, Yu Qing

    2016-01-01

    An endophytic bacterium was isolated from the Chinese medicinal plant Artemisia annua L. The phylogenetic and physiological characterization indicated that the isolate, strain SZ-1, was Bacillus cereus. The endophyte could produce an exopolysaccharide (EPS) at 46 mg/L. The 1,1-diphenyl-2-picrylhydracyl (DPPH) radical scavenging activity of the EPS reached more than 50% at 3-5 mg/mL. The EPS was also effective in scavenging superoxide radical in a concentration dependent fashion with an EC50 value of 2.6 mg/mL. The corresponding EC50 for scavenging hydroxyl radical was 3.1 mg/mL. Moreover, phenanthroline-copper complex-mediated chemiluminescent emission of DNA damage was both inhibited and delayed by EPS. The EPS at 0.7-1.7 mg/mL also protected supercoiled DNA strands in plasmid pBR322 against scission induced by Fenton-mediated hydroxyl radical. The preincubation of PC12 cells with the EPS prior to H₂O₂ exposure increased the cell survival and glutathione (GSH) level and catalase (CAT) activities, and decreased the level of malondialdehyde (MDA) and lactate dehydrogenase (LDH) activity in a dose-dependent manner, suggesting a pronounced protective effect against H₂O₂-induced cytotoxicity. Our study indicated that the EPS could be useful for preventing oxidative DNA damage and cellular oxidation in pharmaceutical and food industries. PMID:26861269

  15. Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases.

    PubMed Central

    Bashir, S; Harris, G; Denman, M A; Blake, D R; Winyard, P G

    1993-01-01

    OBJECTIVES--To estimate the extent of genomic DNA damage and killing of lymphocytes by reactive oxygen intermediates in autoimmune diseases. METHODS--8-Oxo-7-hydrodeoxyguanosine (8-oxodG), a promutagenic DNA lesion induced by reactive oxygen intermediates, was measured by high performance liquid chromatography, coupled with electrochemical detection, in hydrolysates of DNA which had been extracted from lymphocyte and polymorphonuclear leucocyte fractions of human blood. In addition, human primary blood lymphocytes stimulated by concanavalin A were assayed for cytotoxicity induced by hydrogen peroxide on day 0, by assessing cell proliferation during seven days of culture. RESULTS--Constitutive 8-oxodG was detectable (mean (2 SEM) moles 8-oxodG/10(6) moles deoxyguanosine) in DNA isolated from normal human blood lymphocytes (68 (8), n = 26) and polymorphonuclear leucocytes (118 (24), n = 24). Lymphocyte DNA from donors with the following inflammatory autoimmune diseases contained significantly higher levels of 8-oxodG than that from healthy donors: rheumatoid arthritis (98 (16)), systemic lupus erythematosus (137 (28)), vasculitis (100 (32)), and Behçet's disease (92 (19)). Lymphocyte 8-oxodG levels in non-autoimmune controls and patients with scleroderma were not significantly different from those of healthy controls. The levels of 8-oxodG were significantly higher in the DNA from normal polymorphonuclear leucocytes than in paired DNA samples from normal lymphocytes, but there were no differences between levels of 8-oxodG in polymorphonuclear leucocytes from normal subjects and the patients studied. Levels of 8-oxodG did not correlate with disease duration, disease severity, or age. Lymphocytes from patients with systemic lupus erythematosus and rheumatoid arthritis, but not those with scleroderma, also showed cellular hypersensitivity to the toxic effects of hydrogen peroxide. CONCLUSION--There was increased genomic DNA damage, and increased susceptibility to

  16. Derinat Protects Skin against Ultraviolet-B (UVB)-Induced Cellular Damage.

    PubMed

    Hsu, Wen-Li; Lu, Jian-He; Noda, Mami; Wu, Ching-Ying; Liu, Jia-Dai; Sakakibara, Manabu; Tsai, Ming-Hsien; Yu, Hsin-Su; Lin, Ming-Wei; Huang, Yaw-Bin; Yan, Shian-Jang; Yoshioka, Tohru

    2015-01-01

    Ultraviolet-B (UVB) is one of the most cytotoxic and mutagenic stresses that contribute to skin damage and aging through increasing intracellular Ca(2+) and reactive oxygen species (ROS). Derinat (sodium deoxyribonucleate) has been utilized as an immunomodulator for the treatment of ROS-associated diseases in clinics. However, the molecular mechanism by which Derinat protects skin cells from UVB-induced damage is poorly understood. Here, we show that Derinat significantly attenuated UVB-induced intracellular ROS production and decreased DNA damage in primary skin cells. Furthermore, Derinat reduced intracellular ROS, cyclooxygenase-2 (COX-2) expression and DNA damage in the skin of the BALB/c-nu mice exposed to UVB for seven days in vivo. Importantly, Derinat blocked the transient receptor potential canonical (TRPC) channels (TRPCs), as demonstrated by calcium imaging. Together, our results indicate that Derinat acts as a TRPCs blocker to reduce intracellular ROS production and DNA damage upon UVB irradiation. This mechanism provides a potential new application of Derinat for the protection against UVB-induced skin damage and aging. PMID:26569211

  17. Toward reliable algorithmic self-assembly of DNA tiles: a fixed-width cellular automaton pattern.

    PubMed

    Fujibayashi, Kenichi; Hariadi, Rizal; Park, Sung Ha; Winfree, Erik; Murata, Satoshi

    2008-07-01

    Bottom-up fabrication of nanoscale structures relies on chemical processes to direct self-assembly. The complexity, precision, and yield achievable by a one-pot reaction are limited by our ability to encode assembly instructions into the molecules themselves. Nucleic acids provide a platform for investigating these issues, as molecular structure and intramolecular interactions can encode growth rules. Here, we use DNA tiles and DNA origami to grow crystals containing a cellular automaton pattern. In a one-pot annealing reaction, 250 DNA strands first assemble into a set of 10 free tile types and a seed structure, then the free tiles grow algorithmically from the seed according to the automaton rules. In our experiments, crystals grew to approximately 300 nm long, containing approximately 300 tiles with an initial assembly error rate of approximately 1.4% per tile. This work provides evidence that programmable molecular self-assembly may be sufficient to create a wide range of complex objects in one-pot reactions.

  18. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    SciTech Connect

    Roper, Katherine; Coverley, Dawn

    2012-03-10

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening

  19. Downregulation of Cellular Protective Factors of Rumen Epithelium in Goats Fed High Energy Diet

    PubMed Central

    Hollmann, Manfred; Miller, Ingrid; Hummel, Karin; Sabitzer, Sonja; Metzler-Zebeli, Barbara U.; Razzazi-Fazeli, Ebrahim; Zebeli, Qendrim

    2013-01-01

    Energy-rich diets can challenge metabolic and protective functions of the rumen epithelial cells, but the underlying factors are unclear. This study sought to evaluate proteomic changes of the rumen epithelium in goats fed a low, medium, or high energy diet. Expression of protein changes were compared by two-dimensional differential gel electrophoresis followed by protein identification with matrix assisted laser desorption ionisation tandem time-of-flight mass spectrometry. Of about 2,000 spots commonly detected in all gels, 64 spots were significantly regulated, which were traced back to 24 unique proteins. Interestingly, the expression profiles of several chaperone proteins with important cellular protective functions such as heat shock cognate 71 kDa protein, peroxiredoxin-6, serpin H1, protein disulfide-isomerase, and selenium-binding protein were collectively downregulated in response to high dietary energy supply. Similar regulation patterns were obtained for some other proteins involved in transport or metabolic functions. In contrast, metabolic enzymes like retinal dehydrogenase 1 and ATP synthase subunit beta, mitochondrial precursor were upregulated in response to high energy diet. Lower expressions of chaperone proteins in the rumen epithelial cells in response to high energy supply may suggest that these cells were less protected against the potentially harmful rumen toxic compounds, which might have consequences for rumen and systemic health. Our findings also suggest that energy-rich diets and the resulting acidotic insult may render rumen epithelial cells more vulnerable to cellular damage by attenuating their cell defense system, hence facilitating the impairment of rumen barrier function, typically observed in energy-rich fed ruminants. PMID:24349094

  20. Prevention of DNA photodamage by vitamin E compounds and sunscreens: roles of ultraviolet absorbance and cellular uptake.

    PubMed

    McVean, M; Liebler, D C

    1999-03-01

    nuclei corresponded temporally with significant protection against DNA photodamage. The kinetics of accumulation of the three tocopherols in whole cells and in nuclei were similar. Although only alphaTH conferred significant protection compared with irradiated controls at 2 h, the differences between individual tocopherols were not statistically significant. This work suggests that incorporation of tocopherol compounds into sunscreen products confers protection against procarcinogenic DNA photodamage and that cellular uptake and distribution of tocopherol compounds is necessary for their optimal photoprotection.

  1. Prevention of DNA photodamage by vitamin E compounds and sunscreens: roles of ultraviolet absorbance and cellular uptake.

    PubMed

    McVean, M; Liebler, D C

    1999-03-01

    nuclei corresponded temporally with significant protection against DNA photodamage. The kinetics of accumulation of the three tocopherols in whole cells and in nuclei were similar. Although only alphaTH conferred significant protection compared with irradiated controls at 2 h, the differences between individual tocopherols were not statistically significant. This work suggests that incorporation of tocopherol compounds into sunscreen products confers protection against procarcinogenic DNA photodamage and that cellular uptake and distribution of tocopherol compounds is necessary for their optimal photoprotection. PMID:10204801

  2. Dinuclear Ruthenium(II) Complexes as Two-Photon, Time-Resolved Emission Microscopy Probes for Cellular DNA**

    PubMed Central

    Baggaley, Elizabeth; Gill, Martin R; Green, Nicola H; Turton, David; Sazanovich, Igor V; Botchway, Stanley W; Smythe, Carl; Haycock, John W; Weinstein, Julia A; Thomas, Jim A

    2014-01-01

    The first transition-metal complex-based two-photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA-bound probes display characteristic emission lifetimes of more than 160 ns, while shorter-lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence-free imaging. PMID:24458590

  3. Improvement of Cellular Uptake and Transfection Ability of pDNA Using α-Cyclodextrin-Polyamidoamine Conjugates as Gene Delivery System.

    PubMed

    Qin, Linghao; Cao, Duanwen; Huang, Huan; Ji, Gangjian; Feng, Min; Chen, Jianhai; Pan, Shirong

    2016-02-01

    Polyamidoamine (PAMAM) dendrimers are a class of unique nanomaterials which attracted attention because of their extraordinary properties, such as highly branched structure and types of terminal primary groups. In addition, development in PAMAM chemical modification has broadened its biological application especially for drug and gene delivery. In this study, PAMAMs are covalently conjugated onto α-Cyclodextrin (α-CD) via amide bonds obtaining the starburst cationic polymers (CD-PG2). The chemical structure and composition of CD-PG2 was characterized by IH NMR. Physicochemical and biological properties of CD-PG2/pDNA polyplex were evaluated by agarose gel retardation, stability test against DNasecñ, MTT assay, DLS measurement, CLSM observation, LDH leakage test, cellular uptake route analysis and in-vitro cell transfection. Results showed that CD-PG2 can efficiently condense pDNA into nanoscale particles with a narrow size distribution, and protect pDNA form DNase I degradation. Compared with free PEI-25K and commercial product Lipofectamine2000, CD-PG2 shows excellent gene transfection efficiency without serum interference as well as relatively low cytotoxicity. Cellular uptake of CD-PG2/pDNA polyplex is mainly through CME and CvME route and further investigations demonstrate that α-CD can regulate CvME pathway to improve polyplex transfection behavior. In conclusion, CD-PG2 can be considered as a versatile tool for gene delivery, especially for gene transfer in-vivo. PMID:27305760

  4. Evidence for direct cellular protective effect of PL-10 substances (synthesized parts of body protection compound, BPC) and their specificity to gastric mucosal cells.

    PubMed

    Bódis, B; Karádi, O; Németh, P; Dohoczky, C; Kolega, M; Mózsik, G

    1997-01-01

    The direct gastric mucosal cellular effect of four PL-10 substances (a synthesized part of human body protection compound, BPC containing 14 or 15 amino acids) was studied on freshly isolated rat gastric mucosal cells and on a mouse myeloma cell line (Sp2/0-Ag14) in an ethanol-induced cell injury model. The examined substances were not toxic for the cells. Two of them proved to be significantly protective against the direct cellular damaging effect of ethanol (PL 10.1.15AK-3 in 5 microg/ml dose and PL 10.1.AK14-2 dose-dependently, ED50=50 ng/ml) on gastric mucosal cells. This cytoprotective effect was failured on mouse myeloma cells. Based on these results a part of the in vivo protection induced by BPC seems to be a direct cellular protective effect to gastric mucosal cells. PMID:9353174

  5. Salvia fruticosa reduces intrinsic cellular and H2O2-induced DNA oxidation in HEK 293 cells; assessment using flow cytometry

    PubMed Central

    Hani, Saleem Bani; Bayachou, Mekki

    2014-01-01

    Objective To investigate the role of water-soluble extract of Salvia fruticosa (Greek sage) (S. fruticosa) leaves in reducing both intrinsic cellular and H2O2-induced DNA oxidation in cultured human embryonic kidney 293 cells. S. fruticosa, native to the Eastern-Mediterranean basin, is widely used as a medicinal herb for treatment of various diseases. Methods Dried leaves of S. fruticosa were extracted in phosphate buffer saline and purified using both vacuum and high pressure filtrations. Each mL of the preparation contained (7.1±1.0) mg of extract. HEK-293 cells were incubated in one set with S. fruticosa extract in the presence of 0.1 mmol/L H2O2, and in the other set with the addition of the extract alone. The DNA oxidation was measured using fluorescence upon fluorescein isothiocyanate derivatization of 8-oxoguanine moieties. The fluorescence was measured using flow cytometry technique. Results Cells incubated 3 h with 150 µL extract and exposed to 0.1 mmol/L H2O2 showed lower intensity of fluorescence, and thus lower DNA oxidation. Moreover, cells incubated 3 h with 100 µL of the extract showed lower intensity of fluorescence, and thus lower intrinsic cellular DNA oxidation compared to control (without S. fruticosa). Conclusions The results from this study suggest that the water-soluble extract of S. fruticosa leaves protects against both H2O2-induced and intrinsic cellular DNA oxidation in human embryonic kidney 293 cells. PMID:25182726

  6. Mechanisms of radiation interaction with DNA: Potential implications for radiation protection

    SciTech Connect

    Not Available

    1988-01-01

    The Office of Health and Environmental Research (OHER) of the US Department of Energy conducts a broad multidisciplinary research program which includes basic biophysics, biophysical chemistry, molecular and cellular biology as well as experimental animal studies and opportunistic human studies. This research is directed at understanding how low levels of radiation of various qualities produce the spectrum of biological effects that are seen for such exposures. This workshop was entitled ''Mechanisms of Radiation Interaction with DNA: Potential Implications for Radiation Protection.'' It ws jointly sponsored by the Department of Energy and the Commission of European Communities. The aim of the workshop was to review the base of knowledge in the area of mechanisms of radiation action at the DNA level, and to explore ways in which this information can be applied to the development of scientifically sound concepts and procedures for use in the field of radiation protection. The overview of research provided by this multidisciplinary group will be helpful to the Office in program planning. This report includes a summary of the presentations, extended abstracts, the meeting agenda, research recommendations, and a list of participants. Individual papers are processed separately for the data base.

  7. Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

    SciTech Connect

    Du, Fengxia; Zhang, Minjie; Li, Xiaohua; Yang, Caiyun; Meng, Hao; Wang, Dong; Chang, Shuang; Xu, Ye; Price, Brendan; Sun, Yingli

    2014-10-03

    Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.

  8. Development of Cross-Protective Influenza A Vaccines Based on Cellular Responses

    PubMed Central

    Soema, Peter Christiaan; van Riet, Elly; Kersten, Gideon; Amorij, Jean-Pierre

    2015-01-01

    Seasonal influenza vaccines provide protection against matching influenza A virus (IAV) strains mainly through the induction of neutralizing serum IgG antibodies. However, these antibodies fail to confer a protective effect against mismatched IAV. This lack of efficacy against heterologous influenza strains has spurred the vaccine development community to look for other influenza vaccine concepts, which have the ability to elicit cross-protective immune responses. One of the concepts that is currently been worked on is that of influenza vaccines inducing influenza-specific T cell responses. T cells are able to lyse infected host cells, thereby clearing the virus. More interestingly, these T cells can recognize highly conserved epitopes of internal influenza proteins, making cellular responses less vulnerable to antigenic variability. T cells are therefore cross-reactive against many influenza strains, and thus are a promising concept for future influenza vaccines. Despite their potential, there are currently no T cell-based IAV vaccines on the market. Selection of the proper antigen, appropriate vaccine formulation and evaluation of the efficacy of T cell vaccines remains challenging, both in preclinical and clinical settings. In this review, we will discuss the current developments in influenza T cell vaccines, focusing on existing protein-based and novel peptide-based vaccine formulations. Furthermore, we will discuss the feasibility of influenza T cell vaccines and their possible use in the future. PMID:26029218

  9. Sublingual Immunization of Trivalent Human Papillomavirus DNA Vaccine in Baculovirus Nanovector for Protection against Vaginal Challenge

    PubMed Central

    Lee, Hee-Jung; Cho, Hansam; Kim, Mi-Gyeong; Heo, Yoon-Ki; Cho, Yeondong; Gwon, Yong-Dae; Park, Ki Hoon; Jin, Hyerim; Kim, Jinyoung; Oh, Yu-Kyoung; Kim, Young Bong

    2015-01-01

    Here, we report the immunogenicity of a sublingually delivered, trivalent human papillomavirus (HPV) DNA vaccine encapsidated in a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus nanovector. The HERV envelope-coated, nonreplicable, baculovirus-based DNA vaccine, encoding HPV16L1, -18L1 and -58L1 (AcHERV-triHPV), was constructed and sublingually administered to mice without adjuvant. Following sublingual (SL) administration, AcHERV-triHPV was absorbed and distributed throughout the body. At 15 minutes and 1 day post-dose, the distribution of AcHERV-triHPV to the lung was higher than that to other tissues. At 30 days post-dose, the levels of AcHERV-triHPV had diminished throughout the body. Six weeks after the first of three doses, 1×108 copies of SL AcHERV-triHPV induced HPV type-specific serum IgG and neutralizing antibodies to a degree comparable to that of IM immunization with 1×109 copies. AcHERV-triHPV induced HPV type-specific vaginal IgA titers in a dose-dependent manner. SL immunization with 1×1010 copies of AcHERV-triHPV induced Th1 and Th2 cellular responses comparable to IM immunization with 1×109 copies. Molecular imaging revealed that SL AcHERV-triHPV in mice provided complete protection against vaginal challenge with HPV16, HPV18, and HPV58 pseudoviruses. These results support the potential of SL immunization using multivalent DNA vaccine in baculovirus nanovector for induction of mucosal, systemic, and cellular immune responses. PMID:25789464

  10. Origin of giant viruses from smaller DNA viruses not from a fourth domain of cellular life.

    PubMed

    Yutin, Natalya; Wolf, Yuri I; Koonin, Eugene V

    2014-10-01

    The numerous and diverse eukaryotic viruses with large double-stranded DNA genomes that at least partially reproduce in the cytoplasm of infected cells apparently evolved from a single virus ancestor. This major group of viruses is known as Nucleocytoplasmic Large DNA Viruses (NCLDV) or the proposed order Megavirales. Among the "Megavirales", there are three groups of giant viruses with genomes exceeding 500kb, namely Mimiviruses, Pithoviruses, and Pandoraviruses that hold the current record of viral genome size, about 2.5Mb. Phylogenetic analysis of conserved, ancestral NLCDV genes clearly shows that these three groups of giant viruses have three distinct origins within the "Megavirales". The Mimiviruses constitute a distinct family that is distantly related to Phycodnaviridae, Pandoraviruses originate from a common ancestor with Coccolithoviruses within the Phycodnaviridae family, and Pithoviruses are related to Iridoviridae and Marseilleviridae. Maximum likelihood reconstruction of gene gain and loss events during the evolution of the "Megavirales" indicates that each group of giant viruses evolved from viruses with substantially smaller and simpler gene repertoires. Initial phylogenetic analysis of universal genes, such as translation system components, encoded by some giant viruses, in particular Mimiviruses, has led to the hypothesis that giant viruses descend from a fourth, probably extinct domain of cellular life. The results of our comprehensive phylogenomic analysis of giant viruses refute the fourth domain hypothesis and instead indicate that the universal genes have been independently acquired by different giant viruses from their eukaryotic hosts.

  11. Arginine starvation-associated atypical cellular death involves mitochondrial dysfunction, nuclear DNA leakage, and chromatin autophagy

    PubMed Central

    Changou, Chun A.; Chen, Yun-Ru; Xing, Li; Yen, Yun; Chuang, Frank Y. S.; Cheng, R. Holland; Bold, Richard J.; Ann, David K.; Kung, Hsing-Jien

    2014-01-01

    Autophagy is the principal catabolic prosurvival pathway during nutritional starvation. However, excessive autophagy could be cytotoxic, contributing to cell death, but its mechanism remains elusive. Arginine starvation has emerged as a potential therapy for several types of cancers, owing to their tumor-selective deficiency of the arginine metabolism. We demonstrated here that arginine depletion by arginine deiminase induces a cytotoxic autophagy in argininosuccinate synthetase (ASS1)-deficient prostate cancer cells. Advanced microscopic analyses of arginine-deprived dying cells revealed a novel phenotype with giant autophagosome formation, nucleus membrane rupture, and histone-associated DNA leakage encaptured by autophagosomes, which we shall refer to as chromatin autophagy, or chromatophagy. In addition, nuclear inner membrane (lamin A/C) underwent localized rearrangement and outer membrane (NUP98) partially fused with autophagosome membrane. Further analysis showed that prolonged arginine depletion impaired mitochondrial oxidative phosphorylation function and depolarized mitochondrial membrane potential. Thus, reactive oxygen species (ROS) production significantly increased in both cytosolic and mitochondrial fractions, presumably leading to DNA damage accumulation. Addition of ROS scavenger N-acetyl cysteine or knockdown of ATG5 or BECLIN1 attenuated the chromatophagy phenotype. Our data uncover an atypical autophagy-related death pathway and suggest that mitochondrial damage is central to linking arginine starvation and chromatophagy in two distinct cellular compartments. PMID:25122679

  12. DNA-encapsulated magnesium phosphate nanoparticles elicit both humoral and cellular immune responses in mice

    PubMed Central

    Bhakta, Gajadhar; Nurcombe, Victor; Maitra, Amarnath; Shrivastava, Anju

    2014-01-01

    The efficacy of pEGFP (plasmid expressing enhanced green fluorescent protein)-encapsulated PEGylated (meaning polyethylene glycol coated) magnesium phosphate nanoparticles (referred to as MgPi-pEGFP nanoparticles) for the induction of immune responses was investigated in a mouse model. MgPi-pEGFP nanoparticles induced enhanced serum antibody and antigen-specific T-lymphocyte responses, as well as increased IFN-? and IL-12 levels compared to naked pEGFP when administered via intravenous, intraperitoneal or intramuscular routes. A significant macrophage response, both in size and activity, was also observed when mice were immunized with the nanoparticle formulation. The response was highly specific for the antigen, as the increase in interaction between macrophages and lymphocytes as well as lymphocyte proliferation took place only when they were re-stimulated with recombinant green fluorescence protein (rGFP). Thus the nanoparticle formulation elicited both humoral as well as cellular responses. Cytokine profiling revealed the induction of Th-1 type responses. The results suggest DNA-encapsulated magnesium phosphate (MgPi) nanoparticles may constitute a safer, more stable and cost-efficient DNA vaccine formulation. PMID:24936399

  13. Origin of giant viruses from smaller DNA viruses not from a fourth domain of cellular life

    PubMed Central

    Yutin, Natalya; Wolf, Yuri I.; Koonin, Eugene V.

    2015-01-01

    The numerous and diverse eukaryotic viruses with large double-stranded DNA genomes that at least partially reproduce in the cytoplasm of infected cells apparently evolved from a single virus ancestor. This major group of viruses is known as Nucleocytoplasmic Large DNA Viruses (NCLDV) or the proposed order Megavirales. Among the “Megavirales”, there are three groups of giant viruses with genomes exceeding 500 kb, namely Mimiviruses, Pithoviruses, and Pandoraviruses that hold the current record of viral genome size, about 2.5 Mb. Phylogenetic analysis of conserved, ancestral NLCDV genes clearly shows that these three groups of giant viruses have three distinct origins within the “Megavirales”. The Mimiviruses constitute a distinct family that is distantly related to Phycodnaviridae, Pandoraviruses originate from a common ancestor with Coccolithoviruses within the Phycodnaviridae family, and Pithoviruses are related to Iridoviridae and Marseilleviridae. Maximum likelihood reconstruction of gene gain and loss events during the evolution of the “Megavirales” indicates that each group of giant viruses evolved from viruses with substantially smaller and simpler gene repertoires. Initial phylogenetic analysis of universal genes, such as translation system components, encoded by some giant viruses, in particular Mimiviruses, has led to the hypothesis that giant viruses descend from a fourth, probably extinct domain of cellular life. The results of our comprehensive phylogenomic analysis of giant viruses refute the fourth domain hypothesis and instead indicate that the universal genes have been independently acquired by different giant viruses from their eukaryotic hosts. PMID:25042053

  14. Arginine starvation-associated atypical cellular death involves mitochondrial dysfunction, nuclear DNA leakage, and chromatin autophagy.

    PubMed

    Changou, Chun A; Chen, Yun-Ru; Xing, Li; Yen, Yun; Chuang, Frank Y S; Cheng, R Holland; Bold, Richard J; Ann, David K; Kung, Hsing-Jien

    2014-09-30

    Autophagy is the principal catabolic prosurvival pathway during nutritional starvation. However, excessive autophagy could be cytotoxic, contributing to cell death, but its mechanism remains elusive. Arginine starvation has emerged as a potential therapy for several types of cancers, owing to their tumor-selective deficiency of the arginine metabolism. We demonstrated here that arginine depletion by arginine deiminase induces a cytotoxic autophagy in argininosuccinate synthetase (ASS1)-deficient prostate cancer cells. Advanced microscopic analyses of arginine-deprived dying cells revealed a novel phenotype with giant autophagosome formation, nucleus membrane rupture, and histone-associated DNA leakage encaptured by autophagosomes, which we shall refer to as chromatin autophagy, or chromatophagy. In addition, nuclear inner membrane (lamin A/C) underwent localized rearrangement and outer membrane (NUP98) partially fused with autophagosome membrane. Further analysis showed that prolonged arginine depletion impaired mitochondrial oxidative phosphorylation function and depolarized mitochondrial membrane potential. Thus, reactive oxygen species (ROS) production significantly increased in both cytosolic and mitochondrial fractions, presumably leading to DNA damage accumulation. Addition of ROS scavenger N-acetyl cysteine or knockdown of ATG5 or BECLIN1 attenuated the chromatophagy phenotype. Our data uncover an atypical autophagy-related death pathway and suggest that mitochondrial damage is central to linking arginine starvation and chromatophagy in two distinct cellular compartments.

  15. Competitive inhibitor of cellular α-glucosidases protects mice from lethal dengue virus infection.

    PubMed

    Chang, Jinhong; Schul, Wouter; Yip, Andy; Xu, Xiaodong; Guo, Ju-Tao; Block, Timothy M

    2011-11-01

    Dengue virus infection causes diseases in people, ranging from the acute febrile illness dengue fever, to life-threatening dengue hemorrhagic fever/dengue shock syndrome. We previously reported that a host cellular α-glucosidases I and II inhibitor, imino sugar CM-10-18, potently inhibited dengue virus replication in cultured cells, and significantly reduced viremia in dengue virus infected AG129 mice. In this report we show that CM-10-18 also significantly protects mice from death and/or disease progress in two mouse models of lethal dengue virus infection. Our results thus provide a strong support for the development of CM-10-18 or its derivatives as antiviral agents to treat servere dengue virus infections.

  16. Activation of WIP1 Phosphatase by HTLV-1 Tax Mitigates the Cellular Response to DNA Damage

    PubMed Central

    Dayaram, Tajhal; Lemoine, Francene J.; Donehower, Lawrence A.; Marriott, Susan J.

    2013-01-01

    Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR) is a common feature of many cancers. The cancer adult T cell leukemia (ATL) can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1), and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G1 phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX) and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G1/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G1 exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome. PMID:23405243

  17. DNA damage by smoke: Protection by turmeric and other inhibitors of ROS

    SciTech Connect

    Srinivas, L.; Shalini, V.K. )

    1991-01-01

    Twigs-dry leaves smoke condensate (TDS), as a source of clastogenic ROS and carcinogenic PAH, was investigated for its in vitro DNA-damaging effect in calf thymus DNA and human peripheral lymphocytes. An aqueous turmeric component--Aq.T--with an established antioxidant activity, was tested as a DNA protectant. TDS induced 13-fold damage to calf thymus DNA as judged by the emergence of a DNA damage specific, fluorescent product (em: 405 nm). Aq.T at 800 ng/microL extended 69% protection to calf thymus DNA and was comparable to the other protectants such as curcumin, BHA, vitamin E, SOD, and CAT. In human peripheral lymphocytes, TDS induced extensive DNA damage in comparison with the tumor promoter TPA, as judged by FADU. Aq.T at 300 ng/microL extended 90% protection to human lymphocyte DNA against TDS-induced damage, and was more effective than the other protectants--DABCO, D-mannitol, sodium benzoate, vitamin E (ROS quenchers), SOD, CAT (antioxidant enzymes), tannic acid, flufenamic acid, BHA, BHT, n-PG, curcumin and quercetin (antioxidants). Aq.T offered 65% protection to human lymphocyte DNA against TPA-induced damage and was comparable to SOD. The above results indicate that TDS induces substantial DNA damage in calf thymus DNA and human lymphocytes and Aq.T is an efficient protectant.

  18. Efficient Cellular Entry of (r-x-r)-Type Carbamate-Plasmid DNA Complexes and Its Implication for Noninvasive Topical DNA Delivery to Skin.

    PubMed

    Vij, Manika; Natarajan, Poornemaa; Yadav, Amit K; Patil, Kiran M; Pandey, Tanuja; Gupta, Nidhi; Santhiya, Deenan; Kumar, Vaijayanti A; Fernandes, Moneesha; Ganguli, Munia

    2016-06-01

    Arginine-rich cell penetrating peptides are powerful tools for in vitro as well as in vivo delivery of a wide plethora of biomolecules. However, presence of consecutive arginine residues leads to enhanced amenability for proteolytic degradation as well as steric hindrances for membrane interactions which compromise its bioavailability. In order to overcome these limitations we previously reported a safe and stable octaarginine based oligomer, i.e., (r-x-r)4-carbamate, where the backbone amide linkages were replaced by carbamate linkages and 6-aminohexanoic acid based spacer moieties were incorporated for better flexibility, hydrophobicity, optimal spacing of guanidinium groups, and protection against proteolytic cleavage; resulting in improved transfection efficiency over its amide counterpart. In the present work we have investigated the mechanism behind this enhanced transfection efficiency and, based on our observations, demonstrate how the synergistic effect of rationalized oligomer designing, complex characteristics, and cell type contributes to overall effective intracellular delivery. Our results indicate that the (r-x-r)4-carbamate-plasmid DNA complexes primarily utilize lipid raft dependent pathway of cellular entry more than other pathways, and this possibly facilitates their increased entry in the lipid raft rich milieu of skin cells. We also emphasize the utility of oligomer (r-x-r)4-carbamate as an efficient carrier for topical delivery of nucleic acids in skin tissue. This carrier can be utilized for safe, efficient, and noninvasive delivery of therapeutically relevant macromolecular hydrophilic cargo like nucleic acids to skin. PMID:27175623

  19. Efficient Cellular Entry of (r-x-r)-Type Carbamate-Plasmid DNA Complexes and Its Implication for Noninvasive Topical DNA Delivery to Skin.

    PubMed

    Vij, Manika; Natarajan, Poornemaa; Yadav, Amit K; Patil, Kiran M; Pandey, Tanuja; Gupta, Nidhi; Santhiya, Deenan; Kumar, Vaijayanti A; Fernandes, Moneesha; Ganguli, Munia

    2016-06-01

    Arginine-rich cell penetrating peptides are powerful tools for in vitro as well as in vivo delivery of a wide plethora of biomolecules. However, presence of consecutive arginine residues leads to enhanced amenability for proteolytic degradation as well as steric hindrances for membrane interactions which compromise its bioavailability. In order to overcome these limitations we previously reported a safe and stable octaarginine based oligomer, i.e., (r-x-r)4-carbamate, where the backbone amide linkages were replaced by carbamate linkages and 6-aminohexanoic acid based spacer moieties were incorporated for better flexibility, hydrophobicity, optimal spacing of guanidinium groups, and protection against proteolytic cleavage; resulting in improved transfection efficiency over its amide counterpart. In the present work we have investigated the mechanism behind this enhanced transfection efficiency and, based on our observations, demonstrate how the synergistic effect of rationalized oligomer designing, complex characteristics, and cell type contributes to overall effective intracellular delivery. Our results indicate that the (r-x-r)4-carbamate-plasmid DNA complexes primarily utilize lipid raft dependent pathway of cellular entry more than other pathways, and this possibly facilitates their increased entry in the lipid raft rich milieu of skin cells. We also emphasize the utility of oligomer (r-x-r)4-carbamate as an efficient carrier for topical delivery of nucleic acids in skin tissue. This carrier can be utilized for safe, efficient, and noninvasive delivery of therapeutically relevant macromolecular hydrophilic cargo like nucleic acids to skin.

  20. The anthocyanidin delphinidin mobilizes endogenous copper ions from human lymphocytes leading to oxidative degradation of cellular DNA.

    PubMed

    Hanif, Sarmad; Shamim, Uzma; Ullah, M F; Azmi, Asfar S; Bhat, Showket H; Hadi, S M

    2008-07-10

    Epidemiological and experimental evidence exists to suggest that pomegranate and its juice possess chemopreventive and anticancer properties. The anthocyanidin delphinidin is a major polyphenol present in pomegranates and has been shown to be responsible for these effects. Plant polyphenols are recognized as naturally occurring antioxidants but also catalyze oxidative DNA degradation of cellular DNA either alone or in the presence of transition metal ions such as copper. In this paper we show that similar to various other classes of polyphenols, delphinidin is also capable of causing oxidative degradation of cellular DNA. Lymphocytes were exposed to various concentrations of delphinidin (10, 20, 50 microM) for 1h and the DNA breakage was assessed using single cell alkaline gel electrophoresis (Comet assay). Inhibition of DNA breakage by several scavengers of reactive oxygen species (ROS) indicated that it is caused by the formation of ROS. Incubation of lymphocytes with neocuproine (a cell membrane permeable Cu(I) chelator) inhibited DNA degradation in intact lymphocytes in a dose dependent manner. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. We have further shown that delphinidin is able to degrade DNA in cell nuclei and that such DNA degradation is also inhibited by neocuproine suggesting that nuclear copper is mobilized in this reaction. These results indicate that the generation of ROS possibly occurs through mobilization of endogenous copper ions. The results are in support of our hypothesis that the prooxidant activity of plant polyphenols may be an important mechanism for their anticancer properties.

  1. Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis.

    PubMed

    Wätjen, Wim; Michels, Gudrun; Steffan, Bärbel; Niering, Petra; Chovolou, Yvonni; Kampkötter, Andreas; Tran-Thi, Quynh-Hoa; Proksch, Peter; Kahl, Regine

    2005-03-01

    Dietary flavonoids possess a wide spectrum of biochemical and pharmacological actions and are assumed to protect human health. These actions, however, can be antagonistic, and some health claims are mutually exclusive. The antiapoptotic actions of flavonoids may protect against neurodegenerative diseases, whereas their proapoptotic actions could be used for cancer chemotherapy. This study was undertaken to determine whether a cytoprotective dose range of flavonoids could be differentiated from a cytotoxic dose range. Seven structurally related flavonoids were tested for their ability to protect H4IIE rat hepatoma cells against H(2)O(2)-induced damage on the one hand and to induce cellular damage on their own on the other hand. All flavonoids proved to be good antioxidants in a cell-free assay. However, their pharmacologic activity did not correlate with in vitro antioxidant potential but rather with cellular uptake. For quercetin and fisetin, which were readily taken up into the cells, protective effects against H(2)O(2)-induced cytotoxicity, DNA strand breaks, and apoptosis were detected at concentrations as low as 10-25 micromol/L. On the other hand, these flavonoids induced cytotoxicity, DNA strand breaks, oligonucleosomal DNA fragmentation, and caspase activation at concentrations between 50 and 250 micromol/L. Published data on quercetin pharmacokinetics in humans suggest that a dietary supplement of 1-2 g of quercetin may result in plasma concentrations between 10 and 50 micromol/L. Our data suggest that cytoprotective concentrations of some flavonoids are lower by a factor of 5-10 than their DNA-damaging and proapoptotic concentrations.

  2. Glucocorticoids Protect Against the Delayed Behavioral and Cellular Effects of Acute Stress on the Amygdala

    PubMed Central

    Rao, Rajnish P.; Anilkumar, Shobha; McEwen, Bruce; Chattarji, Sumantra

    2013-01-01

    Background A single episode of acute immobilization stress has previously been shown to trigger a delayed onset of anxiety-like behavior and spinogenesis in the basolateral amygdala (BLA) of rats. Spurred on by a seemingly paradoxical observation in which even a modest increase in corticosterone (CORT), caused by a single vehicle injection before stress, could dampen the delayed effects of stress, we hypothesized a protective role for glucocorticoids against stress. Methods We tested this hypothesis by analyzing how manipulations in CORT levels modulate delayed increase in anxiety-like behavior of rats on the elevated plus-maze 10 days after acute stress. We also investigated the cellular correlates of different levels of anxiety under different CORT conditions by quantifying spine density on Golgi-stained BLA principal neurons. Results CORT in drinking water for 12 hours preceding acute stress prevented delayed increase in anxiety rather than exacerbating it. Conversely, vehicle injection failed to prevent the anxiogenic effect of stress in bilaterally adrenalectomized rats. However, when CORT was restored in adrenalectomized rats by injection, the delayed anxiogenic effect of stress was once again blocked. Finally, high and low anxiety states were accompanied by high and low levels of BLA spine density. Conclusions Our findings suggest that the presence of elevated levels of CORT at the time of acute stress confers protection against the delayed enhancing effect of stress on BLA synaptic connectivity and anxiety-like behavior. These observations are consistent with clinical reports on the protective effects of glucocorticoids against the development of posttraumatic symptoms triggered by traumatic stress. PMID:22572034

  3. Carvacrol and rosemary essential oil manifest cytotoxic, DNA-protective and pro-apoptotic effect having no effect on DNA repair.

    PubMed

    Melusova, M; Slamenova, D; Kozics, K; Jantova, S; Horvathova, E

    2014-01-01

    For several thousand years natural products were successfully used to treat a variety of diseases and to maintain health in humans, but until now it is not fully known what causes these medicinal effects. In our study we assessed the cytotoxic, DNA-protective and pro-apoptotic effect of two frequently occurring natural compounds, carvacrol and rosemary essential oil, on human hepatoma HepG2 cells. In addition we examined the in vitro incision repair activity of liver cell extracts prepared from hepatocytes isolated from Sprague-Dawley (SD) rats fed with water containing carvacrol or rosemary oil. Using conventional and modified single cell gel electrophoresis we proved that incubation of HepG2 cells with selected concentrations of carvacrol and rosemary oil significantly protected cellular DNA against two dangerous oxidative agents, hydrogen peroxide (H(2)O(2)) and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). It is interesting that despite this DNA protection, the addition of both volatiles to the drinking water of SD rats had no effect on incision repair capacity of hepatocyte extracts. In this paper we also showed that carvacrol and rosemary oil can trigger apoptotic cell death pathways in HepG2 cells, which is probably connected with their cytotoxicity.

  4. Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

    PubMed

    Rönnberg, Tuomas; Jääskeläinen, Kirsi; Blot, Guillaume; Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

    2012-01-01

    Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

  5. Searching for Cellular Partners of Hantaviral Nonstructural Protein NSs: Y2H Screening of Mouse cDNA Library and Analysis of Cellular Interactome

    PubMed Central

    Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

    2012-01-01

    Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically. PMID:22506017

  6. Punicalagin exerts protective effect against high glucose-induced cellular stress and neural tube defects.

    PubMed

    Zhong, Jianxiang; Reece, E Albert; Yang, Peixin

    2015-11-13

    Maternal diabetes-induced birth defects remain a significant health problem. Studying the effect of natural compounds with antioxidant properties and minimal toxicities on diabetic embryopathy may lead to the development of new and safe dietary supplements. Punicalagin is a primary polyphenol found in pomegranate juice, which possesses antioxidant, anti-inflammatory and anti-tumorigenic properties, suggesting a protective effect of punicalagin on diabetic embryopathy. Here, we examined whether punicalagin could reduce high glucose-induced neural tube defects (NTDs), and if this rescue occurs through blockage of cellular stress and caspase activation. Embryonic day 8.5 (E8.5) mouse embryos were cultured for 24 or 36 h with normal (5 mM) glucose or high glucose (16.7 mM), in presence or absence of 10 or 20 μM punicalagin. 10 μM punicalagin slightly reduced NTD formation under high glucose conditions; however, 20 μM punicalagin significantly inhibited high glucose-induced NTD formation. Punicalagin suppressed high glucose-induced lipid peroxidation marker 4-hydroxynonenal, nitrotyrosine-modified proteins, and lipid peroxides. Moreover, punicalagin abrogated endoplasmic reticulum stress by inhibiting phosphorylated protein kinase ribonucleic acid (RNA)-like ER kinase (p-PERK), phosphorylated inositol-requiring protein-1α (p-IRE1α), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), C/EBP-homologous protein (CHOP), binding immunoglobulin protein (BiP) and x-box binding protein 1 (XBP1) mRNA splicing. Additionally, punicalagin suppressed high glucose-induced caspase 3 and caspase 8 cleavage. Punicalagin reduces high glucose-induced NTD formation by blocking cellular stress and caspase activation. These observations suggest punicalagin supplements could mitigate the teratogenic effects of hyperglycemia in the developing embryo, and possibly prevent diabetes-induced NTDs.

  7. Protective Effect of Folic Acid on Oxidative DNA Damage

    PubMed Central

    Guo, Xiaojuan; Cui, Huan; Zhang, Haiyang; Guan, Xiaoju; Zhang, Zheng; Jia, Chaonan; Wu, Jia; Yang, Hui; Qiu, Wenting; Zhang, Chuanwu; Yang, Zuopeng; Chen, Zhu; Mao, Guangyun

    2015-01-01

    < 0.001). Test of interaction between hypercholesterolemia and FA supplementation on urinary 8-OHdG reduction was significant (P = 0.001). The present study demonstrates that FA fortification is independently linked to the reduction of urinary 8-OHdG/Cr in a dose-related pattern, which suggests that FA is beneficial to protect against oxidative damage to DNA. This effect is apparently stronger in those with hypercholesterolemia. The authors provide a new insight into the prevention and reversal of oxidative DNA damage. PMID:26559255

  8. Inhibition of Influenza Virus Replication by DNA Aptamers Targeting a Cellular Component of Translation Initiation

    PubMed Central

    Rodriguez, Paloma; Pérez-Morgado, M Isabel; Gonzalez, Víctor M; Martín, M Elena; Nieto, Amelia

    2016-01-01

    The genetic diversity of the influenza virus hinders the use of broad spectrum antiviral drugs and favors the appearance of resistant strains. Single-stranded DNA aptamers represent an innovative approach with potential application as antiviral compounds. The mRNAs of influenza virus possess a 5′cap structure and a 3′poly(A) tail that makes them structurally indistinguishable from cellular mRNAs. However, selective translation of viral mRNAs occurs in infected cells through a discriminatory mechanism, whereby viral polymerase and NS1 interact with components of the translation initiation complex, such as the eIF4GI and PABP1 proteins. We have studied the potential of two specific aptamers that recognize PABP1 (ApPABP7 and ApPABP11) to act as anti-influenza drugs. Both aptamers reduce viral genome expression and the production of infective influenza virus particles. The interaction of viral polymerase with the eIF4GI translation initiation factor is hindered by transfection of infected cells with both PABP1 aptamers, and ApPABP11 also inhibits the association of NS1 with PABP1 and eIF4GI. These results indicate that aptamers targeting the host factors that interact with viral proteins may potentially have a broad therapeutic spectrum, reducing the appearance of escape mutants and resistant subtypes. PMID:27070300

  9. Potential Mechanisms for Cancer Resistance in Elephants and Comparative Cellular Response to DNA Damage in Humans

    PubMed Central

    Abegglen, Lisa M.; Caulin, Aleah F.; Chan, Ashley; Lee, Kristy; Robinson, Rosann; Campbell, Michael S.; Kiso, Wendy K.; Schmitt, Dennis L.; Waddell, Peter J; Bhaskara, Srividya; Jensen, Shane T.; Maley, Carlo C.; Schiffman, Joshua D.

    2016-01-01

    IMPORTANCE Evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology. OBJECTIVE To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS). DESIGN, SETTING, AND PARTICIPANTS A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n = 644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n = 8), patients with LFS (n = 10), and age-matched human controls (n = 11). Human samples were collected at the University of Utah between June 2014 and July 2015. EXPOSURES Ionizing radiation and doxorubicin. MAIN OUTCOMES AND MEASURES Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes. RESULTS Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95%CI, 0%–5%]; African wild dog, 8%[95%CI, 0%–16%]; lion, 2%[95%CI, 0% –7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95%CI, 3.14%–6.49%), compared with humans, who have 11% to 25%cancer mortality. While humans have 1 copy (2 alleles) of TP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain

  10. Vaccines displaying mycobacterial proteins on biopolyester beads stimulate cellular immunity and induce protection against tuberculosis.

    PubMed

    Parlane, Natalie A; Grage, Katrin; Mifune, Jun; Basaraba, Randall J; Wedlock, D Neil; Rehm, Bernd H A; Buddle, Bryce M

    2012-01-01

    New improved vaccines are needed for control of both bovine and human tuberculosis. Tuberculosis protein vaccines have advantages with regard to safety and ease of manufacture, but efficacy against tuberculosis has been difficult to achieve. Protective cellular immune responses can be preferentially induced when antigens are displayed on small particles. In this study, Escherichia coli and Lactococcus lactis were engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which displayed a fusion protein of Mycobacterium tuberculosis, antigen 85A (Ag85A)-early secreted antigenic target 6-kDa protein (ESAT-6). L. lactis was chosen as a possible production host due its extensive use in the food industry and reduced risk of lipopolysaccharide contamination. Mice were vaccinated with PHB bead vaccines with or without displaying Ag85A-ESAT-6, recombinant Ag85A-ESAT-6, or M. bovis BCG. Separate groups of mice were used to measure immune responses and assess protection against an aerosol M. bovis challenge. Increased amounts of antigen-specific gamma interferon, interleukin-17A (IL-17A), IL-6, and tumor necrosis factor alpha were produced from splenocytes postvaccination, but no or minimal IL-4, IL-5, or IL-10 was produced, indicating Th1- and Th17-biased T cell responses. Decreased lung bacterial counts and less extensive foci of inflammation were observed in lungs of mice receiving BCG or PHB bead vaccines displaying Ag85A-ESAT-6 produced in either E. coli or L. lactis compared to those observed in the lungs of phosphate-buffered saline-treated control mice. No differences between those receiving wild-type PHB beads and those receiving recombinant Ag85A-ESAT-6 were observed. This versatile particulate vaccine delivery system incorporates a relatively simple production process using safe bacteria, and the results show that it is an effective delivery system for a tuberculosis protein vaccine. PMID:22072720

  11. The bright side of plasmonic gold nanoparticles; activation of Nrf2, the cellular protective pathway

    NASA Astrophysics Data System (ADS)

    Goldstein, Alona; Soroka, Yoram; Frušić-Zlotkin, Marina; Lewis, Aaron; Kohen, Ron

    2016-06-01

    Plasmonic gold nanoparticles (AuNPs) are widely investigated for cancer therapy, due to their ability to strongly absorb light and convert it to heat and thus selectively destroy tumor cells. In this study we shed light on a new aspect of AuNPs and their plasmonic excitation, wherein they can provide anti-oxidant and anti-inflammatory protection by stimulating the cellular protective Nrf2 pathway. Our study was carried out on cells of the immune system, macrophages, and on skin cells, keratinocytes. A different response to AuNPs was noted in the two types of cells, explained by their distinct uptake profiles. In keratinocytes, the exposure to AuNPs, even at low concentrations, was sufficient to activate the Nrf2 pathway, without any irradiation, due to the presence of free AuNPs inside the cytosol. In contrast, in macrophages, the plasmonic excitation of the AuNPs by a low, non-lethal irradiation dose was required for their release from the constraining vesicles. The mechanism by which AuNPs activate the Nrf2 pathway was studied. Direct and indirect activation were suggested, based on the inherent ability of the AuNPs to react with thiol groups and to generate reactive oxygen species, in particular, under plasmonic excitation. The ability of AuNPs to directly activate the Nrf2 pathway renders them good candidates for treatment of disorders in which the up-regulation of Nrf2 is beneficial, specifically for topical treatment of inflammatory skin diseases.

  12. Effects of Spaceflight on Molecular and Cellular Responses to Bleomycin-Induced DNA Damages in Confluent Human Fibroblasts

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2016-01-01

    Spaceflights expose human beings to various risk factors. Among them are microgravity related physiological stresses in immune, cytoskeletal, and cardiovascular systems, and space radiation related elevation of cancer risk. Cosmic radiation consists of energetic protons and other heavier charged particles that induce DNA damages. Effective DNA damage response and repair mechanism is important to maintain genomic integrity and reduce cancer risk. There were studies on effects of spaceflight and microgravity on DNA damage response in cell and animal models, but the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on molecular and cellular responses to DNA damages, bleomycin, an anti-cancer drug and radiomimetic reagent, was used to induce DNA damages in confluent human fibroblasts flown to the International Space Station (ISS) and on ground. After exposure to 1.0 µg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by foci and pattern counting of phosphorylated histone protein H2AX (?-H2AX). The cells on the ISS showed modestly increased average foci counts per nucleus while the distribution of patterns was similar to that on the ground. PCR array analysis showed that expressions of several genes, including CDKN1A and PCNA, were significantly changed in response to DNA damages induced by bleomycin in both flight and ground control cells. However, there were no significant differences in the overall expression profile of DNA damage response genes between the flight and ground samples. Analysis of cellular proliferation status with Ki-67 staining showed a slightly higher proliferating population in cells on the ISS than those on ground. Our results suggested that the difference in ?-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not significantly affect

  13. DNA-based vaccine against La Crosse virus: protective immune response mediated by neutralizing antibodies and CD4+ T cells.

    PubMed

    Schuh, T; Schultz, J; Moelling, K; Pavlovic, J

    1999-07-01

    La Crosse virus (LACV)-mediated encephalitis is the most frequently reported arboviral disease in the United States, but to date no vaccine against this virus is available. We have established a new animal model, genetically targeted mice lacking a functional interferon type I receptor (IFNAR-1). These mice show an age-independent susceptibility to LACV and develop an acute encephalitis within 6 days of infection, thereby allowing the evaluation of vaccines against LACV. Taking advantage of this knockout mouse model, we have assessed the feasibility of DNA vaccination against this viral disease. Plasmid DNAs, encoding either the virus surface glycoproteins G1 and G2 or the internal nucleocapsid protein N, were used to immunize IFNAR-1-deficient mice. Mice vaccinated with DNA encoding the glycoproteins G1 and G2 produced neutralizing antibodies and exhibited a high degree of protection against challenge with high doses of LACV. Depletion of CD4+ T cells in mice vaccinated with DNA encoding G1/G2 reduced their capacity to control the infection. Virus titration and immunohistological analysis revealed that the protected mice showed no evidence of LACV particles in the brain. This indicates that the vaccine-induced immune response efficiently blocked viral spreading from the primary replication site to the brain. In contrast, immunization with DNA encoding protein N yielded only a partial protective effect that can be attributed to the cellular immune response. Taken together, this study shows that DNA vaccines can be designed to efficiently induce a protective immune response based on neutralizing antibodies and CD4+ T cells.

  14. Dose-dependent inhibition of Gag cellular immunity by Env in SIV/HIV DNA vaccinated macaques

    PubMed Central

    Valentin, Antonio; Li, Jinyao; Rosati, Margherita; Kulkarni, Viraj; Patel, Vainav; Jalah, Rashmi; Alicea, Candido; Reed, Steven; Sardesai, Niranjan; Berkower, Ira; Pavlakis, George N; Felber, Barbara K

    2015-01-01

    The induction of a balanced immune response targeting the major structural proteins, Gag and Env of HIV, is important for the development of an efficacious vaccine. The use of DNA plasmids expressing different antigens offers the opportunity to test in a controlled manner the influence of different vaccine components on the magnitude and distribution of the vaccine-induced cellular and humoral immune responses. Here, we show that increasing amounts of env DNA results in greatly enhanced Env antibody titers without significantly affecting the levels of anti-Env cellular immune responses. Co-immunization with Env protein further increased antibody levels, indicating that vaccination with DNA only is not sufficient for eliciting maximal humoral responses against Env. In contrast, under high env:gag DNA plasmid ratio, the development of Gag cellular responses was significantly reduced by either SIV or HIV Env, whereas Gag humoral responses were not affected. Our data indicate that a balanced ratio of the 2 key HIV/SIV vaccine components, Gag and Env, is important to avoid immunological interference and to achieve both maximal humoral responses against Env to prevent virus acquisition and maximal cytotoxic T cell responses against Gag to prevent virus spread. PMID:26125521

  15. The prooxidant action of dietary antioxidants leading to cellular DNA breakage and anticancer effects: implications for chemotherapeutic action against cancer.

    PubMed

    Ullah, M F; Ahmad, Aamir; Khan, Husain Y; Zubair, H; Sarkar, Fazlul H; Hadi, S M

    2013-11-01

    Plant-derived dietary antioxidants have attracted considerable interest in recent past for their ability to induce apoptosis and regression of tumors in animal models. While it is believed that the antioxidant properties of these agents may contribute to lowering the risk of cancer induction by impeding oxidative injury to DNA, it could not account for apoptosis induction and chemotherapeutic observations. In this article, we show that dietary antioxidants can alternatively switch to a prooxidant action in the presence of transition metals such as copper. Such a prooxidant action leads to strand breaks in cellular DNA and growth inhibition in cancer cells. Further, the cellular DNA breakage and anticancer effects were found to be significantly enhanced in the presence of copper ions. Moreover, inhibition of antioxidant-induced DNA strand breaks and oxidative stress by Cu(I)-specific chelators bathocuproine and neocuproine demonstrated the role of endogenous copper in the induction of the prooxidant mechanism. Since it is well established that tissue, cellular, and serum copper levels are considerably elevated in various malignancies, such a prooxidant cytotoxic mechanism better explains the anticancer activity of dietary antioxidants against cancer cells.

  16. Sirtuin 7 promotes cellular survival following genomic stress by attenuation of DNA damage, SAPK activation and p53 response

    SciTech Connect

    Kiran, Shashi; Oddi, Vineesha; Ramakrishna, Gayatri

    2015-02-01

    Maintaining the genomic integrity is a constant challenge in proliferating cells. Amongst various proteins involved in this process, Sirtuins play a key role in DNA damage repair mechanisms in yeast as well as mammals. In the present work we report the role of one of the least explored Sirtuin viz., SIRT7, under conditions of genomic stress when treated with doxorubicin. Knockdown of SIRT7 sensitized osteosarcoma (U2OS) cells to DNA damage induced cell death by doxorubicin. SIRT7 overexpression in NIH3T3 delayed cell cycle progression by causing delay in G1 to S transition. SIRT7 overexpressing cells when treated with low dose of doxorubicin (0.25 µM) showed delayed onset of senescence, lesser accumulation of DNA damage marker γH2AX and lowered levels of growth arrest markers viz., p53 and p21 when compared to doxorubicin treated control GFP expressing cells. Resistance to DNA damage following SIRT7 overexpression was also evident by EdU incorporation studies where cellular growth arrest was significantly delayed. When treated with higher dose of doxorubicin (>1 µM), SIRT7 conferred resistance to apoptosis by attenuating stress activated kinases (SAPK viz., p38 and JNK) and p53 response thereby shifting the cellular fate towards senescence. Interestingly, relocalization of SIRT7 from nucleolus to nucleoplasm together with its co-localization with SAPK was an important feature associated with DNA damage. SIRT7 mediated resistance to doxorubicin induced apoptosis and senescence was lost when p53 level was restored by nutlin treatment. Overall, we propose SIRT7 attenuates DNA damage, SAPK activation and p53 response thereby promoting cellular survival under conditions of genomic stress. - Highlights: • Knockdown of SIRT7 sensitized cells to DNA damage induced apoptosis. • SIRT7 delayed onset of premature senescence by attenuating DNA damage response. • Overexpression of SIRT7 delayed cell cycle progression by delaying G1/S transition. • Upon DNA damage SIRT

  17. Induction of a Protective Response in Mice by the Dengue Virus NS3 Protein Using DNA Vaccines

    PubMed Central

    Costa, Simone M.; Yorio, Anna Paula; Gonçalves, Antônio J. S.; Vidale, Mariana M.; Costa, Emmerson C. B.; Mohana-Borges, Ronaldo; Motta, Marcia A.; Freire, Marcos S.; Alves, Ada M. B.

    2011-01-01

    The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection. PMID:22031819

  18. Induction of a protective response in mice by the dengue virus NS3 protein using DNA vaccines.

    PubMed

    Costa, Simone M; Yorio, Anna Paula; Gonçalves, Antônio J S; Vidale, Mariana M; Costa, Emmerson C B; Mohana-Borges, Ronaldo; Motta, Marcia A; Freire, Marcos S; Alves, Ada M B

    2011-01-01

    The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

  19. More than 10% of yeast genes are related to genome stability and influence cellular senescence via rDNA maintenance

    PubMed Central

    Saka, Kimiko; Takahashi, Akihiro; Sasaki, Mariko; Kobayashi, Takehiko

    2016-01-01

    Genome instability triggers cellular senescence and is a common cause of cancer. The ribosomal RNA genes (rDNA), due to their repetitive structure, form a fragile site with frequent rearrangements. To identify eukaryotic factors that connect reduced genome stability to senescence we screened 4,876 strains of a Saccharomyces cerevisiae deletion library for aberrant rDNA and found 708 genes that contribute to its upkeep. 28 mutants caused abnormalities in non-rDNA chromosomes and among them 12 mutants have abnormalities both in rDNA and in non-rDNA chromosomes. Many mutated genes have not previously been implicated with genome maintenance nor their homologues with tumorigenesis in mammals. The link between rDNA state and senescence was broken after deletion of factors related with DNA polymerase ϵ. These mutations also suppressed the short lifespan phenotype of a sir2 mutant, suggesting a model in which molecular events at the heart of the replication fork induce abnormal rDNA recombination and are responsible for the emergence of an aging signal. PMID:26912831

  20. Partial Protection of PC12 Cells from Cellular Stress by Low-Dose Sodium Nitroprusside Pre-treatment.

    PubMed

    Varga, Judit; Bátor, Judit; Nádasdi, Gergő; Árvai, Zita; Schipp, Renáta; Szeberényi, József

    2016-10-01

    The PC12 rat pheochromocytoma cell line is an in vitro model system widely used for the investigation of intracellular signaling events contributing to neuronal differentiation and cell death. We found earlier that the nitric oxide donor compound sodium nitroprusside (SNP) induced apoptosis of PC12 cells if it was applied in high concentration (400 µM). Yoshioka et al. (J Pharmacol Sci 101:126-134, 2006) reported that cell death evoked by cytotoxic concentrations of SNP could be prevented by a 100 µM SNP pre-treatment in a murine macrophage cell line. The apoptosis caused by toxic-dose SNP treatment (400 µM) could be partially overcome in PC12 cells as well by the low-dose SNP pre-treatment. The partial inhibition of apoptosis was accompanied by reduced phosphorylation of certain proteins (such as stress-activated protein kinases, the p53, and the eIF2α proteins), decreased caspase activation, and less intense internucleosomal DNA fragmentation. The 100 µM SNP pre-treatment reduced the pro-apoptotic potential of certain other stress stimuli (serum withdrawal, cisplatin and tunicamycin treatments) as well, although the underlying biochemical changes were not entirely uniform. On the contrary, the 100 µM SNP pre-treatment was unable to prevent cell death caused by the protein synthesis inhibitor anisomycin. Further clarification of the above-mentioned processes may be important in understanding the mechanisms by which mild nitrosative stress protects cells against certain forms of cellular stress conditions.

  1. Induction of protection against porcine cysticercosis in growing pigs by DNA vaccination.

    PubMed

    Guo, Aijiang; Jin, Zhizhong; Zheng, Yadong; Hai, Gang; Yuan, Gailing; Li, Hailong; Cai, Xuepeng

    2007-01-01

    A DNA vaccine, pcDNA3-B, was developed by using the nucleotide sequence of Taenia solium B antigen and cloning into pcDNA3.1 plasmid. The growing pigs were vaccinated by one intramuscular infection of 200 or 1000 microg pcDNA3-B. The immunization with 1000 microg of pcDNA3-B showed 92.6% protection when the pigs were challenged by T. solium eggs and four of the five pigs vaccinated had no viable cysts. The results provide encouraging information on the use of pcDNA3-B vaccination for the prevention of cysticercosis.

  2. Normal Cellular Prion Protein Protects against Manganese-induced Oxidative Stress and Apoptotic Cell Death

    PubMed Central

    Choi, Christopher J.; Anantharam, Vellareddy; Saetveit, Nathan J.; Houk, Robert. S.; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

    2012-01-01

    The normal prion protein is abundantly expressed in the CNS, but its biological function remains unclear. The prion protein has octapeptide repeat regions that bind to several divalent metals, suggesting that the prion proteins may alter the toxic effect of environmental neurotoxic metals. In the present study, we systematically examined whether prion protein modifies the neurotoxicity of manganese (Mn) by comparing the effect of Mn on mouse neural cells expressing prion protein (PrPC -cells) and prion-knockout (PrPKO -cells). Exposure to Mn (10 μM-1 mM) for 24 hr produced a dose-dependent cytotoxic response in both PrPC -cells and PrPKO -cells. Interestingly, PrPC -cells (EC50 117.6μM) were more resistant to Mn-induced cytotoxicity, as compared to PrPKO -cells (EC50 59.9μM), suggesting a protective role for PrPC against Mn neurotoxicity. Analysis of intracellular Mn levels showed less Mn accumulation in PrPC -cells as compared to PrPKO -cells. Furthermore, Mn-induced mitochondrial depolarization and ROS generation were significantly attenuated in PrPC -cells as compared to PrPKO -cells. Measurement of antioxidant status revealed similar basal levels of glutathione (GSH) in PrPC -cells and PrPKO -cells; however, Mn treatment caused greater depletion of GSH in PrPKO -cells. Mn-induced mitochondrial depolarization and ROS production were followed by time- and dose-dependent activation of the apoptotic cell death cascade involving caspase-9 and -3. Notably, DNA fragmentation induced by both Mn treatment and oxidative stress-inducer hydrogen peroxide (100μM) was significantly suppressed in PrPC -cells as compared to PrPKO -cells. Together, these results demonstrate that prion protein interferes with divalent metal Mn uptake and protects against Mn-induced oxidative stress and apoptotic cell death. PMID:17483122

  3. Modulation of cellular immune response against hepatitis C virus nonstructural protein 3 by cationic liposome encapsulated DNA immunization.

    PubMed

    Jiao, Xuanmao; Wang, Richard Y-H; Feng, Zhiming; Alter, Harvey J; Shih, James Wai-Kuo

    2003-02-01

    A vaccine strategy directed to increase Th1 cellular immune responses, particularly to hepatitis C virus (HCV) nonstructural protein 3 (NS3), has considerable potential to overcome the infection with HCV. DNA vaccination can induce both humoral and cellular immune responses, but it became apparent that the cellular uptake of naked DNA injected into muscle was not very efficient, as much of the DNA is degraded by interstitial nucleases before it reaches the nucleus for transcription. In this paper, cationic liposomes composed of different cationic lipids, such as dimethyl-dioctadecylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), or 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOEPC), were used to improve DNA immunization in mice, and their efficiencies were compared. It was found that cationic liposome-mediated DNA immunization induced stronger HCV NS3-specific immune responses than immunization with naked DNA alone. Cationic liposomes composed of DDAB and equimolar of a neutral lipid, egg yolk phosphatidylcholine (EPC), induced the strongest antigen-specific Th1 type immune responses among the cationic liposome investigated, whereas the liposomes composed of 2 cationic lipids, DDAB and DOEPC, induced an antigen-specific Th2 type immune response. All cationic liposomes used in this study triggered high-level, nonspecific IL-12 production in mice, a feature important for the development of maximum Th1 immune responses. In conclusion, the cationic liposome-mediated gene delivery is a viable HCV vaccine strategy that should be further tested in the chimpanzee model. PMID:12540796

  4. Protective activity of C-geranylflavonoid analogs from Paulownia tomentosa against DNA damage in 137Cs irradiated AHH-1 cells.

    PubMed

    Moon, Hyung-In; Jeong, Min Ho; Jo, Wol Soon

    2014-09-01

    Radiotherapy is an important form of treatment for a wide range of cancers, but it can damage DNA and cause adverse effects. We investigated if the diplacone analogs of P. tomentosa were radio-protective in a human lymphoblastoid cell line (AHH-1). Four geranylated flavonoids, diplacone, 3'-O-methyl-5'-hydroxydiplacone, 3'-O-methyl-5'-O-methyldiplacone and 3'-O-methyldiplacol, were tested for their antioxidant and radio-protective effects. Diplacone analogs effectively scavenged free radicals and inhibited radiation-induced DNA strand breaks in vitro. They significantly decreased levels of reactive oxygen species and cellular DNA damage in 2 Gy-irradiated AHH-1 cells. Glutathione levels and superoxide dismutase activity in irradiated AHH-1 cells increased significantly after treatment with these analogs. The enhanced biological anti-oxidant activity and radioprotective activity of diplacone analogs maintained the survival of irradiated AHH-1 cells in a clonogenic assay. These data suggest that diplacone analogs may protect healthy tissue surrounding tumor cells during radiotherapy to ensure better control of radiotherapy and allow higher doses of radiotherapy to be employed. PMID:25918796

  5. Does cytotoxicity of metallointercalators correlate with cellular uptake or DNA affinity?

    PubMed

    Davis, Kimberley J; Carrall, Judith A; Lai, Barry; Aldrich-Wright, Janice R; Ralph, Stephen F; Dillon, Carolyn T

    2012-08-21

    The cytotoxicity of the metallointercalators, [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1R,2R-diaminocyclohexane)](2+) ([56MERR]) and [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1S,2S-diaminocyclohexane)](2+) ([56MESS]), towards A549 human lung cancer cells was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) value obtained following exposure of A549 cells to [56MESS] for 4 h was approximately three times smaller than that obtained when [56MERR] was administered under the same conditions, indicating that the former complex displayed greater cytotoxicity. Both IC(50) values were greater than that obtained after exposure of A549 cells to cisplatin, demonstrating that the latter compound was the most cytotoxic of the three examined. Microprobe synchrotron radiation X-ray fluorescence (SR-XRF) analyses of metallointercalator-treated A549 cells showed that platinum became localised in DNA-rich regions of the nucleus. In contrast, when the same cells were treated with cisplatin the metal became distributed throughout the cell. Determination of the maximum concentration of platinum present inside the cells using graphite furnace atomic absorption spectrophotometry (GFAAS) of platinum-treated cells suggested that there was greater uptake of [56MERR] compared to [56MESS] by the A549 cells, and that platinum uptake did not account for the greater toxicity of [56MESS], as assessed by the MTT assay. Electrospray ionization mass spectrometric (ESI-MS) and circular dichroism (CD) spectroscopic studies of solutions containing either [56MERR] or [56MESS], and a duplex hexadecamer molecule, showed the two metallointercalators displayed very similar affinity towards the nucleic acid. Overall these results indicate that the difference in cytotoxicity of the two platinum metallointercalators is probably the result of variations in their interactions with other cellular components. PMID:22740039

  6. NF-κB-HOTAIR axis links DNA damage response, chemoresistance and cellular senescence in ovarian cancer

    PubMed Central

    Özeş, Ali R.; Miller, David F.; Özeş, Osman N.; Fang, Fang; Liu, Yunlong; Matei, Daniela; Huang, Tim; Nephew, Kenneth P.

    2016-01-01

    The transcription factor nuclear factor kappa B (NF-κB) and the long non-coding RNA (lncRNA) HOTAIR (HOX transcript antisense RNA) play diverse functional roles in cancer. In this study, we show that upregulation of HOTAIR induced platinum resistance in ovarian cancer, and increased HOTAIR levels were observed in recurrent platinum-resistant ovarian tumors vs. primary ovarian tumors. To investigate the role of HOTAIR during DNA damage induced by platinum, we monitored double-strand breaks and show that HOTAIR expression results in sustained activation of DNA damage response after platinum treatment. We demonstrate that ectopic expression of HOTAIR induces NF-κB activation during DNA damage response and MMP-9 and IL-6 expression, both key NF-κB target genes. We show that HOTAIR regulates activation of NF-κB by decreasing Iκ-Bα (NF-κB inhibitor) and establish that by inducing prolonged NF-κB activation and expression of NF-κB target genes during DNA damage, HOTAIR plays a critical role in cellular senescence and platinum sensitivity. Our findings suggest that a NF-κB-HOTAIR axis drives a positive-feedback loop cascade during DNA damage response and contributes to cellular senescence and chemotherapy resistance in ovarian and other cancers. PMID:27041570

  7. Specific humoral and cellular immunity induced by Trypanosoma cruzi DNA immunization in a canine model

    PubMed Central

    2013-01-01

    Chagas disease has a high incidence in Mexico and other Latin American countries. Because one of the most important known methods of prevention is vector control, which has been effective only in certain areas of South America, the development of a vaccine to protect people at risk has been proposed. In this study, we assessed the cellular and humoral immune response generated following immunization with pBCSP and pBCSSP4 plasmids containing the genes encoding a trans-sialidase protein (present in all three forms of T. cruzi) and an amastigote specific glycoprotein, respectively, in a canine model. Thirty-five beagle dogs were divided randomly into 5 groups (n = 7) and were immunized twice intramuscularly with 500 μg of pBCSSP4, pBCSP, pBk-CMV (empty plasmid) or saline solution. Fifteen days after the last immunization the 4 groups were infected intraperitoneally with 500 000 metacyclic trypomastigotes. The fifth group was unimmunized/infected. The parasitaemia in the immunized/infected dogs was for a shorter period (14 vs. 29 days) and the parasite load was lower. The concentration of IgG1 (0.612 ± 0.019 O.D.) and IgG2 (1.167 ± 0.097 O.D.) subclasses was measured (absorbance) 15 days after the last immunization with both recombinant plasmids, the majority of which were IgG2. The treatment of parasites using the serum from dogs immunized with pBCSP and pBCSSP4 plasmids produced 54% (± 11.8) and 68% (± 21.4) complement-mediated lysis, respectively. At 12 h post immunization, an increase in cytokines was not observed; however, vaccination with pBCSSP4 significantly increased the levels of IFN-γ and IL-10 at 9 months post-infection. The recombinant plasmid immunization stimulated the spleen cell proliferation showing a positive stimulatory index above 2.0. In conclusion, immunization using both genes effectively induces a humoral and cellular immune response. PMID:23497041

  8. Specific humoral and cellular immunity induced by Trypanosoma cruzi DNA immunization in a canine model.

    PubMed

    Arce-Fonseca, Minerva; Ballinas-Verdugo, Martha A; Zenteno, Emma R Abreu; Suárez-Flores, Davinia; Carrillo-Sánchez, Silvia C; Alejandre-Aguilar, Ricardo; Rosales-Encina, José Luis; Reyes, Pedro A; Rodríguez-Morales, Olivia

    2013-01-01

    Chagas disease has a high incidence in Mexico and other Latin American countries. Because one of the most important known methods of prevention is vector control, which has been effective only in certain areas of South America, the development of a vaccine to protect people at risk has been proposed. In this study, we assessed the cellular and humoral immune response generated following immunization with pBCSP and pBCSSP4 plasmids containing the genes encoding a trans-sialidase protein (present in all three forms of T. cruzi) and an amastigote specific glycoprotein, respectively, in a canine model. Thirty-five beagle dogs were divided randomly into 5 groups (n=7) and were immunized twice intramuscularly with 500 μg of pBCSSP4, pBCSP, pBk-CMV (empty plasmid) or saline solution. Fifteen days after the last immunization the 4 groups were infected intraperitoneally with 500,000 metacyclic trypomastigotes. The fifth group was unimmunized/infected. The parasitaemia in the immunized/infected dogs was for a shorter period (14 vs. 29 days) and the parasite load was lower. The concentration of IgG1 (0.612±0.019 O.D.) and IgG2 (1.167±0.097 O.D.) subclasses was measured (absorbance) 15 days after the last immunization with both recombinant plasmids, the majority of which were IgG2. The treatment of parasites using the serum from dogs immunized with pBCSP and pBCSSP4 plasmids produced 54% (±11.8) and 68% (±21.4) complement-mediated lysis, respectively. At 12 h post immunization, an increase in cytokines was not observed; however, vaccination with pBCSSP4 significantly increased the levels of IFN-γ and IL-10 at 9 months post-infection. The recombinant plasmid immunization stimulated the spleen cell proliferation showing a positive stimulatory index above 2.0. In conclusion, immunization using both genes effectively induces a humoral and cellular immune response. PMID:23497041

  9. In Vivo Bystander Effect: Cranial X-Irradiation Leads to Elevated DNA Damage, Altered Cellular Proliferation and Apoptosis, and Increased p53 Levels in Shielded Spleen

    SciTech Connect

    Koturbash, Igor; Loree, Jonathan; Kutanzi, Kristy; Koganow, Clayton; Pogribny, Igor; Kovalchuk, Olga

    2008-02-01

    Purpose: It is well accepted that irradiated cells may 'forward' genome instability to nonirradiated neighboring cells, giving rise to the 'bystander effect' phenomenon. Although bystander effects were well studied by using cell cultures, data for somatic bystander effects in vivo are relatively scarce. Methods and Materials: We set out to analyze the existence and molecular nature of bystander effects in a radiation target-organ spleen by using a mouse model. The animal's head was exposed to X-rays while the remainder of the body was completely protected by a medical-grade shield. Using immunohistochemistry, we addressed levels of DNA damage, cellular proliferation, apoptosis, and p53 protein in the spleen of control animals and completely exposed and head-exposed/body bystander animals. Results: We found that localized head radiation exposure led to the induction of bystander effects in the lead-shielded distant spleen tissue. Namely, cranial irradiation led to increased levels of DNA damage and p53 expression and also altered levels of cellular proliferation and apoptosis in bystander spleen tissue. The observed bystander changes were not caused by radiation scattering and were observed in two different mouse strains; C57BL/6 and BALB/c. Conclusion: Our study proves that bystander effects occur in the distant somatic organs on localized exposures. Additional studies are required to characterize the nature of an enigmatic bystander signal and analyze the long-term persistence of these effects and possible contribution of radiation-induced bystander effects to secondary radiation carcinogenesis.

  10. Water extracts of tree Hypericum sps. protect DNA from oxidative and alkylating damage and enhance DNA repair in colon cells.

    PubMed

    Ramos, Alice A; Marques, Filipe; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

    2013-01-01

    Diet may induce colon carcinogenesis through oxidative or alkylating DNA damage. However, diet may also contain anticarcinogenic compounds that contribute to cancer prevention. DNA damage prevention and/or induction of repair are two important mechanisms involved in cancer chemoprevention by dietary compounds. Hypericum sps. are widely used in traditional medicine to prepare infusions due to their beneficial digestive and neurologic effects. In this study, we investigated the potential of water extracts from three Hypericum sps. and some of their main phenolic compounds to prevent and repair oxidative and alkylating DNA damage in colon cells. The results showed that water extracts of Hypericum perforatum, Hypericum androsaemum, Hypericum undulatum, quercetin and rutin have protective effect against oxidative DNA damage in HT29 cells. Protective effect was also observed against alkylating DNA damage induced by methyl-methanesulfonate, except for H. androsaemum. With regard to alkylating damage repair H. perforatum, H. androsaemum and chlorogenic acid increased repair of alkylating DNA damage by base excision repair pathway. No effect was observed on nucleotide excision repair pathway. Antigenotoxic effects of Hypericum sps. may contribute to colon cancer prevention and the high amount of phenolic compounds present in Hypericum sps. play an important role in DNA protective effects.

  11. Extracellular reduction of uranium via Geobacter conductive pili as a protective cellular mechanism

    PubMed Central

    Cologgi, Dena L.; Lampa-Pastirk, Sanela; Speers, Allison M.; Kelly, Shelly D.; Reguera, Gemma

    2011-01-01

    The in situ stimulation of Fe(III) oxide reduction by Geobacter bacteria leads to the concomitant precipitation of hexavalent uranium [U(VI)] from groundwater. Despite its promise for the bioremediation of uranium contaminants, the biological mechanism behind this reaction remains elusive. Because Fe(III) oxide reduction requires the expression of Geobacter's conductive pili, we evaluated their contribution to uranium reduction in Geobacter sulfurreducens grown under pili-inducing or noninducing conditions. A pilin-deficient mutant and a genetically complemented strain with reduced outer membrane c-cytochrome content were used as controls. Pili expression significantly enhanced the rate and extent of uranium immobilization per cell and prevented periplasmic mineralization. As a result, pili expression also preserved the vital respiratory activities of the cell envelope and the cell's viability. Uranium preferentially precipitated along the pili and, to a lesser extent, on outer membrane redox-active foci. In contrast, the pilus-defective strains had different degrees of periplasmic mineralization matching well with their outer membrane c-cytochrome content. X-ray absorption spectroscopy analyses demonstrated the extracellular reduction of U(VI) by the pili to mononuclear tetravalent uranium U(IV) complexed by carbon-containing ligands, consistent with a biological reduction. In contrast, the U(IV) in the pilin-deficient mutant cells also required an additional phosphorous ligand, in agreement with the predominantly periplasmic mineralization of uranium observed in this strain. These findings demonstrate a previously unrecognized role for Geobacter conductive pili in the extracellular reduction of uranium, and highlight its essential function as a catalytic and protective cellular mechanism that is of interest for the bioremediation of uranium-contaminated groundwater. PMID:21896750

  12. The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system.

    PubMed

    Tinker, Andrew; Aziz, Qadeer; Thomas, Alison

    2014-01-01

    ATP-sensitive potassium channels (K(ATP)) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated K(ATP) channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system.

  13. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

    SciTech Connect

    Zheng Min; Jin Ningyi; Liu Qi; Huo Xiaowei; Li Yang; Hu Bo; Ma Haili; Zhu Zhanbo; Cong Yanzhao; Li Xiao; Jin Minglan; Zhu Guangze

    2009-08-15

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  14. Potent induction of cellular antioxidants and phase 2 enzymes by resveratrol in cardiomyocytes: protection against oxidative and electrophilic injury.

    PubMed

    Cao, Zhuoxiao; Li, Yunbo

    2004-04-01

    Resveratrol is known to be protective against oxidative cardiovascular disorders. However, the underlying mechanisms remain unclear. This study was undertaken to determine if resveratrol could increase endogenous antioxidants and phase 2 enzymes in cardiomyocytes, and if such increased cellular defenses could provide protection against oxidative and electrophilic cell injury. Incubation of cardiac H9C2 cells with low micromolar resveratrol resulted in a significant induction of a scope of cellular antioxidants and phase 2 enzymes in a concentration- and/or time-dependent fashion. To investigate the protective effects of the resveratrol-induced cellular defenses on oxidative and electrophilic cell injury, H9C2 cells were first incubated with resveratrol, and then exposed to xanthine oxidase (XO)/xanthine, 4-hydroxy-2-nonenal or doxorubicin. We observed that resveratrol pretreatment afforded a marked protection against the above agent-mediated cytotoxicity in H9C2 cells. Moreover, the resveratrol pretreatment led to a great reduction in XO/xanthine-induced intracellular accumulation of ROS. Taken together, this study demonstrates that resveratrol induces antioxidants and phase 2 enzymes in cardiomyocytes, which is accompanied by increased resistance to oxidative and electrophilic cell injury.

  15. Interactions of DNA with clay minerals and soil colloidal particles and protection against degradation by DNase.

    PubMed

    Cai, Peng; Huang, Qiao-Yun; Zhang, Xue-Wen

    2006-05-01

    Adsorption, desorption, and degradation by nucleases of DNA on four different colloidal fractions from a Brown soil and clay minerals were studied. The adsorption of DNase I and the structures of native DNA, adsorbed and desorbed, were also investigated by Fourier Transform Infrared (FTIR), circular dichroism (CD), and fluorescence spectroscopy, to determine the protection mechanism of DNA molecules by soil colloids and minerals against enzymatic degradation. Kaolinite exhibited the highest adsorption affinity for DNA among the examined soil colloids and clay minerals. In comparison with organomineral complexes (organic clays), DNA was tightly adsorbed by H2O2-treated clays (inorganic clays). FTIR spectra showed that the binding of DNA on kaolinite and inorganic clays changed its conformation from the B-form to the Z-form, whereas montmorillonite and organic clays retained the original B-form of DNA. A structural change from the B- to the C-form in DNA molecules desorbed from kaolinite was observed by CD spectroscopy and confirmed by fluorescence spectroscopy. The presence of soil colloids and minerals provided protection to DNA against degradation by DNase I. The higher level of protection was found with montmorillonite and organic clays compared to kaolinite and inorganic clays. The protection of DNA against nuclease degradation by soil colloids and minerals is apparently not controlled by the adsorption affinity of DNA molecules for the colloids and the conformational change of bound DNA. The higher stability of DNA seemed to be attributed mainly to the presence of organic matter in the system and the adsorption of nucleases on soil colloids and minerals. The information obtained in this study is of fundamental significance for the understanding of the behavior of extracellular DNA in soil environment.

  16. Nanoparticle formulation enhanced protective immunity provoked by PYGPI8p-transamidase related protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.

    PubMed

    Cherif, Mahamoud Sama; Shuaibu, Mohammed Nasir; Kodama, Yukinobu; Kurosaki, Tomoaki; Helegbe, Gideon Kofi; Kikuchi, Mihoko; Ichinose, Akitoyo; Yanagi, Tetsuo; Sasaki, Hitoshi; Yui, Katsuyuki; Tien, Nguyen Huy; Karbwang, Juntra; Hirayama, Kenji

    2014-04-01

    We have previously reported the new formulation of polyethylimine (PEI) with gamma polyglutamic acid (γ-PGA) nanoparticle (NP) to have provided Plasmodium yoelii merozoite surface protein-1 (PyMSP-1) plasmid DNA vaccine with enhanced protective cellular and humoral immunity in the lethal mouse malaria model. PyGPI8p-transamidase-related protein (PyTAM) was selected as a possible candidate vaccine antigen by using DNA vaccination screening from 29 GPI anchor and signal sequence motif positive genes picked up using web-based bioinformatics tools; though the observed protection was not complete. Here, we observed augmented protective effect of PyTAM DNA vaccine by using PEI and γ-PGA complex as delivery system. NP-coated PyTAM plasmid DNA immunized mice showed a significant survival rate from lethal P. yoelii challenge infection compared with naked PyTAM plasmid or with NP-coated empty plasmid DNA group. Antigen-specific IgG1 and IgG2b subclass antibody levels, proportion of CD4 and CD8T cells producing IFN-γ in the splenocytes and IL-4, IFN-γ, IL-12 and TNF-α levels in the sera and in the supernatants from ex vivo splenocytes culture were all enhanced by the NP-coated PyTAM DNA vaccine. These data indicates that NP augments PyTAM protective immune response, and this enhancement was associated with increased DC activation and concomitant IL-12 production.

  17. Identification of an mtDNA mutation hot spot in UV-induced mouse skin tumors producing altered cellular biochemistry.

    PubMed

    Jandova, Jana; Eshaghian, Alex; Shi, Mingjian; Li, Meiling; King, Lloyd E; Janda, Jaroslav; Sligh, James E

    2012-02-01

    There is increasing awareness of the role of mtDNA alterations in the development of cancer, as mtDNA point mutations are found at high frequency in a variety of human tumors. To determine the biological effects of mtDNA mutations in UV-induced skin tumors, hairless mice were irradiated to produce tumors, and the tumor mtDNAs were screened for single-nucleotide changes using temperature gradient capillary electrophoresis (TGCE), followed by direct sequencing. A mutation hot spot (9821insA) in the mitochondrially encoded tRNA arginine (mt-Tr) locus (tRNA(Arg)) was discovered in approximately one-third of premalignant and malignant skin tumors. To determine the functional relevance of this particular mutation in vitro, cybrid cell lines containing different mt-Tr (tRNA(Arg)) alleles were generated. The resulting cybrid cell lines contained the same nuclear genotype and differed only in their mtDNAs. The biochemical analysis of the cybrids revealed that the mutant haplotype is associated with diminished levels of complex I protein (CI), resulting in lower levels of baseline oxygen consumption and lower cellular adenosine triphosphate (ATP) production. We hypothesize that this specific mtDNA mutation alters cellular biochemistry, supporting the development of keratinocyte neoplasia.

  18. Immune response and protection elicited by DNA immunisation against Taenia cysticercosis.

    PubMed

    Wang, Qing-min; Sun, Shu-han; Hu, Zhen-lin; Wu, Dan; Wang, Zhong-chuan

    2003-04-01

    The study evaluated DNA vaccination in Taenia solium cysticercosis prevention by using cDNA of an antigen (cC1) from T. solium metacestode. pcDNA3-cC1 DNA vaccine was constructed by inserting the cDNA into the eukaryotic expression plasmid pcDNA3. Positive expression of the pcDNA3-cC1 product was confirmed by its transfection into COS7 cell and enzyme-linked immunoabsorbent assay using serum of pigs infected with T. solium metacestode. Immunisation of BALB/c mice with three injections of pcDNA3-cC1 induced antigen-specific immune responses of the Th1 phenotype. Inoculation of new-born pigs induced protection against challenge with T. solium by 73.3% reduction of the metacestode number. Antibodies elicited by DNA immunisation with pcDNA3-cC1 specifically reacted with native cC1 protein, which was mainly restricted to the cyst wall of T. solium metacestode. Positive apoptosis signals were also detected in the cyst wall cells of metacestode slices from pigs immunised with pcDNA3-cC1 by TUNEL staining method. Those suggested that apoptosis played a role in protecting pigs immunised with pcDNA3-cC1 nucleic acid vaccine from pathogen challenge.

  19. DNA Vaccination Elicits Protective Immune Responses against Pandemic and Classic Swine Influenza Viruses in Pigs ▿ †

    PubMed Central

    Gorres, J. Patrick; Lager, Kelly M.; Kong, Wing-Pui; Royals, Michael; Todd, John-Paul; Vincent, Amy L.; Wei, Chih-Jen; Loving, Crystal L.; Zanella, Eraldo L.; Janke, Bruce; Kehrli, Marcus E.; Nabel, Gary J.; Rao, Srinivas S.

    2011-01-01

    Swine influenza is a highly contagious viral infection in pigs that significantly impacts the pork industry due to weight loss and secondary infections. There is also the potential of a significant threat to public health, as was seen in 2009 when the pandemic H1N1 influenza virus strain emerged from reassortment events among avian, swine, and human influenza viruses within pigs. As classic and pandemic H1N1 strains now circulate in swine, an effective vaccine may be the best strategy to protect the pork industry and public health. Current inactivated-virus vaccines available for swine influenza protect only against viral strains closely related to the vaccine strain, and egg-based production of these vaccines is insufficient to respond to large outbreaks. DNA vaccines are a promising alternative since they can potentially induce broad-based protection with more efficient production methods. In this study we evaluated the potentials of monovalent and trivalent DNA vaccine constructs to (i) elicit both humoral and gamma interferon (IFN-γ) responses and (ii) protect pigs against viral shedding and lung disease after challenge with pandemic H1N1 or classic swine H1N1 influenza virus. We also compared the efficiency of a needle-free vaccine delivery method to that of a conventional needle/syringe injection. We report that DNA vaccination elicits robust serum antibody and cellular responses after three immunizations and confers significant protection against influenza virus challenge. Needle-free delivery elicited improved antibody responses with the same efficiency as conventional injection and should be considered for development as a practical alternative for vaccine administration. PMID:21918118

  20. CSR, a scavenger receptor-like protein with a protective role against cellular damage causedby UV irradiation and oxidative stress.

    PubMed

    Han, H J; Tokino, T; Nakamura, Y

    1998-06-01

    Oxidative stress is a pathogenic condition that causes cellular damage and, in a normally functioning cell, several transcription factors respond to this threat by modulating expression of genes whose products ameliorate the altered redox status in some way. We have isolated a novel macrophage scavenger receptor-like gene, CSR (cellular stress response), whose transcription in normal fibroblasts was significantly elevated by exposure to UV radiation or hydrogen peroxide, and pre-treatment with antioxidants prevented induction of CSR . Under conditions of oxidative stress, reactive oxygen species were significantly depleted in CSR -overexpressing cells, indicating that the CSR product protects cells by scavenging oxidative molecules or harmful products of oxidation. Further investigations into the regulation and function of CSR should open a way to understanding the cellular response and the pathogenic processes caused by oxidative stress.

  1. An electrochemical DNA biosensor for evaluating the effect of mix anion in cellular fluid on the antioxidant activity of CeO2 nanoparticles.

    PubMed

    Zhai, Yanwu; Zhang, Yan; Qin, Fei; Yao, Xin

    2015-08-15

    CeO2 nanoparticles are of particular interest as a novel antioxidant for scavenging free radicals. However, some studies showed that they could cause cell damage or death by generating reactive oxygen species (ROS). Up to now, it is not well understood about these paradoxical phenomena. Therefore, many attentions have been paid to the factors that could affect the antioxidant activity of CeO2 nanoparticles. CeO2 nanoparticles would inevitably encounter body fluid environment for its potential medical application. In this work the antioxidant activity behavior of CeO2 nanoparticles is studied in simulated cellular fluid, which contains main body anions (HPO4(2-), HCO3(-), Cl(-) and SO4(2-)), by a method of electrochemical DNA biosensor. We found that in the solution of Cl(-) and SO4(2-), CeO2 nanoparticles can protect DNA from damage by hydroxyl radicals, while in the presence of HPO4(2-) and HCO3(-), CeO2 nanoparticles lose the antioxidant activity. This can be explained by the cerium phosphate and cerium carbonate formed on the surface of the nanoparticles, which interfere with the redox cycling between Ce(3+) and Ce(4+). These results not only add basic knowledge to the antioxidant activity of CeO2 nanoparticles under different situations, but also pave the way for practical applications of nanoceria. Moreover, it also shows electrochemical DNA biosensor is an effective method to explore the antioxidant activity of CeO2 nanoparticles.

  2. A prime-boost immunization with Tc52 N-terminal domain DNA and the recombinant protein expressed in Pichia pastoris protects against Trypanosoma cruzi infection.

    PubMed

    Matos, Marina N; Sánchez Alberti, Andrés; Morales, Celina; Cazorla, Silvia I; Malchiodi, Emilio L

    2016-06-14

    We have previously reported that the N-terminal domain of the antigen Tc52 (NTc52) is the section of the protein that confers the strongest protection against Trypanosoma cruzi infection. To improve vaccine efficacy, we conducted here a prime-boost strategy (NTc52PB) by inoculating two doses of pcDNA3.1 encoding the NTc52 DNA carried by attenuated Salmonella (SNTc52), followed by two doses of recombinant NTc52 expressed in Picchia pastoris plus ODN-CpG as adjuvant. This strategy was comparatively analyzed with the following protocols: (1) two doses of NTc52+ODN-CpG by intranasal route followed by two doses of NTc52+ODN-CpG by intradermal route (NTc52CpG); (2) four doses of SNTc52; and (3) a control group with four doses of Salmonella carrying the empty plasmid. All immunized groups developed a predominant Th1 cellular immune response but with important differences in antibody development and protection against infection. Thus, immunization with just SNTc52 induces a strong specific cellular response, a specific systemic antibody response that is weak yet functional (considering lysis of trypomastigotes and inhibition of cell invasion), and IgA mucosal immunity, protecting in both the acute and chronic stages of infection. The group that received only recombinant protein (NTc52CpG) developed a strong antibody immune response but weaker cellular immunity than the other groups, and the protection against infection was clear in the acute phase of infection but not in chronicity. The prime-boost strategy, which combines DNA and protein vaccine and both mucosal and systemic immunizations routes, was the best assayed protocol, inducing strong cellular and humoral responses as well as specific mucosal IgA, thus conferring better protection in the acute and chronic stages of infection.

  3. A prime-boost immunization with Tc52 N-terminal domain DNA and the recombinant protein expressed in Pichia pastoris protects against Trypanosoma cruzi infection.

    PubMed

    Matos, Marina N; Sánchez Alberti, Andrés; Morales, Celina; Cazorla, Silvia I; Malchiodi, Emilio L

    2016-06-14

    We have previously reported that the N-terminal domain of the antigen Tc52 (NTc52) is the section of the protein that confers the strongest protection against Trypanosoma cruzi infection. To improve vaccine efficacy, we conducted here a prime-boost strategy (NTc52PB) by inoculating two doses of pcDNA3.1 encoding the NTc52 DNA carried by attenuated Salmonella (SNTc52), followed by two doses of recombinant NTc52 expressed in Picchia pastoris plus ODN-CpG as adjuvant. This strategy was comparatively analyzed with the following protocols: (1) two doses of NTc52+ODN-CpG by intranasal route followed by two doses of NTc52+ODN-CpG by intradermal route (NTc52CpG); (2) four doses of SNTc52; and (3) a control group with four doses of Salmonella carrying the empty plasmid. All immunized groups developed a predominant Th1 cellular immune response but with important differences in antibody development and protection against infection. Thus, immunization with just SNTc52 induces a strong specific cellular response, a specific systemic antibody response that is weak yet functional (considering lysis of trypomastigotes and inhibition of cell invasion), and IgA mucosal immunity, protecting in both the acute and chronic stages of infection. The group that received only recombinant protein (NTc52CpG) developed a strong antibody immune response but weaker cellular immunity than the other groups, and the protection against infection was clear in the acute phase of infection but not in chronicity. The prime-boost strategy, which combines DNA and protein vaccine and both mucosal and systemic immunizations routes, was the best assayed protocol, inducing strong cellular and humoral responses as well as specific mucosal IgA, thus conferring better protection in the acute and chronic stages of infection. PMID:27177947

  4. Specificity of cellular DNA-binding sites of microbial populations in a Florida reservoir

    SciTech Connect

    Paul, J.H.; Pichard, S.L. )

    1989-11-01

    The substrate specificity of the DNA-binding mechanism(s) of bacteria in a Florida reservoir was investigated in short- and long-term uptake studies with radiolabeled DNA and unlabeled competitors. Thymine oligonucleotides ranging in size from 2 base pairs to 19 to 24 base pairs inhibited DNA binding in 20-min incubations by 43 to 77%. Deoxynucleoside monophosphates, thymidine, and thymine had little effect on short-term DNA binding, although several of these compounds inhibited the uptake of the radiolabel from DNA in 4-h incubations. Inorganic phosphate and glucose-1-phosphate inhibited neither short- nor long-term binding of ({sup 3}H)- or ({sup 32}P)DNA, indicating that DNA was not utilized as a phosphorous source in this reservoir. RNA inhibited both short- and long-term radiolabeled DNA uptake as effectively as unlabeled DNA. Collectively these results indicate that aquatic bacteria possess a generalized nuclei acid uptake/binding mechanism specific for compounds containing phosphodiester bonds and capable of recognizing oligonucleotides as short as dinucleotides. This binding site is distinct from nucleoside-, nucleotide-, phosphomonoester-, and inorganic phosphate-binding sites. Such a nucleic acid-binding mechanism may have evolved for the utilization of extracellular DNA (and perhaps RNA), which is abundant in many marine and freshwater environments.

  5. Damage to cellular DNA from particulate radiations, the efficacy of its processing and the radiosensitivity of mammalian cells. Emphasis on DNA double strand breaks and chromatin breaks

    NASA Technical Reports Server (NTRS)

    Lett, J. T.

    1992-01-01

    For several years, it has been evident that cellular radiation biology is in a necessary period of consolidation and transition (Lett 1987, 1990; Lett et al. 1986, 1987). Both changes are moving apace, and have been stimulated by studies with heavy charged particles. From the standpoint of radiation chemistry, there is now a consensus of opinion that the DNA hydration shell must be distinguished from bulk water in the cell nucleus and treated as an integral part of DNA (chromatin) (Lett 1987). Concomitantly, sentiment is strengthening for the abandonment of the classical notions of "direct" and "indirect" action (Fielden and O'Neill 1991; O'Neill 1991; O'Neill et al. 1991; Schulte-Frohlinde and Bothe 1991 and references therein). A layer of water molecules outside, or in the outer edge of, the DNA (chromatin) hydration shell influences cellular radiosensitivity in ways not fully understood. Charge and energy transfer processes facilitated by, or involving, DNA hydration must be considered in rigorous theories of radiation action on cells. The induction and processing of double stand breaks (DSBs) in DNA (chromatin) seem to be the predominant determinants of the radiotoxicity of normally radioresistant mammalian cells, the survival curves of which reflect the patterns of damage induced and the damage present after processing ceases, and can be modelled in formal terms by the use of reaction (enzyme) kinetics. Incongruities such as sublethal damage are neither scientifically sound nor relevant to cellular radiation biology (Calkins 1991; Lett 1990; Lett et al. 1987a). Increases in linear energy transfer (LET infinity) up to 100-200 keV micron-1 cause increases in the extents of neighboring chemical and physical damage in DNA denoted by the general term DSB. Those changes are accompanied by decreasing abilities of cells normally radioresistant to sparsely ionizing radiations to process DSBs in DNA and chromatin and to recover from radiation exposure, so they make

  6. Gene Expression Profile Changes and Cellular Responses to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Kidane, Yared; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Rohde, Larry; Wu, Honglu

    2016-01-01

    Living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. In addition, DNA in space can be damaged by toxic chemicals or reactive oxygen species generated due to increased levels of environmental and psychological stresses. Understanding the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage affects the accuracy of the radiation risk assessment for astronauts and the mutation rate in microorganisms. Although possible synergistic effects of space radiation and microgravity have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate the effects of spaceflight on cellular responses to DNA damage, confluent human fibroblast cells (AG1522) flown on the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB). Damages in the DNA were quantified by immunofluorescence staining for ?-H2AX, which showed similar percentages of different types of stained cells between flight and ground. However, there was a slight shift in the distribution of the ?-H2AX foci number in the flown cells with countable foci. Comparison of the cells in confluent and in exponential growth conditions indicated that the proliferation rate between flight and the ground may be responsible for such a shift. A microarray analysis of gene expressions in response to bleomycin treatment was also performed. Comparison of the responsive pathways between the flown and ground cells showed similar responses with the p53 network being the top upstream regulator. Similar responses at the RNA level between different gravity conditions were also observed with a PCR array analysis containing a set of genes involved in DNA damage signaling; with BBC3, CDKN1A, PCNA and PPM1D being significantly

  7. Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery.

    PubMed

    Tuteja, Narendra; Tuteja, Renu

    2004-05-01

    DNA helicases are ubiquitous molecular motor proteins which harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of energetically stable duplex DNA, and thus play important roles in nearly all aspects of nucleic acid metabolism, including replication, repair, recombination, and transcription. They break the hydrogen bonds between the duplex helix and move unidirectionally along the bound strand. All helicases are also translocases and DNA-dependent ATPases. Most contain conserved helicase motifs that act as an engine to power DNA unwinding. All DNA helicases share some common properties, including nucleic acid binding, NTP binding and hydrolysis, and unwinding of duplex DNA in the 3' to 5' or 5' to 3' direction. The minichromosome maintenance (Mcm) protein complex (Mcm4/6/7) provides a DNA-unwinding function at the origin of replication in all eukaryotes and may act as a licensing factor for DNA replication. The RecQ family of helicases is highly conserved from bacteria to humans and is required for the maintenance of genome integrity. They have also been implicated in a variety of human genetic disorders. Since the discovery of the first DNA helicase in Escherichia coli in 1976, and the first eukaryotic one in the lily in 1978, a large number of these enzymes have been isolated from both prokaryotic and eukaryotic systems, and the number is still growing. In this review we cover the historical background of DNA helicases, helicase assays, biochemical properties, prokaryotic and eukaryotic DNA helicases including Mcm proteins and the RecQ family of helicases. The properties of most of the known DNA helicases from prokaryotic and eukaryotic systems, including viruses and bacteriophages, are summarized in tables.

  8. Reovirus inhibition of cellular DNA synthesis: role of the S1 gene.

    PubMed

    Sharpe, A H; Fields, B N

    1981-04-01

    Type 3 reovirus inhibits L cell DNA synthesis, whereas type 1 reovirus exerts little or no effect on L cell DNA synthesis. By using recombinant viruses containing both type 1 and type 3 double-standard RNA segments, we determined that one double-stranded RNA segment, the reovirus type 3 S1 double-stranded RNA segment which encodes the viral hemagglutinin, segregates with and is responsible for the capacity of reovirus type 3 to inhibit L cell DNA synthesis.

  9. Effects of Spaceflight on Molecular and Cellular Responses to Bleomycin-induced DNA Damages in Confluent Human Fibroblasts

    NASA Astrophysics Data System (ADS)

    Lu, Tao; Wu, Honglu; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Wong, Michael

    2016-07-01

    Spaceflights expose human beings to various risk factors. Among them are microgravity related physiological stresses in immune, cytoskeletal, and cardiovascular systems, and space radiation related elevation of cancer risk. Cosmic radiation consists of energetic protons and other heavier charged particles that induce DNA damages. Effective DNA damage response and repair mechanism is important to maintain genomic integrity and reduce cancer risk. There were studies on effects of spaceflight and microgravity on DNA damage response in cell and animal models, but the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on molecular and cellular responses to DNA damages, bleomycin, an anti-cancer drug and radiomimetic reagent, was used to induce DNA damages in confluent human fibroblasts flown to the International Space Station (ISS) and on ground. After exposure to 1.0 mg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by focus pattern and focus number counting of phosphorylated histone protein H2AX (γg-H2AX). The cells on the ISS showed modestly increased average focus counts per nucleus while the distribution of patterns was similar to that on the ground. PCR array analysis showed that expressions of several genes, including CDKN1A and PCNA, were significantly changed in response to DNA damages induced by bleomycin in both flight and ground control cells. However, there were no significant differences in the overall expression profiles of DNA damage response genes between the flight and ground samples. Analysis of cellular proliferation status with Ki-67 staining showed a slightly higher proliferating population in cells on the ISS than those on ground. Our results suggested that the difference in γg-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not

  10. Hybridization of potato spindle tuber viroid to cellular DNA of normal plants.

    PubMed

    Hadidi, A; Jones, D M; Gillespie, D H; Wong-Staal, F; Diener, T O

    1976-07-01

    Molecular hybridization experiments of (125)I-labeled potato spindle tuber viroid (PSTV) with DNA from uninfected or PSTV-infected tomato plants showed that infrequent DNA sequences complementary to PSTV exist in both uninfected and infected cells. DNA titration experiments revealed that at least 60% of PSTV is represented by sequences in DNA of several normal solanaceous host species. Phylogenetically diverse plants contain sequences related to less of the PSTV. PSTV-infected tomato or Gynura aurantiaca plants did not possess new PSTV sequences at detectable levels. These results support the hypothesis that PSTV may have originated from genes in normal solanaceous plants.

  11. Sequence-specific interactions between a cellular DNA-binding protein and the simian virus 40 origin of DNA replication

    SciTech Connect

    Traut, W.; Fanning, E.

    1988-02-01

    The core origin of simian virus 40 (SV40) DNA replication is composed of a 64-base-pair sequence encompassing T-antigen-binding site II and adjacent sequences on either side. A 7-base-pair sequence to the early side of T-antigen-binding site II which is conserved among the papovavirus genomes SV40, BK, JC and SA12 was recently shown to be part of a 10-base-pair sequence required for origin activity, but its functional role was not defined. In the present report, the authors used gel retention assays to identify a monkey cell factor that interacts specifically with double-stranded DNA carrying this sequence and also binds to single-stranded DNA. DNA-protein complexes formed with extracts from primate cells are more abundant and display electrophoretic mobilities distinct from those formed with rodent cell extracts. The binding activity of the factor on mutant templates is correlate with the replication activity of the origin. The results suggest that the monkey cell factor may be involved in SV40 DNA replication.

  12. Enhanced Immune Response to DNA Vaccine Encoding Bacillus anthracis PA-D4 Protects Mice against Anthrax Spore Challenge

    PubMed Central

    Kim, Na Young; Chang, Dong Suk; Kim, Yeonsu; Kim, Chang Hwan; Hur, Gyeung Haeng; Yang, Jai Myung; Shin, Sungho

    2015-01-01

    Anthrax has long been considered the most probable bioweapon-induced disease. The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of anthrax. In the current study, we evaluated the efficiency of a genetic vaccination with the fourth domain (D4) of PA, which is responsible for initial binding of the anthrax toxin to the cellular receptor. The eukaryotic expression vector was designed with the immunoglobulin M (IgM) signal sequence encoding for PA-D4, which contains codon-optimized genes. The expression and secretion of recombinant protein was confirmed in vitro in 293T cells transfected with plasmid and detected by western blotting, confocal microscopy, and enzyme-linked immunosorbent assay (ELISA). The results revealed that PA-D4 protein can be efficiently expressed and secreted at high levels into the culture medium. When plasmid DNA was given intramuscularly to mice, a significant PA-D4-specific antibody response was induced. Importantly, high titers of antibodies were maintained for nearly 1 year. Furthermore, incorporation of the SV40 enhancer in the plasmid DNA resulted in approximately a 15-fold increase in serum antibody levels in comparison with the plasmid without enhancer. The antibodies produced were predominantly the immunoglobulin G2 (IgG2) type, indicating the predominance of the Th1 response. In addition, splenocytes collected from immunized mice produced PA-D4-specific interferon gamma (IFN-γ). The biodistribution study showed that plasmid DNA was detected in most organs and it rapidly cleared from the injection site. Finally, DNA vaccination with electroporation induced a significant increase in immunogenicity and successfully protected the mice against anthrax spore challenge. Our approach to enhancing the immune response contributes to the development of DNA vaccines against anthrax and other biothreats. PMID:26430894

  13. DNA and virus particle vaccination protects against acquisition and confers control of viremia upon heterologous simian immunodeficiency virus challenge.

    PubMed

    Patel, Vainav; Jalah, Rashmi; Kulkarni, Viraj; Valentin, Antonio; Rosati, Margherita; Alicea, Candido; von Gegerfelt, Agneta; Huang, Wensheng; Guan, Yongjun; Keele, Brandon F; Bess, Julian W; Piatak, Michael; Lifson, Jeffrey D; Williams, William T; Shen, Xiaoying; Tomaras, Georgia D; Amara, Rama R; Robinson, Harriet L; Johnson, Welkin; Broderick, Kate E; Sardesai, Niranjan Y; Venzon, David J; Hirsch, Vanessa M; Felber, Barbara K; Pavlakis, George N

    2013-02-19

    We have previously shown that macaques vaccinated with DNA vectors expressing SIVmac239 antigens developed potent immune responses able to reduce viremia upon high-dose SIVmac251 challenge. To further improve vaccine-induced immunity and protection, we combined the SIVmac239 DNA vaccine with protein immunization using inactivated SIVmac239 viral particles as protein source. Twenty-six weeks after the last vaccination, the animals were challenged intrarectally at weekly intervals with a titrated dose of the heterologous SIVsmE660. Two of DNA-protein coimmunized macaques did not become infected after 14 challenges, but all controls were infected by 11 challenges. Vaccinated macaques showed modest protection from SIVsmE660 acquisition compared with naïve controls (P = 0.050; stratified for TRIM5α genotype). Vaccinees had significantly lower peak (1.6 log, P = 0.0048) and chronic phase viremia (P = 0.044), with 73% of the vaccinees suppressing viral replication to levels below assay detection during the 40-wk follow-up. Vaccine-induced immune responses associated significantly with virus control: binding antibody titers and the presence of rectal IgG to SIVsmE660 Env correlated with delayed SIVsmE660 acquisition; SIV-specific cytotoxic T cells, prechallenge CD4(+) effector memory, and postchallenge CD8(+) transitional memory cells correlated with control of viremia. Thus, SIVmac239 DNA and protein-based vaccine protocols were able to achieve high, persistent, broad, and effective cellular and humoral immune responses able to delay heterologous SIVsmE660 infection and to provide long-term control of viremia. These studies support a role of DNA and protein-based vaccines for development of an efficacious HIV/AIDS vaccine.

  14. DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding.

    PubMed

    Li, Chun Mei; Zheng, Lin Ling; Yang, Xiao Xi; Wan, Xiao Yan; Wu, Wen Bi; Zhen, Shu Jun; Li, Yuan Fang; Luo, Ling Fei; Huang, Cheng Zhi

    2016-01-01

    Viral infections have caused numerous diseases and deaths worldwide. Due to the emergence of new viruses and frequent virus variation, conventional antiviral strategies that directly target viral or cellular proteins are limited because of the specificity, drug resistance and rapid clearance from the human body. Therefore, developing safe and potent antiviral agents with activity against viral infection at multiple points in the viral life cycle remains a major challenge. In this report, we propose a new modality to inhibit viral infection by fabricating DNA conjugated gold nanoparticle (DNA-AuNP) networks on cell membranes as a protective barrier. The DNA-AuNPs networks were found, via a plaque formation assay and viral titers, to have potent antiviral ability and protect host cells from human respiratory syncytial virus (RSV). Confocal immunofluorescence image analysis showed 80 ± 3.8% of viral attachment, 91.1 ± 0.9% of viral entry and 87.9 ± 2.8% of viral budding were inhibited by the DNA-AuNP networks, which were further confirmed by real-time fluorescence imaging of the RSV infection process. The antiviral activity of the networks may be attributed to steric effects, the disruption of membrane glycoproteins and limited fusion of cell membrane bilayers, all of which play important roles in viral infection. Therefore, our results suggest that the DNA-AuNP networks have not only prophylactic effects to inhibit virus attachment and entry, but also therapeutic effects to inhibit viral budding and cell-to-cell spread. More importantly, this proof-of-principle study provides a pathway for the development of a universal, broad-spectrum antiviral therapy.

  15. Intradermal Gene Immunization: The Possible Role of DNA Uptake in the Induction of Cellular Immunity to Viruses

    NASA Astrophysics Data System (ADS)

    Raz, Eyal; Carson, Dennis A.; Parker, Suezanne E.; Parr, Tyler B.; Abai, Anna M.; Aichinger, Gerald; Gromkowski, Stanislaw H.; Singh, Malini; Lew, Denise; Yankauckas, Michelle A.; Baird, Stephen M.; Rhodes, Gary H.

    1994-09-01

    The skin and mucous membranes are the anatomical sites where most viruses are first encountered by the immune system. Previous experiments have suggested that striated muscle cells are unique among mammalian cell types in their capacity to take up and express free DNA in the absence of a viral vector or physical carrier. However, we have found that mice injected into the superficial skin with free (naked) plasmid DNA encoding the influenza nucleoprotein gene had discrete foci of epidermal and dermal cells, including cells with dendritic morphology, that contained immunoreactive nucleoprotein antigen. A single intradermal administration of 0.3-15 μ g of free plasmid DNA induced anti-nucleoprotein-specific antibody and cytotoxic T lymphocytes that persisted for at least 68-70 weeks after vaccination. Intradermal gene administration induced higher antibody titers than did direct gene injection into skeletal muscle and did not cause local inflammation or necrosis. Compared with control animals, the gene-injected mice were resistant to challenge with a heterologous strain of influenza virus. These results indicate that the cells of the skin can take up and express free foreign DNA and induce cellular and humoral immune responses against the encoded protein. We suggest that DNA uptake by the skin-associated lymphoid tissues may play a role in the induction of cytotoxic T cells against viruses and other intracellular pathogens.

  16. Immunostimulant Adjuvant Patch Enhances Humoral and Cellular Immune Responses to DNA Immunization

    PubMed Central

    Mkrtichyan, Mikayel; Ghochikyan, Anahit; Movsesyan, Nina; Karapetyan, Adrine; Begoyan, Gayane; Yu, Jianmei; Glenn, Gregory M.; Ross, Ted M.; Agadjanyan, Michael G.; Cribbs, David H.

    2008-01-01

    The focus of this report is on the development of an improved DNA immunization protocol, which takes advantage of the strengths of DNA immunization, as well as those associated with adjuvant delivered by transcutaneous immunostimulatory (IS) patches. Because transcutaneous delivery of adjuvants to the skin at the vaccination site has been shown to amplify the immune response to protein antigens, we hypothesized that the same IS patch when placed on the skin at the site of DNA injection could further enhance the immune response to a DNA influenza vaccine. We have combined an influenza DNA vaccine, hemagglutinin fused with three copies of complement C3d, to enhance uptake and antigen presentation, with an IS patch containing heat-labile enterotoxin from Escherichia coli. Coadministration of a potent adjuvant in IS patches placed on the skin at the site of DNA vaccination dramatically amplifies anti-influenza antibody immune response. Supplementing DNA vaccines with IS patches may be a particularly valuable strategy because DNA vaccines can be rapidly modified in response to mutations in pathogens, and individuals with compromised immune systems such as transplant patients and the elderly will benefit from the enhanced antibody response induced by the IS patches. PMID:17961074

  17. Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression.

    PubMed

    Sakurai, F; Inoue, R; Nishino, Y; Okuda, A; Matsumoto, O; Taga, T; Yamashita, F; Takakura, Y; Hashida, M

    2000-05-15

    In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MBT-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [32P]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA

  18. Immunization with non-replicating E. coli minicells delivering both protein antigen and DNA protects mice from lethal challenge with lymphocytic choriomeningitis virus

    PubMed Central

    Giacalone, Matthew J.; Zapata, Juan C.; Berkley, Neil L.; Sabbadini, Roger A.; Chu, Yen-Lin; Salvato, Maria S.; McGuire, Kathleen L.

    2008-01-01

    In the midst of new investigations into the mechanisms of both delivery and protection of new vaccines and vaccine carriers, it has become clear that immunization with delivery mechanisms that do not involve living, replicating organisms are vastly preferred. In this report, non-replicating bacterial minicells simultaneously co-delivering the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) and the corresponding DNA vaccine were tested for the ability to generate protective cellular immune responses in mice. It was found that good protection (89%) was achieved after intramuscular administration, moderate protection (31%) was achieved after intranasal administration, and less protection (7%) was achieved following gastric immunization. These results provide a solid foundation on which to pursue the use of bacterial minicells as a non-replicating vaccine delivery platform. PMID:17258845

  19. DNA Vaccines: Protective Immunizations by Parenteral, Mucosal, and Gene-Gun Inoculations

    NASA Astrophysics Data System (ADS)

    Fynan, Ellen F.; Webster, Robert G.; Fuller, Deborah H.; Haynes, Joel R.; Santoro, Joseph C.; Robinson, Harriet L.

    1993-12-01

    Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 μg of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens have been protected against a lethal influenza challenge. Parenteral routes of inoculation that achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis. In mice, 95% protection was achieved by two immunizations with beads loaded with as little as 0.4 μg of DNA. The breadth of routes supporting successful DNA immunizations, coupled with the very small amounts of DNA required for gene-gun immunizations, highlight the potential of this remarkably simple technique for the development of subunit vaccines.

  20. Possible prebiotic significance of polyamines in the condensation, protection, encapsulation, and biological properties of DNA

    NASA Technical Reports Server (NTRS)

    Baeza, I.; Ibanez, M.; Wong, C.; Chavez, P.; Gariglio, P.; Oro, J.

    1991-01-01

    Some properties of DNA condensed with spermidine have been compared with the properties of DNA condensed with Co3+(NH3)6 to determine whether condensation of DNA with these trivalent cations protects DNA against the action of DNase I and increases transcription and encapsulation of DNA into liposomes. It was shown that DNA condensed with Co3+(NH3)6 was resistant to the action of the endonuclease DNase I such as DNA condensed with spermidine was. However, DNA condensed with Co3+(NH3)6 was significantly less active in transcription with the E. coli RNA polymerase than DNA-spermidine condensed forms. In addition, it was demonstrated that both compacted forms of DNA were more efficiently encapsulated into neutral liposomes; however, negatively, charged liposomes were scarcely formed in the presence of DNA condensed with Co3+(NH3)6. These experiments and the well documented properties of polyamines increasing the resistance to radiations and hydrolysis of nucleic acids, as well as their biological activities, such as replication, transcription, and translation, together with the low concentration of Co3+ in the environment, lead us to propose spermidine as a plausible prebiotic DNA condensing agent rather than Co3+ and the basic proteins proposed by other authors. Then, we consider the possible role and relevance of the polyamine-nucleic acids complexes in the evolution of life.

  1. Chiral Ruthenium(II) Polypyridyl Complexes: Stabilization of G-Quadruplex DNA, Inhibition of Telomerase Activity and Cellular Uptake

    PubMed Central

    Yu, Qianqian; Liu, Yanan; Wang, Chuan; Sun, Dongdong; Yang, Xingcheng; Liu, Yanyu; Liu, Jie

    2012-01-01

    Two ruthenium(II) complexes, Λ-[Ru(phen)2(p-HPIP)]2+ and Δ-[Ru(phen)2(p-HPIP)]2+, were synthesized and characterized via proton nuclear magnetic resonance spectroscopy, electrospray ionization-mass spectrometry, and circular dichroism spectroscopy. This study aims to clarify the anticancer effect of metal complexes as novel and potent telomerase inhibitors and cellular nucleus target drug. First, the chiral selectivity of the compounds and their ability to stabilize quadruplex DNA were studied via absorption and emission analyses, circular dichroism spectroscopy, fluorescence-resonance energy transfer melting assay, electrophoretic mobility shift assay, and polymerase chain reaction stop assay. The two chiral compounds selectively induced and stabilized the G-quadruplex of telomeric DNA with or without metal cations. These results provide new insights into the development of chiral anticancer agents for G-quadruplex DNA targeting. Telomerase repeat amplification protocol reveals the higher inhibitory activity of Λ-[Ru(phen)2(p-HPIP)]2+ against telomerase, suggesting that Λ-[Ru(phen)2(p-HPIP)]2+ may be a potential telomerase inhibitor for cancer chemotherapy. MTT assay results show that these chiral complexes have significant antitumor activities in HepG2 cells. More interestingly, cellular uptake and laser-scanning confocal microscopic studies reveal the efficient uptake of Λ-[Ru(phen)2(p-HPIP)]2+ by HepG2 cells. This complex then enters the cytoplasm and tends to accumulate in the nucleus. This nuclear penetration of the ruthenium complexes and their subsequent accumulation are associated with the chirality of the isomers as well as with the subtle environment of the ruthenium complexes. Therefore, the nucleus can be the cellular target of chiral ruthenium complexes for anticancer therapy. PMID:23236402

  2. DNA vaccination with KMP11 and Lutzomyia longipalpis salivary protein protects hamsters against visceral leishmaniasis

    PubMed Central

    da Silva, Robson A.A.; Tavares, Natália M.; Costa, Dirceu; Pitombo, Maiana; Barbosa, Larissa; Fukutani, Kyioshi; Miranda, Jose C.; de Oliveira, Camila I.; Valenzuela, Jesus G.; Barral, Aldina; Soto, Manuel; Barral-Netto, Manoel; Brodskyn, Cláudia

    2013-01-01

    It was recently shown that immunization of hamsters with DNA plasmids coding LJM19, a sand fly salivary protein, partially protected against a challenge with Leishmania chagasi, whereas immunization with KMP11 DNA plasmid, a Leishmania antigen, induced protection against L. donovani infection. In the present study, we evaluated the protective effect of immunization with both LJM19 and KMP11 DNA plasmid together. Concerning the protection against an infection by L. chagasi, immunization with DNA plasmids coding LJM19 or KMP11, as well as with both plasmids combined, induced IFN-γ production in draining lymph nodes at 7, 14 and 21 days post-immunization. Immunized hamsters challenged with L. chagasi plus Salivary Gland Sonicate (SGS) from Lutzomyia longipalpis showed an enhancement of IFN-γ/IL-10 and IFN-γ/TGF-β in draining lymph nodes after 7 and 14 days of infection. Two and five months after challenge, immunized animals showed reduced parasite load in the liver and spleen, as well as increased IFN-γ/IL-10 and IFN-γ/TGF-β ratios in the spleen. Furthermore, immunized animals remained with a normal hematological profile even five months after the challenge, whereas L. chagasi in unimmunized hamsters lead to a significant anemia. The protection observed with LJM19 or KMP11 DNA plasmids used alone was very similar to the protection obtained by the combination of both plasmids. PMID:21875567

  3. Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction.

    PubMed

    Genelhu, M S; Zanini, M S; Veloso, I F; Carneiro, A M; Lopes, M T; Salas, C E

    1998-09-01

    We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 microgram = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25 degrees C) for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.

  4. Immunogenicity and Protective Efficacy of Brugia malayi Heavy Chain Myosin as Homologous DNA, Protein and Heterologous DNA/Protein Prime Boost Vaccine in Rodent Model

    PubMed Central

    Gupta, Jyoti; Pathak, Manisha; Misra, Sweta; Misra-Bhattacharya, Shailja

    2015-01-01

    We earlier demonstrated the immunoprophylactic efficacy of recombinant heavy chain myosin (Bm-Myo) of Brugia malayi (B. malayi) in rodent models. In the current study, further attempts have been made to improve this efficacy by employing alternate approaches such as homologous DNA (pcD-Myo) and heterologous DNA/protein prime boost (pcD-Myo+Bm-Myo) in BALB/c mouse model. The gene bm-myo was cloned in a mammalian expression vector pcDNA 3.1(+) and protein expression was confirmed in mammalian Vero cell line. A significant degree of protection (79.2%±2.32) against L3 challenge in pcD-Myo+Bm-Myo immunized group was observed which was much higher than that exerted by Bm-Myo (66.6%±2.23) and pcD-Myo (41.6%±2.45). In the heterologous immunized group, the percentage of peritoneal leukocytes such as macrophages, neutrophils, B cells and T cells marginally increased and their population augmented further significantly following L3 challenge. pcD-Myo+Bm-Myo immunization elicited robust cellular and humoral immune responses as compared to pcD-Myo and Bm-Myo groups as evidenced by an increased accumulation of CD4+, CD8+ T cells and CD19+ B cells in the mouse spleen and activation of peritoneal macrophages. Though immunized animals produced antigen-specific IgG antibodies and isotypes, sera of mice receiving pcD-Myo+Bm-Myo or Bm-Myo developed much higher antibody levels than other groups and there was profound antibody-dependent cellular adhesion and cytotoxicity (ADCC) to B. malayi infective larvae (L3). pcD-Myo+Bm-Myo as well as Bm-Myo mice generated a mixed T helper cell phenotype as evidenced by the production of both pro-inflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines. Mice receiving pcD-Myo on contrary displayed a polarized pro-inflammatory immune response. The findings suggest that the priming of animals with DNA followed by protein booster generates heightened and mixed pro- and anti-inflammatory immune responses that are capable of providing

  5. Immunogenicity and Protective Efficacy of Brugia malayi Heavy Chain Myosin as Homologous DNA, Protein and Heterologous DNA/Protein Prime Boost Vaccine in Rodent Model.

    PubMed

    Gupta, Jyoti; Pathak, Manisha; Misra, Sweta; Misra-Bhattacharya, Shailja

    2015-01-01

    We earlier demonstrated the immunoprophylactic efficacy of recombinant heavy chain myosin (Bm-Myo) of Brugia malayi (B. malayi) in rodent models. In the current study, further attempts have been made to improve this efficacy by employing alternate approaches such as homologous DNA (pcD-Myo) and heterologous DNA/protein prime boost (pcD-Myo+Bm-Myo) in BALB/c mouse model. The gene bm-myo was cloned in a mammalian expression vector pcDNA 3.1(+) and protein expression was confirmed in mammalian Vero cell line. A significant degree of protection (79.2%±2.32) against L3 challenge in pcD-Myo+Bm-Myo immunized group was observed which was much higher than that exerted by Bm-Myo (66.6%±2.23) and pcD-Myo (41.6%±2.45). In the heterologous immunized group, the percentage of peritoneal leukocytes such as macrophages, neutrophils, B cells and T cells marginally increased and their population augmented further significantly following L3 challenge. pcD-Myo+Bm-Myo immunization elicited robust cellular and humoral immune responses as compared to pcD-Myo and Bm-Myo groups as evidenced by an increased accumulation of CD4+, CD8+ T cells and CD19+ B cells in the mouse spleen and activation of peritoneal macrophages. Though immunized animals produced antigen-specific IgG antibodies and isotypes, sera of mice receiving pcD-Myo+Bm-Myo or Bm-Myo developed much higher antibody levels than other groups and there was profound antibody-dependent cellular adhesion and cytotoxicity (ADCC) to B. malayi infective larvae (L3). pcD-Myo+Bm-Myo as well as Bm-Myo mice generated a mixed T helper cell phenotype as evidenced by the production of both pro-inflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines. Mice receiving pcD-Myo on contrary displayed a polarized pro-inflammatory immune response. The findings suggest that the priming of animals with DNA followed by protein booster generates heightened and mixed pro- and anti-inflammatory immune responses that are capable of providing

  6. Stress-induced rearrangements of cellular networks: Consequences for protection and drug design.

    PubMed

    Szalay, Máté S; Kovács, István A; Korcsmáros, Tamás; Böde, Csaba; Csermely, Péter

    2007-07-31

    The complexity of the cells can be described and understood by a number of networks such as protein-protein interaction, cytoskeletal, organelle, signalling, gene transcription and metabolic networks. All these networks are highly dynamic producing continuous rearrangements in their links, hubs, network-skeleton and modules. Here we describe the adaptation of cellular networks after various forms of stress causing perturbations, congestions and network damage. Chronic stress decreases link-density, decouples or even quarantines modules, and induces an increased competition between network hubs and bridges. Extremely long or strong stress may induce a topological phase transition in the respective cellular networks, which switches the cell to a completely different mode of cellular function. We summarize our initial knowledge on network restoration after stress including the role of molecular chaperones in this process. Finally, we discuss the implications of stress-induced network rearrangements in diseases and ageing, and propose therapeutic approaches both to increase the robustness and help the repair of cellular networks. PMID:17433306

  7. Linkage between reovirus-induced apoptosis and inhibition of cellular DNA synthesis: role of the S1 and M2 genes.

    PubMed Central

    Tyler, K L; Squier, M K; Brown, A L; Pike, B; Willis, D; Oberhaus, S M; Dermody, T S; Cohen, J J

    1996-01-01

    The mammalian reoviruses are capable of inhibiting cellular DNA synthesis and inducing apoptosis. Reovirus strains type 3 Abney (T3A) and type 3 Dearing (T3D) inhibit cellular DNA synthesis and induce apoptosis to a substantially greater extent than strain type 1 Lang (T1L). We used T1L x T3A and T1L x T3D reassortant viruses to identify viral genes associated with differences in the capacities of reovirus strains to elicit these cellular responses to viral infection. We found that the S1 and M2 genome segments determine differences in the capacities of both T1L x T3A and T1L x T3D reassortant viruses to inhibit cellular DNA synthesis and to induce apoptosis. These genes encode viral outer-capsid proteins that play important roles in viral attachment and disassembly. To extend these findings, we used field isolate strains of reovirus to determine whether the strain-specific differences in inhibition of cellular DNA synthesis and induction of apoptosis are also associated with viral serotype, a property determined by the S1 gene. In these experiments, type 3 field isolate strains were found to inhibit cellular DNA synthesis and to induce apoptosis to a greater extent than type 1 field isolate strains. Statistical analysis of these data indicate a significant correlation between the capacity of T1L x T3A and T1L x T3D reassortant viruses and field isolate strains to inhibit cellular DNA synthesis and to induce apoptosis. These findings suggest that reovirus-induced inhibition of cellular DNA synthesis and induction of apoptosis are linked and that both phenomena are induced by early steps in the viral replication cycle. PMID:8892922

  8. Correlated analysis of cellular DNA, membrane antigens and light scatter of human lymphoid cells

    SciTech Connect

    Braylan, R.C.; Benson, N.A.; Nourse, V.; Kruth, H.S.

    1982-03-01

    Flow cytometric correlated analysis of membrane antigens, DNA, and light scatter was performed on human lymphoid cells using fluorescein (FITC)-conjugated antibodies to label B- and T-cell antigens and propidium iodide (PI) to stain DNA after ethanol fixation and RNase treatment. A FACS II flow cytometer was modified to obtain digitized measurements of two color fluorescence and light scatter emissions, simultaneously. Software was written to allow single parameter analysis or correlated analysis of any two of the three parameters acquired. Ethanol fixation preserved FITC surface labeling for at least 15 weeks, but produced marked changes in light scatter. No changes in FITC distributions were observed after RNase treatment and PI staining, and the presence of FITC labeling did not affect DNA distributions. Within heterogeneous cell populations, the DNA distribution of cell subpopulations identified by a membrane antigen was clearly demonstrated.

  9. Cellular immunostimulation by CpG-sequence-coated DNA origami structures.

    PubMed

    Schüller, Verena J; Heidegger, Simon; Sandholzer, Nadja; Nickels, Philipp C; Suhartha, Nina A; Endres, Stefan; Bourquin, Carole; Liedl, Tim

    2011-12-27

    To investigate the potential of DNA origami constructs as programmable and noncytotoxic immunostimulants, we tested the immune responses induced by hollow 30-helix DNA origami tubes covered with up to 62 cytosine-phosphate-guanine (CpG) sequences in freshly isolated spleen cells. Unmethylated CpG sequences that are highly specific for bacterial DNA are recognized by a specialized receptor of the innate immune system localized in the endosome, the Toll-like receptor 9 (TLR9). When incubated with oligonucleotides containing CpGs, immune cells are stimulated through TLR9 to produce and secrete cytokine mediators such as interleukin-6 (IL-6) and interleukin-12p70 (IL-12p70), a process associated with the initiation of an immune response. In our studies, the DNA origami tube built from an 8634 nt long variant of the commonly used single-stranded DNA origami scaffold M13mp18 and 227 staple oligonucleotides decorated with 62 CpG-containing oligonucleotides triggered a strong immune response, characterized by cytokine production and immune cell activation, which was entirely dependent on TLR9 stimulation. Such decorated origami tubes also triggered higher immunostimulation than equal amounts of CpG oligonucleotides associated with a standard carrier system such as Lipofectamine. In the absence of CpG oligonucleotides, cytokine production induced by the origami tubes was low and was not related to TLR9 recognition. Fluorescent microscopy revealed localization of CpG-containing DNA origami structures in the endosome. The DNA constructs showed in contrast to Lipofectamine no detectable toxicity and did not affect the viability of splenocytes. We thus demonstrate that DNA origami constructs represent a delivery system for CpG oligonucleotides that is both efficient and nontoxic. PMID:22092186

  10. Anti-oxidative cellular protection effect of fasting-induced autophagy as a mechanism for hormesis.

    PubMed

    Moore, Michael N; Shaw, Jennifer P; Ferrar Adams, Dawn R; Viarengo, Aldo

    2015-06-01

    The aim of this investigation was to test the hypothesis that fasting-induced augmented lysosomal autophagic turnover of cellular proteins and organelles will reduce potentially harmful lipofuscin (age-pigment) formation in cells by more effectively removing oxidatively damaged proteins. An animal model (marine snail--common periwinkle, Littorina littorea) was used to experimentally test this hypothesis. Snails were deprived of algal food for 7 days to induce an augmented autophagic response in their hepatopancreatic digestive cells (hepatocyte analogues). This treatment resulted in a 25% reduction in the cellular content of lipofuscin in the digestive cells of the fasting animals in comparison with snails fed ad libitum on green alga (Ulva lactuca). Similar findings have previously been observed in the digestive cells of marine mussels subjected to copper-induced oxidative stress. Additional measurements showed that fasting significantly increased cellular health based on lysosomal membrane stability, and reduced lipid peroxidation and lysosomal/cellular triglyceride. These findings support the hypothesis that fasting-induced augmented autophagic turnover of cellular proteins has an anti-oxidative cytoprotective effect by more effectively removing damaged proteins, resulting in a reduction in the formation of potentially harmful proteinaceous aggregates such as lipofuscin. The inference from this study is that autophagy is important in mediating hormesis. An increase was demonstrated in physiological complexity with fasting, using graph theory in a directed cell physiology network (digraph) model to integrate the various biomarkers. This was commensurate with increased health status, and supportive of the hormesis hypothesis. The potential role of enhanced autophagic lysosomal removal of damaged proteins in the evolutionary acquisition of stress tolerance in intertidal molluscs is discussed and parallels are drawn with the growing evidence for the involvement of

  11. Transcriptomal profiling of the cellular response to DNA damage mediated by Slug (Snai2)

    PubMed Central

    Pérez-Caro, M; Bermejo-Rodríguez, C; González-Herrero, I; Sánchez-Beato, M; Piris, M A; Sánchez-García, I

    2008-01-01

    Snai2-deficient cells are radiosensitive to DNA damage. The function of Snai2 in response to DNA damage seems to be critical for its function in normal development and cancer. Here, we applied a functional genomics approach that combined gene-expression profiling and computational molecular network analysis to obtain global dissection of the Snai2-dependent transcriptional response to DNA damage in primary mouse embryonic fibroblasts (MEFs), which undergo p53-dependent growth arrest in response to DNA damage. Although examination of the response showed that overall expression of p53 target gene expression patterns was similarly altered in both control and Snai2-deficient cells, we have identified and validated candidate Snai2 target genes linked to Snai2 gene function in response to DNA damage. This work defines for the first time the effect of Snai2 on p53 target genes in cells undergoing growth arrest, elucidates the Snai2-dependent molecular network induced by DNA damage, points to novel putative Snai2 targets, and suggest a mechanistic model, which has implications for cancer management. PMID:18182996

  12. Analysis of 7,8-Dihydro-8-oxo-2′-deoxyguanosine in Cellular DNA during Oxidative Stress

    PubMed Central

    2009-01-01

    Analysis of cellular 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dGuo) as a biomarker of oxidative DNA damage has been fraught with numerous methodological problems. This is primarily due to artifactual oxidation of dGuo that occurs during DNA isolation and hydrolysis. Therefore, it has become necessary to rely on using the comet assay, which is not necessarily specific for 8-oxo-dGuo. A highly specific and sensitive method based on immunoaffinity purification and stable isotope dilution liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) that avoids artifact formation has now been developed. Cellular DNA was isolated using cold DNAzol (a proprietary product that contains guanidine thiocyanate) instead of chaotropic- or phenol-based methodology. Chelex-treated buffers were used to prevent Fenton chemistry-mediated generation of reactive oxygen species (ROS) and artifactual oxidation of DNA bases. Deferoxamine was also added to all buffers in order to complex any residual transition metal ions remaining after Chelex treatment. The LC-MRM/MS method was used to determine that the basal 8-oxo-dGuo level in DNA from human bronchoalveolar H358 cells was 2.2 ± 0.4 8-oxo-dGuo/107 dGuo (mean ± standard deviation) or 5.5 ± 1.0 8-oxo-dGuo/108 nucleotides. Similar levels were observed in human lung adenocarcinoma A549 cells, mouse hepatoma Hepa-1c1c7 cells, and human HeLa cervical epithelial adenocarcinoma cells. These values are an order of magnitude lower than is typically reported for basal 8-oxo-dGuo levels in DNA as determined by other MS- or chromatography-based assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO3) as a positive control or with the methylating agent methyl methanesulfonate (MMS) as a negative control. A linear dose−response for 8-oxo-dGuo formation (r2 = 0.962) was obtained with increasing concentrations of KBrO3 in the range of 0.05 mM to 2.50 mM. In contrast, no 8-oxo-dGuo was

  13. Decreased cellular permeability to H2O2 protects Saccharomyces cerevisiae cells in stationary phase against oxidative stress.

    PubMed

    Sousa-Lopes, A; Antunes, F; Cyrne, L; Marinho, H S

    2004-12-01

    The higher resistance of stationary-phase Saccharomyces cerevisiae to H2O2 when compared with exponential phase is well characterized, but the molecular mechanisms underlying it remain mostly unknown. By applying the steady-state H2O2-delivery model, we show that (a) cellular permeability to H2O2 is five times lower in stationary--than in exponential phase; (b) cell survival to H2O2 correlates with H2O2 cellular gradients for a variety of cells; and, (c) cells in stationary phase are predicted to be more susceptible to intracellular H2O2 than in exponential phase. In conclusion, limiting H2O2 diffusion into cells is a key protective mechanism against extracellular H2O2.

  14. RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA.

    PubMed

    Wolf, Christine; Rapp, Alexander; Berndt, Nicole; Staroske, Wolfgang; Schuster, Max; Dobrick-Mattheuer, Manuela; Kretschmer, Stefanie; König, Nadja; Kurth, Thomas; Wieczorek, Dagmar; Kast, Karin; Cardoso, M Cristina; Günther, Claudia; Lee-Kirsch, Min Ae

    2016-01-01

    Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA. PMID:27230542

  15. RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA

    PubMed Central

    Wolf, Christine; Rapp, Alexander; Berndt, Nicole; Staroske, Wolfgang; Schuster, Max; Dobrick-Mattheuer, Manuela; Kretschmer, Stefanie; König, Nadja; Kurth, Thomas; Wieczorek, Dagmar; Kast, Karin; Cardoso, M. Cristina; Günther, Claudia; Lee-Kirsch, Min Ae

    2016-01-01

    Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA. PMID:27230542

  16. Prediction of cellular radiosensitivity from DNA damage induced by gamma-rays and carbon ion irradiation in canine tumor cells.

    PubMed

    Wada, Seiichi; Van Khoa, Tran; Kobayashi, Yasuhiko; Funayama, Tomoo; Ogihara, Kikumi; Ueno, Shunji; Ito, Nobuhiko

    2005-11-01

    Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.

  17. Temsirolimus promotes autophagic clearance of amyloid-β and provides protective effects in cellular and animal models of Alzheimer's disease.

    PubMed

    Jiang, Teng; Yu, Jin-Tai; Zhu, Xi-Chen; Tan, Meng-Shan; Wang, Hui-Fu; Cao, Lei; Zhang, Qiao-Quan; Shi, Jian-Quan; Gao, Li; Qin, Hao; Zhang, Ying-Dong; Tan, Lan

    2014-03-01

    Accumulation of amyloid-β peptides (Aβ) within brain is a major pathogenic hallmark of Alzheimer's disease (AD). Emerging evidence suggests that autophagy, an important intracellular catabolic process, is involved in Aβ clearance. Here, we investigated whether temsirolimus, a newly developed compound approved by Food and Drug Administration and European Medicines Agency for renal cell carcinoma treatment, would promote autophagic clearance of Aβ and thus provide protective effects in cellular and animal models of AD. HEK293 cells expressing the Swedish mutant of APP695 (HEK293-APP695) were treated with vehicle or 100nM temsirolimus for 24h in the presence or absence of 3-methyladenine (5mM) or Atg5-siRNA, and intracellular Aβ levels as well as autophagy biomarkers were measured. Meanwhile, APP/PS1 mice received intraperitoneal injection of temsirolimus (20mg/kg) every 2 days for 60 days, and brain Aβ burden, autophagy biomarkers, cellular apoptosis in hippocampus, and spatial cognitive functions were assessed. Our results showed that temsirolimus enhanced Aβ clearance in HEK293-APP695 cells and in brain of APP/PS1 mice in an autophagy-dependent manner. Meanwhile, temsirolimus attenuated cellular apoptosis in hippocampus of APP/PS1 mice, which was accompanied by an improvement in spatial learning and memory abilities. In conclusion, our study provides the first evidence that temsirolimus promotes autophagic Aβ clearance and exerts protective effects in cellular and animal models of AD, suggesting that temsirolimus administration may represent a new therapeutic strategy for AD treatment. Meanwhile, these findings emphasize the notion that many therapeutic agents possess pleiotropic actions aside from their main applications. PMID:24602800

  18. Calcitriol-copper interaction leads to non enzymatic, reactive oxygen species mediated DNA breakage and modulation of cellular redox scavengers in hepatocellular carcinoma.

    PubMed

    Rizvi, Asim; Farhan, Mohd; Naseem, Imrana; Hadi, S M

    2016-09-01

    Calcitriol is the metabolically active form of Vitamin D and is known to kill cancer cells. Using the rat model of DEN induced hepatocellular carcinoma we show that there is a marked increase in cellular levels of copper in hepatocellular carcinoma and that calcitriol-copper interaction leads to reactive oxygen species mediated DNA breakage selectively in hepatocellular carcinoma cells. In vivo studies show that calcitriol selectively induces severe fluctuations in cellular enzymatic and non enzymatic scavengers of reactive oxygen species in the malignant tissue. Lipid peroxidation, a well established marker of oxidative stress, was found to increase, and substantial cellular DNA breakage was observed. We propose that calcitriol is a proxidant in the cellular milieu of hepatocellular carcinoma cells, and this copper mediated prooxidant action of calcitriol causes selective DNA breakage in malignant cells, while sparing normal (non malignant) cells.

  19. Calcitriol-copper interaction leads to non enzymatic, reactive oxygen species mediated DNA breakage and modulation of cellular redox scavengers in hepatocellular carcinoma.

    PubMed

    Rizvi, Asim; Farhan, Mohd; Naseem, Imrana; Hadi, S M

    2016-09-01

    Calcitriol is the metabolically active form of Vitamin D and is known to kill cancer cells. Using the rat model of DEN induced hepatocellular carcinoma we show that there is a marked increase in cellular levels of copper in hepatocellular carcinoma and that calcitriol-copper interaction leads to reactive oxygen species mediated DNA breakage selectively in hepatocellular carcinoma cells. In vivo studies show that calcitriol selectively induces severe fluctuations in cellular enzymatic and non enzymatic scavengers of reactive oxygen species in the malignant tissue. Lipid peroxidation, a well established marker of oxidative stress, was found to increase, and substantial cellular DNA breakage was observed. We propose that calcitriol is a proxidant in the cellular milieu of hepatocellular carcinoma cells, and this copper mediated prooxidant action of calcitriol causes selective DNA breakage in malignant cells, while sparing normal (non malignant) cells. PMID:27343126

  20. Protective Effect of Thymoquinone against Cyclophosphamide-Induced Hemorrhagic Cystitis through Inhibiting DNA Damage and Upregulation of Nrf2 Expression.

    PubMed

    Gore, Prashant R; Prajapati, Chaitali P; Mahajan, Umesh B; Goyal, Sameer N; Belemkar, Sateesh; Ojha, Shreesh; Patil, Chandragouda R

    2016-01-01

    Cyclophosphamide (CYP) induced hemorrhagic cystitis is a dose-limiting side effect involving increased oxidative stress, inflammatory cytokines and suppressed activity of nuclear factor related erythroid 2-related factor (Nrf2). Thymoquinone (TQ), an active constituent of Nigella sativa seeds, is reported to increase the expression of Nrf2, exert antioxidant action, and anti-inflammatory effects in the experimental animals. The present study was designed to explore the effects of TQ on CYP-induced hemorrhagic cystitis in Balb/c mice. Cystitis was induced by a single intraperitoneal injection of CYP (200 mg/kg). TQ was administered intraperitoneally at 5, 10 and 20 mg/kg doses twice a day, for three days before and three days after the CYP administration. The efficacy of TQ was determined in terms of the protection against the CYP-induced histological perturbations in the bladder tissue, reduction in the oxidative stress, and inhibition of the DNA fragmentation. Immunohistochemistry was performed to examine the expression of Nrf2. TQ protected against CYP-induced oxidative stress was evident from significant reduction in the lipid peroxidation, restoration of the levels of reduced glutathione, catalase and superoxide dismutase activities. TQ treatment significantly reduced the DNA damage evident as reduced DNA fragmentation. A significant decrease in the cellular infiltration, edema, epithelial denudation and hemorrhage were observed in the histological observations. There was restoration and rise in the Nrf2 expression in the bladder tissues of mice treated with TQ. These results confirm that, TQ ameliorates the CYP-induced hemorrhagic cystitis in mice through reduction in the oxidative stress, inhibition of the DNA damage and through increased expression of Nrf2 in the bladder tissues. PMID:27489498

  1. Protective Effect of Thymoquinone against Cyclophosphamide-Induced Hemorrhagic Cystitis through Inhibiting DNA Damage and Upregulation of Nrf2 Expression

    PubMed Central

    Gore, Prashant R.; Prajapati, Chaitali P.; Mahajan, Umesh B.; Goyal, Sameer N.; Belemkar, Sateesh; Ojha, Shreesh; Patil, Chandragouda R.

    2016-01-01

    Cyclophosphamide (CYP) induced hemorrhagic cystitis is a dose-limiting side effect involving increased oxidative stress, inflammatory cytokines and suppressed activity of nuclear factor related erythroid 2-related factor (Nrf2). Thymoquinone (TQ), an active constituent of Nigella sativa seeds, is reported to increase the expression of Nrf2, exert antioxidant action, and anti-inflammatory effects in the experimental animals. The present study was designed to explore the effects of TQ on CYP-induced hemorrhagic cystitis in Balb/c mice. Cystitis was induced by a single intraperitoneal injection of CYP (200 mg/kg). TQ was administered intraperitoneally at 5, 10 and 20 mg/kg doses twice a day, for three days before and three days after the CYP administration. The efficacy of TQ was determined in terms of the protection against the CYP-induced histological perturbations in the bladder tissue, reduction in the oxidative stress, and inhibition of the DNA fragmentation. Immunohistochemistry was performed to examine the expression of Nrf2. TQ protected against CYP-induced oxidative stress was evident from significant reduction in the lipid peroxidation, restoration of the levels of reduced glutathione, catalase and superoxide dismutase activities. TQ treatment significantly reduced the DNA damage evident as reduced DNA fragmentation. A significant decrease in the cellular infiltration, edema, epithelial denudation and hemorrhage were observed in the histological observations. There was restoration and rise in the Nrf2 expression in the bladder tissues of mice treated with TQ. These results confirm that, TQ ameliorates the CYP-induced hemorrhagic cystitis in mice through reduction in the oxidative stress, inhibition of the DNA damage and through increased expression of Nrf2 in the bladder tissues. PMID:27489498

  2. Taenia crassiceps cysticercosis: humoral immune response and protection elicited by DNA immunization.

    PubMed

    Rosas, G; Cruz-Revilla, C; Fragoso, G; López-Casillas, F; Pérez, A; Bonilla, M A; Rosales, R; Sciutto, E

    1998-06-01

    The purpose of this study was to evaluate DNA vaccination in cysticercosis prevention by using a Taenia crassiceps cDNA of a recombinant antigen (KETc7) that has been reported as protective against murine cysticercosis. The KETc7 cDNA was cloned into the pcDNA3 plasmid alone or with the betaglycan signal peptide sequence (pTc-7 and pTc-sp7, respectively). Positive expression of the pTc-sp7 product was confirmed by transfection of C33 cells and immunofluorescence using sera of mice infected with T. crassiceps. Immunization of mice with 3 injections of pTc-sp7 DNA at the higher dose (200 microg) was the most effective to induce antibody with or without bupivacaine. Immunization with pTc-sp7 induced protection against challenge with T. crassiceps cysticerci as successfully as previously observed with the KETc7 recombinant protein. Antibodies elicited by DNA immunization with pTc-sp7 specifically reacted with the native protein of 56 kDa previously reported, which is immunolocalized in the tegument of T. crassiceps cysticerci. The 56-kDa antigen is also present in Taenia solium oncospheres, cysticerci, and adult tissue. The protection induced in DNA-immunized mice and the observation that the injected plasmid remains as an episomic form within muscle cells, encouraged us to continue testing this procedure to prevent T. solium cysticercosis.

  3. A review on hemeoxygenase-2: focus on cellular protection and oxygen response.

    PubMed

    Muñoz-Sánchez, Jorge; Chánez-Cárdenas, María Elena

    2014-01-01

    Hemeoxygenase (HO) system is responsible for cellular heme degradation to biliverdin, iron, and carbon monoxide. Two isoforms have been reported to date. Homologous HO-1 and HO-2 are microsomal proteins with more than 45% residue identity, share a similar fold and catalyze the same reaction. However, important differences between isoforms also exist. HO-1 isoform has been extensively studied mainly by its ability to respond to cellular stresses such as hemin, nitric oxide donors, oxidative damage, hypoxia, hyperthermia, and heavy metals, between others. On the contrary, due to its apparently constitutive nature, HO-2 has been less studied. Nevertheless, its abundance in tissues such as testis, endothelial cells, and particularly in brain, has pointed the relevance of HO-2 function. HO-2 presents particular characteristics that made it a unique protein in the HO system. Since attractive results on HO-2 have been arisen in later years, we focused this review in the second isoform. We summarize information on gene description, protein structure, and catalytic activity of HO-2 and particular facts such as its cellular impact and activity regulation. Finally, we call attention on the role of HO-2 in oxygen sensing, discussing proposed hypothesis on heme binding motifs and redox/thiol switches that participate in oxygen sensing as well as evidences of HO-2 response to hypoxia. PMID:25136403

  4. Investigations of DNA damage induction and repair resulting from cellular exposure to high dose-rate pulsed proton beams

    NASA Astrophysics Data System (ADS)

    Renis, M.; Borghesi, M.; Favetta, M.; Malfa, G.; Manti, L.; Romano, F.; Schettino, G.; Tomasello, B.; Cirrone, G. A. P.

    2013-07-01

    Studies regarding the radiobiological effects of low dose radiation, microbeam irradiation services have been developed in the world and today laser acceleration of protons and heavy ions may be used in radiation therapy. The application of different facilities is essential for studying bystander effects and relating signalling phenomena in different cells or tissues. In particular the use of ion beams results advantageous in cancer radiotherapy compared to more commonly used X-rays, since the ability of ions in delivering lethal amount of doses into the target tumour avoiding or limiting damage to the contiguous healthy tissues. At the INFN-LNS in Catania, a multidisciplinary radiobiology group is strategically structured aimed to develop radiobiological research, finalised to therapeutic applications, compatible with the use of high dose laser-driven ion beams. The characteristic non-continuous dose rates with several orders of magnitude of laser-driven ion beams makes this facility very interesting in the cellular systems' response to ultra-high dose rates with non-conventional pulse time intervals cellular studies. Our group have projected to examine the effect of high dose laser-driven ion beams on two cellular types: foetal fibroblasts (normal control cells) and DU145 (prostate cancer cells), studying the modulation of some different bio-molecular parameters, in particular cell proliferation and viability, DNA damage, redox cellular status, morphological alterations of both the cytoskeleton components and some cell organelles and the possible presence of apoptotic or necrotic cell death. Our group performed preliminary experiments with high energy (60 MeV), dose rate of 10 Gy/min, doses of 1, 2, 3 Gy and LET 1 keV/μm on human foetal fibroblasts (control cells). We observed that cell viability was not influenced by the characteristics of the beam, the irradiation conditions or the analysis time. Conversely, DNA damage was present at time 0, immediately

  5. Investigations of DNA damage induction and repair resulting from cellular exposure to high dose-rate pulsed proton beams

    SciTech Connect

    Renis, M.; Malfa, G.; Tomasello, B.; Borghesi, M.; Schettino, G.; Favetta, M.; Romano, F.; Cirrone, G. A. P.; Manti, L.

    2013-07-26

    Studies regarding the radiobiological effects of low dose radiation, microbeam irradiation services have been developed in the world and today laser acceleration of protons and heavy ions may be used in radiation therapy. The application of different facilities is essential for studying bystander effects and relating signalling phenomena in different cells or tissues. In particular the use of ion beams results advantageous in cancer radiotherapy compared to more commonly used X-rays, since the ability of ions in delivering lethal amount of doses into the target tumour avoiding or limiting damage to the contiguous healthy tissues. At the INFN-LNS in Catania, a multidisciplinary radiobiology group is strategically structured aimed to develop radiobiological research, finalised to therapeutic applications, compatible with the use of high dose laser-driven ion beams. The characteristic non-continuous dose rates with several orders of magnitude of laser-driven ion beams makes this facility very interesting in the cellular systems' response to ultra-high dose rates with non-conventional pulse time intervals cellular studies. Our group have projected to examine the effect of high dose laser-driven ion beams on two cellular types: foetal fibroblasts (normal control cells) and DU145 (prostate cancer cells), studying the modulation of some different bio-molecular parameters, in particular cell proliferation and viability, DNA damage, redox cellular status, morphological alterations of both the cytoskeleton components and some cell organelles and the possible presence of apoptotic or necrotic cell death. Our group performed preliminary experiments with high energy (60 MeV), dose rate of 10 Gy/min, doses of 1, 2, 3 Gy and LET 1 keV/μm on human foetal fibroblasts (control cells). We observed that cell viability was not influenced by the characteristics of the beam, the irradiation conditions or the analysis time. Conversely, DNA damage was present at time 0, immediately

  6. Protection from radiation-induced mitochondrial and genomic DNA damage by an extract of Hippophae rhamnoides.

    PubMed

    Shukla, Sandeep Kumar; Chaudhary, Pankaj; Kumar, Indracanti Prem; Samanta, Namita; Afrin, Farhat; Gupta, Manju Lata; Sharma, Upendra Kumar; Sinha, Arun Kumar; Sharma, Yogendra Kumar; Sharma, Rakesh Kumar

    2006-12-01

    Hippophae rhamnoides or seabuckthorn is used extensively in Indian and Tibetan traditional medicine for the treatment of circulatory disorders, ischemic heart disease, hepatic injury, and neoplasia. In the present study, we have evaluated the radioprotective potential of REC-1001, a fraction isolated from the berries of H. rhamnoides. Chemical analysis of the extract indicated that REC-1001 was approximately 68% by weight polyphenols, and contained kaempferol, isorhamnetin, and quercetin. The effect of REC-1001 on modulating radiation-induced DNA damage was determined in murine thymocytes by measuring nonspecific nuclear DNA damage at the whole genome level using the alkaline halo assay and by measuring sequence/gene-specific DNA damage both in nuclear DNA (beta-globin gene) and in mitochondrial DNA using a quantitative polymerase chain reaction. Treatment with 10 Gy resulted in a significant amount of DNA damage in the halo assay and reductions in the amplification of both the beta-globin gene and mitochondrial DNA. REC-1001 dose-dependently reduced the amount of damage detected in each assay, with the maximum protective effects observed at the highest REC-1001 dose evaluated (250 micro g/ml). Studies measuring the nicking of naked plasmid DNA further established the radioprotective effect of REC-1001. To elucidate possible mechanisms of action, the antioxidant properties and the free-radical scavenging activities of REC-1001 were evaluated. REC-1001 dose-dependently scavenged radiation-induced hydroxyl radicals, chemically-generated superoxide anions, stabilized DPPH radicals, and reduced Fe(3+) to Fe(2+). The results of the study indicate that the REC-1001 extract of H. rhamnoides protects mitochondrial and genomic DNA from radiation-induced damage. The polyphenols/flavonoids present in the extract might be responsible for the free radical scavenging and DNA protection afforded by REC-1001. PMID:16948057

  7. Intranasal DNA Vaccine for Protection against Respiratory Infectious Diseases: The Delivery Perspectives

    PubMed Central

    Xu, Yingying; Yuen, Pak-Wai; Lam, Jenny Ka-Wing

    2014-01-01

    Intranasal delivery of DNA vaccines has become a popular research area recently. It offers some distinguished advantages over parenteral and other routes of vaccine administration. Nasal mucosa as site of vaccine administration can stimulate respiratory mucosal immunity by interacting with the nasopharyngeal-associated lymphoid tissues (NALT). Different kinds of DNA vaccines are investigated to provide protection against respiratory infectious diseases including tuberculosis, coronavirus, influenza and respiratory syncytial virus (RSV) etc. DNA vaccines have several attractive development potential, such as producing cross-protection towards different virus subtypes, enabling the possibility of mass manufacture in a relatively short time and a better safety profile. The biggest obstacle to DNA vaccines is low immunogenicity. One of the approaches to enhance the efficacy of DNA vaccine is to improve DNA delivery efficiency. This review provides insight on the development of intranasal DNA vaccine for respiratory infections, with special attention paid to the strategies to improve the delivery of DNA vaccines using non-viral delivery agents. PMID:25014738

  8. The protective effect of clay minerals against damage to adsorbed DNA induced by cadmium and mercury.

    PubMed

    Hou, Yakun; Wu, Pingxiao; Zhu, Nengwu

    2014-01-01

    The adsorption of Salmon Sperm DNA on three kinds of raw clay (rectorite, montmorillonite and sericite) was investigated as a function of pH, ionic strength and the concentrations of DNA and phosphate ions in solution. The DNA adsorption was reduced in the following order: rectorite>montmorillonite>sericite. Based on these findings, there is a strong evidence that the mechanisms for DNA adsorption on clay involve electrostatic forces, cation bridging and ligand exchange. Cyclic voltammetry (CV) and UV-vis absorption and fluorescence spectroscopy were used to compare the properties of unbound DNA and the absorbed DNA on rectorite, both in the absence and presence of Cd(2+) and Hg(2+) inaqueous solutions. The interaction of heavy metals with the unbound DNA was evidenced by the disappearance of reduction peaks in CV, a small bathochromic shift in UV-vis spectroscopy and an incomplete quenching in the emission spectra. Such changes were not observed in the DNA-rectorite hybrids, which is evidence that adsorption on the clay can reduce the extent of the DNA damage caused by heavy metals. Therefore, in these experience the rectorite played an important role in protecting DNA against Cd(2+) and Hg(2+) induced damage.

  9. Cellular and molecular mechanisms of endothelial ischemia/reperfusion injury: perspectives and implications for postischemic myocardial protection

    PubMed Central

    Yang, Qin; He, Guo-Wei; Underwood, Malcolm John; Yu, Cheuk-Man

    2016-01-01

    Ischemia/reperfusion (I/R) injury is a major cause of myocardial damage. Despite continuous efforts, minimizing I/R injury still represents a great challenge in standard medical treatments of ischemic heart disease, i.e., thrombolytic therapy, primary percutaneous coronary intervention, and coronary arterial bypass grafting. Development of effective interventions and strategies to prevent or reduce myocardial I/R injury is therefore of great clinical significance. Endothelial dysfunction plays a significant role in myocardial I/R injury, which renders endothelial cells an attractive target for postischemic myocardial protection. The rapidly evolving knowledge of the mechanisms of endothelial I/R injury helps broaden perspective for future development of novel strategies targeting endothelium for alleviating myocardial I/R damage. This review provides a comprehensive summary of the cellular and molecular mechanisms of endothelial I/R injury. Current perspectives and future directions for developing endothelium targeting therapeutics for postischemic myocardial protection are further discussed. PMID:27158368

  10. A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge

    NASA Astrophysics Data System (ADS)

    Deliyannis, Georgia; Boyle, Jefferey S.; Brady, Jamie L.; Brown, Lorena E.; Lew, Andrew M.

    2000-06-01

    Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs in vivo). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2-24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines.

  11. TRF2-Mediated Control of Telomere DNA Topology as a Mechanism for Chromosome-End Protection.

    PubMed

    Benarroch-Popivker, Delphine; Pisano, Sabrina; Mendez-Bermudez, Aaron; Lototska, Liudmyla; Kaur, Parminder; Bauwens, Serge; Djerbi, Nadir; Latrick, Chrysa M; Fraisier, Vincent; Pei, Bei; Gay, Alexandre; Jaune, Emilie; Foucher, Kevin; Cherfils-Vicini, Julien; Aeby, Eric; Miron, Simona; Londoño-Vallejo, Arturo; Ye, Jing; Le Du, Marie-Hélène; Wang, Hong; Gilson, Eric; Giraud-Panis, Marie-Josèphe

    2016-01-21

    The shelterin proteins protect telomeres against activation of the DNA damage checkpoints and recombinational repair. We show here that a dimer of the shelterin subunit TRF2 wraps ∼ 90 bp of DNA through several lysine and arginine residues localized around its homodimerization domain. The expression of a wrapping-deficient TRF2 mutant, named Top-less, alters telomeric DNA topology, decreases the number of terminal loops (t-loops), and triggers the ATM checkpoint, while still protecting telomeres against non-homologous end joining (NHEJ). In Top-less cells, the protection against NHEJ is alleviated if the expression of the TRF2-interacting protein RAP1 is reduced. We conclude that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ. The presence of RAP1 at telomeres appears as a backup mechanism to prevent NHEJ when topology-mediated telomere protection is impaired.

  12. Translocation of cellular prion protein to non-lipid rafts protects human prion-mediated neuronal damage.

    PubMed

    Jeong, Jae-Kyo; Moon, Myung-Hee; Lee, You-Jin; Seol, Jae-Won; Park, Sang-Youel

    2012-03-01

    Prions are the causative agents of transmissible spongiform encephalopathies, such as variant Creutzfeldt-Jakob disease in humans. Cellular prion proteins (PrPC) connect with cholesterol- and glycosphingolipid-rich lipid rafts through association of their glycosyl-phosphatidylinositol (GPI) anchor with saturated raft lipids and interaction of their N-terminal regions. Our previous study showed that cellular cholesterol enrichment prevented PrP(106-126)-induced neuronal death. We have now studied the influence of membrane cholesterol in PrP(106-126)-mediated neurotoxicity and identified membrane domains involved in this activity. We found that PrPC is normally distributed in lipid rafts, but high membrane cholesterol levels as a result of cholesterol treatment led to the translocation of PrPC from lipid rafts to non-lipid rafts. Moreover, cholesterol-mediated PrPC translocation protects PrP(106-126)-mediated apoptosis and p-38 activation and caspase-3 activation. In a mitochondrial functional assay including mitochondrial transmembrane potential, cholesterol treatment prevented the loss of mitochondrial potential, translocation of Bax and cytochrome c by prion protein fragment. Our results indicate that modulation of the PrPC location appears to protect against neuronal cell death caused by prion peptides. The results of this study suggest that regulation of membrane cholesterol affects the translocation of PrPC, which in turn regulates PrP(106-126)-induced mitochondrial dysfunction and neurotoxicity.

  13. Protection of DNA during oxidative stress and cytotoxic potential of Artemisia absinthium.

    PubMed

    Ali, Abid; Rahman, Khalilur; Jahan, Nazish; Jamil, Amer; Rashid, Abid; Shah, Syed Muhammad Ali

    2016-01-01

    Medicinal plants are rich in secondary metabolites (alkoloids, glycosides, coumarins, flavonides, steroids, etc.) and considered to be more effective and a safer alternative source to manage a variety of diseases related to liver, heart and kidney disordered. This study determines in vitro antioxidant and in vivo toxicological profile including hemolytic, brine shrimp lethality and mutagenicity of aerial parts of Artemisia absinthium. DNA protection assay was performed on pUC19 plasmid vector using H(2)O(2) as oxidative agent. Total phenolic and flavonoid content were determined using colorimetric methods. Toxicity of the plant was evaluated by brine shrimp lethality, hemolytic and mutagenic activity. DNA protection assay of the plant showed concentration dependent protective effect and at concentration 10μL/mL revealed complete protective effect against H(2)O(2) induced DNA damage. Highest phenolic and flavonoid content was found to be 167.3 (mg GAE 100g DW(-1)) and 14 (mg CE 100g DW(-1)) respectively. Results showed that A. absinthium is potent against standard toxicological procedures, that indicates the presence of bioactive components in the plant and possess antioxidant activity that protects DNA against H(2)O(2) induced oxidative damage. Thus the results showed/support that A. absinthium provides significant health benefits. PMID:27005506

  14. Sequence-selective DNA recognition and enhanced cellular up-take by peptide-steroid conjugates.

    PubMed

    Ruiz García, Yara; Iyer, Abhishek; Van Lysebetten, Dorien; Pabon, Y Vladimir; Louage, Benoit; Honcharenko, Malgorzata; De Geest, Bruno G; Smith, C I Edvard; Strömberg, Roger; Madder, Annemieke

    2015-12-25

    Several GCN4 bZIP TF models have previously been designed and synthesized. However, the synthetic routes towards these constructs are typically tedious and difficult. We here describe the substitution of the Leucine zipper domain of the protein by a deoxycholic acid derivative appending the two GCN4 binding region peptides through an optimized double azide-alkyne cycloaddition click reaction. In addition to achieving sequence specific dsDNA binding, we have investigated the potential of these compounds to enter cells. Confocal microscopy and flow cytometry show the beneficial influence of the steroid on cell uptake. This unique synthetic model of the bZIP TF thus combines sequence specific dsDNA binding properties with enhanced cell-uptake. Given the unique properties of deoxycholic acid and the convergent nature of the synthesis, we believe this work represents a key achievement in the field of TF mimicry.

  15. Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes.

    PubMed

    Kim, Hye Kyung

    2016-01-01

    Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1β and IL-6, senescence associated-β-galactosidase (SA-β-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1β) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-β-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes.

  16. Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes

    PubMed Central

    Kim, Hye Kyung

    2016-01-01

    Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1β and IL-6, senescence associated-β-galactosidase (SA-β-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1β) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-β-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes. PMID:27483310

  17. Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes.

    PubMed

    Kim, Hye Kyung

    2016-01-01

    Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1β and IL-6, senescence associated-β-galactosidase (SA-β-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1β) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-β-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes. PMID:27483310

  18. Protection of repetitive DNA borders from self-induced meiotic instability

    PubMed Central

    Vader, Gerben; Blitzblau, Hannah G.; Tame, Mihoko A.; Falk, Jill E.; Curtin, Lisa; Hochwagen, Andreas

    2011-01-01

    DNA double strand breaks (DSBs) in repetitive sequences are a potent source of genomic instability, due to the possibility of non-allelic homologous recombination (NAHR). Repetitive sequences are especially at risk during meiosis, when numerous programmed DSBs are introduced into the genome to initiate meiotic recombination 1. Within the budding yeast repetitive ribosomal (r)DNA array, meiotic DSB formation is prevented in part through Sir2-dependent heterochromatin 2,3. Here, we demonstrate that the edges of the rDNA array are exceptionally susceptible to meiotic DSBs, revealing an inherent heterogeneity within the rDNA array. We find that this localised DSB susceptibility necessitates a border-specific protection system consisting of the meiotic ATPase Pch2 and the origin recognition complex subunit Orc1. Upon disruption of these factors, DSB formation and recombination specifically increased in the outermost rDNA repeats, leading to NAHR and rDNA instability. Strikingly, the Sir2-dependent heterochromatin of the rDNA itself was responsible for the induction of DSBs at the rDNA borders in pch2Δ cells. Thus, while Sir2 activity globally prevents meiotic DSBs within the rDNA, it creates a highly permissive environment for DSB formation at the heterochromatin/euchromatin junctions. Heterochromatinised repetitive DNA arrays are abundantly present in most eukaryotic genomes. Our data define the borders of such chromatin domains as distinct high-risk regions for meiotic NAHR, whose protection may be a universal requirement to prevent meiotic genome rearrangements associated with genomic diseases and birth defects. PMID:21822291

  19. Plasmodium falciparum synthetic LbL microparticle vaccine elicits protective neutralizing antibody and parasite-specific cellular immune responses

    PubMed Central

    Powell, Thomas J.; Tang, Jie; DeRome, Mary E.; Mitchell, Robert A.; Jacobs, Andrea; Deng, Yanhong; Palath, Naveen; Cardenas, Edwin; Boyd, James G.; Nardin, Elizabeth

    2013-01-01

    Epitopes of the circumsporozoite (CS) protein of Plasmodium falciparum, the most pathogenic species of the malaria parasite, have been shown to elicit protective immunity in experimental animals and human volunteers. The mechanisms of immunity include parasite-neutralizing antibodies that can inhibit parasite motility in the skin at the site of infection and in the bloodstream during transit to the hepatocyte host cell and also block interaction with host cell receptors on hepatocytes. In addition, specific CD4+ and CD8+ cellular mechanisms target the intracellular hepatic forms, thus preventing release of erythrocytic stage parasites from the infected hepatocyte and the ensuing blood stage cycle responsible for clinical disease. An innovative method for producing particle vaccines, layer-by-layer (LbL) fabrication of polypeptide films on solid CaCO3 cores, was used to produce synthetic malaria vaccines containing a tri-epitope CS peptide T1BT* comprising the antibody epitope of the CS repeat region (B) and two T-cell epitopes, the highly conserved T1 epitope and the universal epitope T*. Mice immunized with microparticles loaded with T1BT* peptide developed parasite-neutralizing antibodies and malaria-specific T-cell responses including cytotoxic effector T-cells. Protection from liver stage infection following challenge with live sporozoites from infected mosquitoes correlated with neutralizing antibody levels. Although some immunized mice with low or undetectable neutralizing antibodies were also protected, depletion of T-cells prior to challenge resulted in the majority of mice remaining resistant to challenge. In addition, mice immunized with microparticles bearing only T-cell epitopes were not protected, demonstrating that cellular immunity alone was not sufficient for protective immunity. Although the microparticles without adjuvant were immunogenic and protective, a simple modification with the lipopeptide TLR2 agonist Pam3Cys increased the potency and

  20. Plasmodium falciparum synthetic LbL microparticle vaccine elicits protective neutralizing antibody and parasite-specific cellular immune responses.

    PubMed

    Powell, Thomas J; Tang, Jie; Derome, Mary E; Mitchell, Robert A; Jacobs, Andrea; Deng, Yanhong; Palath, Naveen; Cardenas, Edwin; Boyd, James G; Nardin, Elizabeth

    2013-04-01

    Epitopes of the circumsporozoite (CS) protein of Plasmodium falciparum, the most pathogenic species of the malaria parasite, have been shown to elicit protective immunity in experimental animals and human volunteers. The mechanisms of immunity include parasite-neutralizing antibodies that can inhibit parasite motility in the skin at the site of infection and in the bloodstream during transit to the hepatocyte host cell and also block interaction with host cell receptors on hepatocytes. In addition, specific CD4+ and CD8+ cellular mechanisms target the intracellular hepatic forms, thus preventing release of erythrocytic stage parasites from the infected hepatocyte and the ensuing blood stage cycle responsible for clinical disease. An innovative method for producing particle vaccines, layer-by-layer (LbL) fabrication of polypeptide films on solid CaCO3 cores, was used to produce synthetic malaria vaccines containing a tri-epitope CS peptide T1BT comprising the antibody epitope of the CS repeat region (B) and two T-cell epitopes, the highly conserved T1 epitope and the universal epitope T. Mice immunized with microparticles loaded with T1BT peptide developed parasite-neutralizing antibodies and malaria-specific T-cell responses including cytotoxic effector T-cells. Protection from liver stage infection following challenge with live sporozoites from infected mosquitoes correlated with neutralizing antibody levels. Although some immunized mice with low or undetectable neutralizing antibodies were also protected, depletion of T-cells prior to challenge resulted in the majority of mice remaining resistant to challenge. In addition, mice immunized with microparticles bearing only T-cell epitopes were not protected, demonstrating that cellular immunity alone was not sufficient for protective immunity. Although the microparticles without adjuvant were immunogenic and protective, a simple modification with the lipopeptide TLR2 agonist Pam3Cys increased the potency and

  1. 3D image copyright protection based on cellular automata transform and direct smart pixel mapping

    NASA Astrophysics Data System (ADS)

    Li, Xiao-Wei; Kim, Seok-Tae; Lee, In-Kwon

    2014-10-01

    We propose a three-dimensional (3D) watermarking system with the direct smart pixel mapping algorithm to improve the resolution of the reconstructed 3D watermark plane images. The depth-converted elemental image array (EIA) is obtained through the computational pixel mapping method. In the watermark embedding process, the depth-converted EIA is first scrambled by using the Arnold transform, which is then embedded in the middle frequency of the cellular automata (CA) transform. Compared with conventional computational integral imaging reconstruction (CIIR) methods, this proposed scheme gives us a higher resolution of the reconstructed 3D plane images by using the quality-enhanced depth-converted EIA. The proposed method, which can obtain many transform planes for embedding watermark data, uses CA transforms with various gateway values. To prove the effectiveness of the proposed method, we present the results of our preliminary experiments.

  2. Peptides mimicking GD2 ganglioside elicit cellular, humoral and tumor-protective immune responses in mice

    PubMed Central

    Wondimu, Assefa; Zhang, Tianqian; Kieber-Emmons, Thomas; Gimotty, Phyllis; Sproesser, Katrin; Somasundaram, Rajasekharan; Ferrone, Soldano; Tsao, Chun-Yen

    2012-01-01

    Introduction Because of its restricted distribution in normal tissues and its high expression on tumors of neuroectodermal origin, GD2 ganglioside is an excellent target for active specific immunotherapy. However, GD2 usually elicits low-titered IgM and no IgG or cellular immune responses, limiting its usefulness as a vaccine for cancer patients. We have previously shown that anti-idiotypic monoclonal antibody mimics of GD2 can induce antigen-specific humoral and cellular immunity in mice, but inhibition of tumor growth by the mimics could not be detected. Methods and results Here, we isolated two peptides from phage display peptide libraries by panning with GD2-specific mAb ME361. The peptides inhibited binding of the mAb to GD2. When coupled to keyhole limpet hemocyanin (KLH) or presented as multiantigenic peptides in QS21 adjuvant, the peptides induced in mice antibodies binding specifically to GD2 and delayed-type hypersensitive lymphocytes reactive specifically with GD2-positive D142.34 mouse melanoma cells. Induction of delayed-type hypersensitivity (DTH) reaction was dependent on CD4-positive lymphocytes. The immunity elicited by the peptides significantly inhibited growth of GD2-positive melanoma cells in mice. Conclusion Our study suggests that immunization with peptides mimicking GD2 ganglioside inhibits tumor growth through antibody and/or CD4-positive T cell-mediated mechanisms. Cytolytic T lymphocytes most likely do not play a role. Our results provide the basis for structural analysis of carbohydrate mimicry by peptides. PMID:18157673

  3. Protective effects of Commiphora erythraea resin constituents against cellular oxidative damage.

    PubMed

    Marcotullio, Maria Carla; Messina, Federica; Curini, Massimo; Macchiarulo, Antonio; Cellanetti, Marco; Ricci, Donata; Giamperi, Laura; Bucchini, Anahi; Minelli, Alba; Mierla, Anna Lisa; Bellezza, Ilaria

    2011-12-14

    By bioguided fractionation of the hexane extract of Commiphora erythraea resin we isolated four furanosesquiterpenoids that were tested for their protective activity against oxidative stress. Furanodienone and 1,10(15)-furanogermacra-dien-6-ones showed to be potent inhibitors of lipid peroxidation (IC(50) of -0.087 μM), being more active than the methoxylated analogues. Furthermore, using BV2 microglial cells, we found that furanodienone from C. erythraea is able to counteract LPS-induced cell death and decrease LPS-induced NO generation thus protecting microglial cells from LPS-induced cytotoxicity. Finally, docking studies were undertaken to gain insight into the possible binding mode of the isolated compounds at 5-LOX binding site.

  4. Effects of cellular differentiation, chromosomal integration and 5-aza-2'-deoxycytidine treatment on human papillomavirus-16 DNA methylation in cultured cell lines.

    PubMed

    Kalantari, Mina; Lee, Denis; Calleja-Macias, Itzel E; Lambert, Paul F; Bernard, Hans-Ulrich

    2008-05-10

    Human papillomavirus-16 (HPV-16) genomes in cell culture and in situ are affected by polymorphic methylation patterns, which can repress the viral transcription. In order to understand some of the underlying mechanisms, we investigated changes of the methylation of HPV-16 DNA in cell cultures in response to cellular differentiation, to recombination with cellular DNA, and to an inhibitor of methylation. Undifferentiated W12E cells, derived from a precancerous lesion, contained extrachromosomal HPV-16 DNA with a sporadically methylated enhancer-promoter segment. Upon W12E cell differentiation, the viral DNA was demethylated, suggesting a link between differentiation and the epigenetic state of HPV-16 DNA. The viral genomes present in two W12I clones, in which individual copies of the HPV-16 genome have integrated into cellular DNA (type 1 integrants), were unmethylated, akin to that seen in the cervical carcinoma cell line SiHa (also a type 1 integrant). This finding is consistent with hypomethylation being necessary for continued viral gene expression. In contrast, two of three type 2 integrant W12I clones, containing concatemers of HPV-16 genomes integrated into the cellular DNA contained hypermethylated viral DNA, as observed in the cervical carcinoma cell line CaSki (also a type 2 integrant). A third, type 2, W12I clone, interestingly with fewer copies of the viral genome, contained unmethylated HPV-16 genomes. Epithelial differentiation of W12I clones did not lead to demethylation of chromosomally integrated viral genomes as was seen for extrachromosomal HPV-16 DNA in W12E clones. Hypomethylation of CaSki cells in the presence of the DNA methylation inhibitor 5-aza-2'-deoxycytidine reduced the cellular viability, possibly as a consequence of toxic effects of an excess of HPV-16 gene products. Our data support a model wherein (i) the DNA methylation state of extrachromosomal HPV16 replicons and epithelial differentiation are inversely coupled during the viral

  5. DNA vaccine protects ornamental koi (Cyprinus carpio koi) against North American spring viremia of carp virus

    USGS Publications Warehouse

    Emmenegger, E.J.; Kurath, G.

    2008-01-01

    The emergence of spring viremia of carp virus (SVCV) in the United States constitutes a potentially serious alien pathogen threat to susceptible fish stocks in North America. A DNA vaccine with an SVCV glycoprotein (G) gene from a North American isolate was constructed. In order to test the vaccine a challenge model utilizing a specific pathogen-free domestic koi stock and a cold water stress treatment was also developed. We have conducted four trial studies demonstrating that the pSGnc DNA vaccine provided protection in vaccinated fish against challenge at low, moderate, and high virus doses of the homologous virus. The protection was significant (p < 0.05) as compared to fish receiving a mock vaccine construct containing a luciferase reporter gene and to non-vaccinated controls in fish ranging in age from 3 to 14 months. In all trials, the SVCV-G DNA immunized fish were challenged 28-days post-vaccination (546 degree-days) and experienced low mortalities varying from 10 to 50% with relative percent survivals ranging from 50 to 88%. The non-vaccinated controls and mock construct vaccinated fish encountered high cumulative percent mortalities ranging from 70 to 100%. This is the first report of a SVCV DNA vaccine being tested successfully in koi. These experiments prove that the SVCV DNA (pSGnc) vaccine can elicit specific reproducible protection and validates its potential use as a prophylactic vaccine in koi and other vulnerable North American fish stocks.

  6. Actin and DNA Protect Histones from Degradation by Bacterial Proteases but Inhibit Their Antimicrobial Activity

    PubMed Central

    Sol, Asaf; Skvirsky, Yaniv; Blotnick, Edna; Bachrach, Gilad; Muhlrad, Andras

    2016-01-01

    Histones are small polycationic proteins located in the cell nucleus. Together, DNA and histones are integral constituents of the nucleosomes. Upon apoptosis, necrosis, and infection – induced cell death, histones are released from the cell. The extracellular histones have strong antimicrobial activity but are also cytotoxic and thought as mediators of cell death in sepsis. The antimicrobial activity of the cationic extracellular histones is inhibited by the polyanionic DNA and F-actin, which also become extracellular upon cell death. DNA and F-actin protect histones from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis. However, though the integrity of the histones is protected, the activity of histones as antibacterial agents is lost. The inhibition of the histone’s antibacterial activity and their protection from proteolysis by DNA and F-actin indicate a tight electrostatic interaction between the positively charged histones and negatively charged DNA and F-actin, which may have physiological significance in maintaining the equilibrium between the beneficial antimicrobial activity of extracellular histones and their cytotoxic effects. PMID:27555840

  7. Actin and DNA Protect Histones from Degradation by Bacterial Proteases but Inhibit Their Antimicrobial Activity.

    PubMed

    Sol, Asaf; Skvirsky, Yaniv; Blotnick, Edna; Bachrach, Gilad; Muhlrad, Andras

    2016-01-01

    Histones are small polycationic proteins located in the cell nucleus. Together, DNA and histones are integral constituents of the nucleosomes. Upon apoptosis, necrosis, and infection - induced cell death, histones are released from the cell. The extracellular histones have strong antimicrobial activity but are also cytotoxic and thought as mediators of cell death in sepsis. The antimicrobial activity of the cationic extracellular histones is inhibited by the polyanionic DNA and F-actin, which also become extracellular upon cell death. DNA and F-actin protect histones from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis. However, though the integrity of the histones is protected, the activity of histones as antibacterial agents is lost. The inhibition of the histone's antibacterial activity and their protection from proteolysis by DNA and F-actin indicate a tight electrostatic interaction between the positively charged histones and negatively charged DNA and F-actin, which may have physiological significance in maintaining the equilibrium between the beneficial antimicrobial activity of extracellular histones and their cytotoxic effects. PMID:27555840

  8. Ru(ii)-polypyridyl surface functionalised gold nanoparticles as DNA targeting supramolecular structures and luminescent cellular imaging agents

    NASA Astrophysics Data System (ADS)

    Martínez-Calvo, Miguel; Orange, Kim N.; Elmes, Robert B. P.; La Cour Poulsen, Bjørn; Williams, D. Clive; Gunnlaugsson, Thorfinnur

    2015-12-01

    The development of Ru(ii) functionalized gold nanoparticles 1-3.AuNP is described. These systems were found to be mono-disperse with a hydrodynamic radius of ca. 15 nm in water but gave rise to the formation of higher order structures in buffered solution. The interaction of 1-3.AuNP with DNA was also studied by spectroscopic and microscopic methods and suggested the formation of large self-assembly structures in solution. The uptake of 1-3.AuNP by cancer cells was studied using both confocal fluorescence as well as transmission electron microscopy (TEM), with the aim of investigating their potential as tools for cellular biology. These systems displaying a non-toxic profile with favourable photophysical properties may have application across various biological fields including diagnostics and therapeutics.The development of Ru(ii) functionalized gold nanoparticles 1-3.AuNP is described. These systems were found to be mono-disperse with a hydrodynamic radius of ca. 15 nm in water but gave rise to the formation of higher order structures in buffered solution. The interaction of 1-3.AuNP with DNA was also studied by spectroscopic and microscopic methods and suggested the formation of large self-assembly structures in solution. The uptake of 1-3.AuNP by cancer cells was studied using both confocal fluorescence as well as transmission electron microscopy (TEM), with the aim of investigating their potential as tools for cellular biology. These systems displaying a non-toxic profile with favourable photophysical properties may have application across various biological fields including diagnostics and therapeutics. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05598a

  9. Spatiotemporal cellular imaging of polymer-pDNA nanocomplexes affords in situ morphology and trafficking trends.

    PubMed

    Ingle, Nilesh P; Xue, Lian; Reineke, Theresa M

    2013-11-01

    Synthetic polymers are ubiquitous in the development of drug and polynucleotide delivery vehicles, offering promise for personalized medicine. However, the polymer structure plays a central yet elusive role in dictating the efficacy, safety, mechanisms, and kinetics of therapeutic transport in a spatial and temporal manner. Here, we decipher the intracellular pathways pertaining to shape, size, location, and mechanism of four structurally divergent polymer vehicles (Tr455, Tr477, jetPEI, and Glycofect) that create colloidal nanoparticles (polyplexes) when complexed with fluorescently labeled plasmid DNA (pDNA). Multiple high resolution tomographic images of whole HeLa (human cervical adenocarcinoma) cells were captured via confocal microscopy at 4, 8, 12, and 24 h. The images were reconstructed to visualize and quantify trends in situ in a four-dimensional spatiotemporal manner. The data revealed heretofore-unseen images of polyplexes in situ and structure-function relationships, i.e., Glycofect polyplexes are trafficked as the smallest polyplex complexes and Tr455 polyplexes have expedited translocation to the perinuclear region. Also, all of the polyplex types appeared to be preferentially internalized and trafficked via early endosomes affiliated with caveolae, a Rab-5-dependent pathway, actin, and microtubules. PMID:24007201

  10. Mutations affecting sensitivity of the cellular slime mold Dictyostelium discoideum to DNA-damaging agents.

    PubMed

    Bronner, C E; Welker, D L; Deering, R A

    1992-09-01

    We describe 22 new mutants of D. discoideum that are sensitive to DNA damage. These mutants were isolated on the basis of sensitivity to either temperature, gamma-rays, or 4-nitroquinolone-1-oxide (4NQO). The doses of gamma-rays, ultraviolet light (UV), and 4NQO required to reduce the survival of colony-forming ability of these mutants to 10% (D10) range from 2% to 100% of the D10s for the nonmutant, parent strains. For most of the mutants, those which are very sensitive to one agent are very sensitive to all agents tested and those which are moderately sensitive to one agent, are moderately sensitive to all agents tested. One mutant is sensitive only to 4NQO. Linkage relationships have been examined for 13 of these mutants. This linkage information was used to design complementation tests to determine allelism with previously characterized complementation groups affecting sensitivity to radiation. 4 of the new mutants fall within previously identified complementation groups and 3 new complementation groups have been identified (radJ, radK and radL). Other new loci probably also exist among these new mutants. This brings the number of characterized mutants of D. discoideum which are sensitive to DNA-damaging agents to 33 and the number of assigned complementation groups to 11. PMID:1380652

  11. Manipulation of cellular DNA damage repair machinery facilitates propagation of human papillomaviruses.

    PubMed

    Wallace, Nicholas A; Galloway, Denise A

    2014-06-01

    In general, the interplay among viruses and DNA damage repair (DDR) pathways can be divided based on whether the interaction promotes or inhibits the viral lifecycle. The propagation of human papillomaviruses is both promoted and inhibited by DDR proteins. As a result, HPV proteins both activate repair pathways, such as the ATM and ATR pathways, and inhibit other pathways, most notably the p53 signaling pathway. Indeed, the role of HPV proteins, with regard to the DDR pathways, can be divided into two broad categories. The first set of viral proteins, HPV E1 and E2 activate a DNA damage response and recruit repair proteins to viral replication centers, where these proteins are likely usurped to replicate the viral genome. Because the activation of the DDR response typically elicits a cell cycle arrest that would impeded the viral lifecycle, the second set of HPV proteins, HPV E6 and E7, prevents the DDR response from pausing cell cycle progression or inducing apoptosis. This review provides a detailed account of the interactions among HPV proteins and DDR proteins that facilitate HPV propagation.

  12. Oxidative stress diverts tRNA synthetase to nucleus for protection against DNA damage.

    PubMed

    Wei, Na; Shi, Yi; Truong, Lan N; Fisch, Kathleen M; Xu, Tao; Gardiner, Elisabeth; Fu, Guangsen; Hsu, Yun-Shiuan Olivia; Kishi, Shuji; Su, Andrew I; Wu, Xiaohua; Yang, Xiang-Lei

    2014-10-23

    Tyrosyl-tRNA synthetase (TyrRS) is known for its essential aminoacylation function in protein synthesis. Here we report a function for TyrRS in DNA damage protection. We found that oxidative stress, which often downregulates protein synthesis, induces TyrRS to rapidly translocate from the cytosol to the nucleus. We also found that angiogenin mediates or potentiates this stress-induced translocalization. The nuclear-localized TyrRS activates transcription factor E2F1 to upregulate the expression of DNA damage repair genes such as BRCA1 and RAD51. The activation is achieved through direct interaction of TyrRS with TRIM28 to sequester this vertebrate-specific epigenetic repressor and its associated HDAC1 from deacetylating and suppressing E2F1. Remarkably, overexpression of TyrRS strongly protects against UV-induced DNA double-strand breaks in zebrafish, whereas restricting TyrRS nuclear entry completely abolishes the protection. Therefore, oxidative stress triggers an essential cytoplasmic enzyme used for protein synthesis to translocate to the nucleus to protect against DNA damage.

  13. Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells.

    PubMed

    Gu, Jijin; Hao, Junguo; Fang, Xiaoling; Sha, Xianyi

    2016-04-01

    Generally, the major obstacles for efficient gene delivery are cellular internalization and endosomal escape of nucleic acid such as plasmid DNA (pDNA) or small interfering RNA (siRNA). We previously developed Pluronic P123 modified polypropyleneimine (PPI)/pDNA (P123-PPI/pDNA) polyplexes as a gene delivery system. The results showed that P123-PPI/pDNA polyplexes revealed higher transfection efficiency than PPI/pDNA polyplexes in multidrug resistant breast cancer cells. As a continued effort, the present investigation on the factors influencing the transfection efficiency, cellular uptake mechanisms, and intracellular fate of P123-PPI/pDNA polyplexes is reported. The presence of P123 was the main factor influencing the transfection efficiency of P123-PPI/pDNA polyplexes in MCF-7/ADR cells, but other parameters, such as N/P ratio, FBS concentration, incubation time and temperature were important as well. The endocytic inhibitors against clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis were involved in the internalization to investigate their effects on the cellular uptake and transfection efficiency of P123-PPI/pDNA polyplexes in vitro. The data showed that the internalization of P123-PPI/pDNA polyplexes was obtained from both CME and CvME. Colocalization experiments with TRITC-transferrin (CME indicator), Alexa Fluor 555-CTB (CvME indicator), monoclonal anti-α-tubulin (microtubule indicator), and LysoTracker Green (Endosome/lysosome indicator) were carried out to confirm the internalization routes. The results showed that both CME and CvME played vital roles in the effective transfection of P123-PPI/pDNA polyplexes. Endosome/lysosome system and skeleton, including actin filament and microtubule, were necessary for the transportation after internalization. PMID:26741268

  14. Protective effects of novel metal-nonoates on the cellular components of the vascular system.

    PubMed

    Monti, Martina; Solito, Raffaella; Puccetti, Luca; Pasotti, Luca; Roggeri, Riccardo; Monzani, Enrico; Casella, Luigi; Morbidelli, Lucia

    2014-12-01

    At the cardiovascular level, nitric oxide (NO) controls smooth muscle functions, maintains vascular integrity, and exerts an antihypertensive effect. Metal-nonoates are a recently discovered class of NO donors, with NO release modulated through the complexation of the N-aminoethylpiperazine N-diazeniumdiolate ligand to metal ions, and thus representing a significant innovation with respect to the drugs traditionally used. In this study, we characterized the vascular protective effects of the most effective compound of this class, Ni(PipNONO)Cl, compared with the commercial N-diazeniumdiolate group derivate, diethylenetriamine/nitric oxide (DETA/NO). Ni(PipNONO)Cl induced a concentration-dependent relaxation of precontracted rat aortic rings. The ED50 was 0.67 µM, compared with 4.3 µM obtained with DETA/NO. When tested on cultured microvascular endothelial cells, Ni(PipNONO)Cl exerted a protective effect on the endothelium, promoting cell proliferation and survival in the picomolar range. The administration of Ni(PipNONO)Cl to vascular smooth muscle cells reduced the cell number, promoting their apoptosis at a high concentration (10 µM). Inhibition of smooth muscle cell migration, a hallmark of atherosclerosis, was accompanied by cytoskeletal rearrangement and loss of lamellipodia. When added to isolated platelets, Ni(PipNONO)Cl significantly reduced ADP-induced aggregation. Since atherosclerosis is accompanied by an inflammatory environment, cultured endothelial cells were exposed to interleukin (IL)-1β. In the presence of IL-1β, Ni(PipNONO)Cl inhibited cyclooxygenase-2 and inducible nitric oxide synthase upregulation, and reduced endothelial permeability and the platelet and monocyte adhesion markers CD31 and CD40 at the plasma membrane. Overall, these data indicate that Ni(PipNONO)Cl exerts vascular protective effects relevant for vascular dysfunction and prevention of atherosclerosis and thrombosis.

  15. Mcl-1 protects prostate cancer cells from cell death mediated by chemotherapy-induced DNA damage

    PubMed Central

    Reiner, Teresita; de las Pozas, Alicia; Parrondo, Ricardo; Palenzuela, Deanna; Cayuso, William; Rai, Priyamvada; Perez-Stable, Carlos

    2015-01-01

    The anti-apoptotic protein Mcl-1 is highly expressed in castration-resistant prostate cancer (CRPC), resulting in resistance to apoptosis and association with poor prognosis. Although predominantly localized in the cytoplasm, there is evidence that Mcl-1 exhibits nuclear localization where it is thought to protect against DNA damage-induced cell death. The role of Mcl-1 in mediating resistance to chemotherapy-induced DNA damage in prostate cancer (PCa) is not known. We show in human PCa cell lines and in TRAMP, a transgenic mouse model of PCa, that the combination of the antimitotic agent ENMD-1198 (analog of 2-methoxyestradiol) with betulinic acid (BA, increases proteotoxic stress) targets Mcl-1 by increasing its proteasomal degradation, resulting in increased γH2AX (DNA damage) and apoptotic/necrotic cell death. Knockdown of Mcl-1 in CRPC cells leads to elevated γH2AX, DNA strand breaks, and cell death after treatment with 1198 + BA- or doxorubicin. Additional knockdowns in PC3 cells suggests that cytoplasmic Mcl-1 protects against DNA damage by blocking the mitochondrial release of apoptosis-inducing factor and thereby preventing its nuclear translocation and subsequent interaction with the cyclophilin A endonuclease. Overall, our results suggest that chemotherapeutic agents that target Mcl-1 will promote cell death in response to DNA damage, particularly in CRPC. PMID:26425662

  16. A novel liquid multi-phytonutrient supplement demonstrates DNA-protective effects.

    PubMed

    Baechler, Benjamin J; Nita, Florina; Jones, Lon; Frestedt, Joy L

    2009-06-01

    This study explored the DNA protective (anti-mutagenic) effects of an oral, liquid, multi-phytonutrient dietary supplement containing a proprietary blend of fruits, vegetables and aloe vera concentrated components in addition to a proprietary catechin complex from green tea (VIBE Cardiac & Life, Eniva Nutraceuticals, Anoka, MN; herein described as "VIBE"). This study tested the hypothesis that VIBE would reduce DNA damage in skin cells exposed to UVR. Human epidermal cells, from the cell line A431NS, were treated with 0% (control), 0.125%, 0.5%, 1% and 2% VIBE, and then exposed to 240 J/m(2) UVR. The amount of DNA damage was assessed using the COMET assay. At each concentration tested, a significantly smaller amount of DNA damage was measured by the COMET assay for the VIBE treated cells compared to the control cells exposed to UVR without VIBE. The dose response curves showed a maximal response at 0.5% VIBE with a threefold reduction in COMET tail density compared to the control samples without VIBE (p < 0.001). Additional research is warranted in human clinical trials to further explore the results of this study which demonstrated the DNA protective and anti-mutagenic effects of VIBE for human skin cells exposed to UVR-induced DNA damage.

  17. The Apollo 5' exonuclease functions together with TRF2 to protect telomeres from DNA repair.

    PubMed

    Lenain, Christelle; Bauwens, Serge; Amiard, Simon; Brunori, Michele; Giraud-Panis, Marie-Josèphe; Gilson, Eric

    2006-07-11

    A major issue in telomere research is to understand how the integrity of chromosome ends is preserved . The human telomeric protein TRF2 coordinates several pathways that prevent checkpoint activation and chromosome fusions. In this work, we identified hSNM1B, here named Apollo, as a novel TRF2-interacting factor. Interestingly, the N-terminal domain of Apollo is closely related to that of Artemis, a factor involved in V(D)J recombination and DNA repair. Both proteins belong to the beta-CASP metallo-beta-lactamase family of DNA caretaker proteins. Apollo appears preferentially localized at telomeres in a TRF2-dependent manner. Reduced levels of Apollo exacerbate the sensitivity of cells to TRF2 inhibition, resulting in severe growth defects and an increased number of telomere-induced DNA-damage foci and telomere fusions. Purified Apollo protein exhibits a 5'-to-3' DNA exonuclease activity. We conclude that Apollo is a novel component of the human telomeric complex and works together with TRF2 to protect chromosome termini from being recognized and processed as DNA damage. These findings unveil a previously undescribed telomere-protection mechanism involving a DNA 5'-to-3' exonuclease.

  18. Protective Polymer Coatings for High-Throughput, High-Purity Cellular Isolation

    PubMed Central

    2016-01-01

    Cell-based therapies are emerging as the next frontier of medicine, offering a plausible path forward in the treatment of many devastating diseases. Critically, current methods for antigen positive cell sorting lack a high throughput method for delivering ultrahigh purity populations, prohibiting the application of some cell-based therapies to widespread diseases. Here we show the first use of targeted, protective polymer coatings on cells for the high speed enrichment of cells. Individual, antigen-positive cells are coated with a biocompatible hydrogel which protects the cells from a surfactant solution, while uncoated cells are immediately lysed. After lysis, the polymer coating is removed through orthogonal photochemistry, and the isolate has >50% yield of viable cells and these cells proliferate at rates comparable to control cells. Minority cell populations are enriched from erythrocyte-depleted blood to >99% purity, whereas the entire batch process requires 1 h and <$2000 in equipment. Batch scale-up is only contingent on irradiation area for the coating photopolymerization, as surfactant-based lysis can be easily achieved on any scale. PMID:26244409

  19. Protection of pigs against Taenia solium cysticercosis using recombinant antigen or in combination with DNA vaccine.

    PubMed

    Guo, Ying-Jun; Sun, Shu-Han; Zhang, Yi; Chen, Zhu-Huan; Wang, Kai-Yu; Huang, Li; Zhang, Shu; Zhang, Hong-Ying; Wang, Qing-Min; Wu, Dan; Zhu, Wei-Jia

    2004-09-28

    In the present study, we investigated the duration of protection afforded to pigs immunized in two different prime-boost regimens: one is homologus priming and boosting with a protein vaccine, and the other is priming with a DNA vaccine and boosting with the protein vaccine. Groups of pigs that received the same vaccination regimen were then challenged with Taenia solium eggs at 6, 12 or 20 weeks post-immunization (wpi), respectively. The results showed that all vaccinated pigs challenged at 6 or 12 wpi showed significant (P < 0.05) reduction in the development of cysts. When challenged at 20 wpi, pigs primed with the DNA vaccine (pcDNA3-cC1) followed by two boosters of the protein vaccine (GST-cC1) showed significant (P < 0.05) protection against the challenge of T. solium eggs, whereas pigs receiving three injections of the protein vaccine showed no significant protection compared to non-vaccinated controls (P > 0.05). Antibody isotype assays showed that DNA prime-protein boost regimen induced a predominantly IgG2 response, compared to an IgG1 biased response for the protein prime-protein boost regimen. In addition, peripheral blood mononuclear cells (PBMC) obtained from the DNA prime-protein boost group proliferated strongly in response to GST-cC1 protein, and this responsiveness persisted until 20 wpi. Taken together, our data suggest that the use of a prime-boost strategy combining DNA and protein vaccines may be better than protein alone for the longevity of protection against the challenge of T. solium eggs.

  20. The p53 network: Cellular and systemic DNA damage responses in aging and cancer

    PubMed Central

    Reinhardt, H. Christian; Schumacher, Björn

    2014-01-01

    Genome instability contributes to cancer development and accelerates age-related pathologies as evidenced by a variety of congenital cancer susceptibility and progeroid syndromes that are caused by defects in genome maintenance mechanisms. DNA damage response pathways that are mediated through the tumor suppressor p53 play an important role in the cell intrinsic responses to genome instability, including a transient cell cycle arrest, senescence and apoptosis. Both senescence and apoptosis are powerful tumor suppressive pathways preventing the uncontrolled proliferation of transformed cells. However, both pathways can potentially deplete stem and progenitor cell pools, thus promoting tissue degeneration and organ failure, which are both hallmarks of aging. p53 signaling is also involved in mediating non-cell autonomous interactions with the innate immune system and in the systemic adjustments during the aging process. The network of p53 target genes thus functions as an important regulator of cancer prevention and the physiology of aging. PMID:22265392

  1. Cellular Tug-of-War: Forces at Work and DNA Stretching in Mitosis

    NASA Astrophysics Data System (ADS)

    Griffin, Brian; Kilfoil, Maria L.

    2013-03-01

    In the microscopic world of the cell dominated by thermal noise, a cell must be able to successfully segregate its DNA with high fidelity in order to pass its genetic information on to its progeny. In this process of mitosis in eukaryotes, driving forces act on the cytoskeleton-based architecture called the mitotic spindle to promote this division. Our preliminary data demonstrates that the dynamics of this process in yeast cells is universal. Moreover, the dynamics suggest an increasing load as the chromosomes are pulled apart. To investigate this, we use three-dimensional imaging to track the dynamics of the poles of this architecture and the points of attachment to chromosomes simultaneously and with high spatial resolution. We analyze the relative motions of chromosomes as they are organized before segregation and as they are pulled apart, using this data to investigate the force-response behavior of this cytoskeleton-chromosome polymer system.

  2. Transcriptional activation and repression by cellular DNA-binding protein C/EBP.

    PubMed Central

    Pei, D Q; Shih, C H

    1990-01-01

    A putative transcription factor, C/EBP, isolated from rat liver nuclei, has been shown to bind to at least two different sequence motifs: the CCAAT promoter domain and a core sequence [GTGG(T/A)(T/A)(T/A)G] common to many viral enhancers, including simian virus 40 and human hepatitis B virus. It has been proposed that C/EBP might function as a positive transcription factor by facilitating the communication between promoter and enhancer elements through its dual binding activities to DNA. Surprisingly, results from three different approaches suggest that C/EBP functions as a transcriptional repressor to hepatitis B virus and simian virus 40. Further investigation indicated that C/EBP can function as both a transcriptional activator and a repressor, depending on the reporter gene system. Images PMID:2157040

  3. Anthracyclines Induce DNA Damage Response-Mediated Protection against Severe Sepsis

    PubMed Central

    Figueiredo, Nuno; Chora, Angelo; Raquel, Helena; Pejanovic, Nadja; Pereira, Pedro; Hartleben, Björn; Neves-Costa, Ana; Moita, Catarina; Pedroso, Dora; Pinto, Andreia; Marques, Sofia; Faridi, Hafeez; Costa, Paulo; Gozzelino, Raffaella; Zhao, Jimmy L.; Soares, Miguel P.; Gama-Carvalho, Margarida; Martinez, Jennifer; Zhang, Qingshuo; Döring, Gerd; Grompe, Markus; Simas, J. Pedro; Huber, Tobias B.; Baltimore, David; Gupta, Vineet; Green, Douglas R.; Ferreira, João A.; Moita, Luis F.

    2014-01-01

    Summary Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fancony Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis. PMID:24184056

  4. Amifostine (WR2721) Confers DNA Protection to In Vivo Cisplatin-Treated Murine Peripheral Blood Leukocytes

    PubMed Central

    Prieto González, E. A.; Fuchs, A. G.; Sánchez, González S.

    2009-01-01

    Amifostine [S-2-3-aminopropil amino ethyl phosphorotioic acid], a modulator agent for antineoplastic drugs involved in free radicals generation has given controversial results in cisplatin treated leukocytes in vitro. We have evaluated the amifostine protection over leukocytes in vivo, using comet assay. Groups of five OF1 male mice were given one of three doses of amifostine (56, 105 and 200 mg/Kg) after a cisplatin single injection (10 mg/Kg). Serum malonyldialdehide levels, catalase and superoxide dismutase activity were also evaluated. Amifostine showed significant DNA protection (p< 0.01) at the two lower doses evaluated. Malonyldildehide decreased in all amifostine treatments with respect to cisplatin while antioxidant enzyme activities remained unchanged. However, DNA migration increased with the highest amifostine dose; in fact highest dose of amifostine did no protect damage caused by cisplatin this result have implications on amifostine treatment schedules in clinical practice. PMID:19809542

  5. Detection of cellular DNA adducts in human fibroblasts treated with A-ring saturated and A-NCR-DMBA

    SciTech Connect

    Kumari, H.L.; Abood, N.; Goswami, S.P.; Bhat, H.B.; Milo, G.E.; Vitiak, D.T.

    1986-05-01

    Previous reports from these laboratories revealed that 1,2,3,4-tetrahydro 7,12-dimethylbenz(a)anthracene (TH-DMBA) and 6,11-dimethylcyclopentano(a)anthracene (CP-DMA) transform human fibroblasts to an abnormal phenotype. The parent PAH 7,12-DMBA does not transform these cells. Since 7,12-DMBA but not TH-DMBA or CP-DMA is anticipated to form active bay region diol epoxides, they investigated whether the A-ring analogues form adducts with cellular DNA, when the cells are treated in S phase of the cell cycle. Treatment for 10 hr was carried out when the cells exhibited S phase entry i.e. 10 hr after Gl release from a nutritionally deficient block. Adducts were isolated using a modification of the /sup 32/P-post-labeling technique. Preliminary results indicate that TH-DMBA formed two different nucleotide adducts where as there were three different adducts identified with CP-DMA. These results suggest the level of modification to be circa 1-2 adducts/10/sup 7/ total nucleotides for TH-DMBA and 2-4 adducts/10/sup 7/ total nucleotides for CP-DMA. The present evidence accumulated to date strongly suggests that alternate mechanisms exist to oxygenate the FAH to reactive intermediates that subsequently form specific DNA adducts.

  6. Investigating the cellular fate of a DNA-targeted platinum-based anticancer agent by orthogonal double-click chemistry.

    PubMed

    Qiao, Xin; Ding, Song; Liu, Fang; Kucera, Gregory L; Bierbach, Ulrich

    2014-03-01

    Confocal fluorescence microscopy was used to study a platinum-based anticancer agent in intact NCI-H460 lung cancer cells. Orthogonal copper-catalyzed azide-alkyne cycloaddition (click) reactions were used to simultaneously determine the cell-cycle-specific localization of the azide-functionalized platinum-acridine agent 1 and monitor its effects on nucleic acid metabolism. Copper-catalyzed postlabeling showed advantages over copper-free click chemistry using a dibenzocyclooctyne (DIBO)-modified reporter dye, which produced high background levels in microscopic images and failed to efficiently label platinum adducts in chromatin. Compound 1 was successfully labeled with the fluorophore DIBO to yield 1* (characterized by in-line high-performance liquid chromatography/electrospray mass spectrometry). 1 and 1* show a high degree of colocalization in the confocal images, but the ability of 1* to target the (compacted) chromatin was markedly reduced, most likely owing to the steric bulk introduced by the DIBO tag. Nuclear platinum levels correlated inversely with the ability of the cells to synthesize DNA and cause cell cycle arrest, as confirmed by bivariate flow cytometry analysis. In addition, a decrease in the level of cellular transcription, shrinkage of the nucleolar regions, and redistribution of RNA into the cytosol were observed. Postlabeling in conjunction with colocalization experiments is a useful tool for studying the cell killing mechanism of this type of DNA-targeted agent.

  7. DOE contractors' workshop: Cellular and molecular aspects of radiation induced DNA damage and repair

    SciTech Connect

    Not Available

    1987-01-01

    For four decades the US Department of Energy and its predecessors have been the lead federal agency in supporting radiation biology research. Over the years emphasis in this program has gradually shifted from dose-effect studies on animals to research on the effects of radiations of various qualities on cells and molecules. Mechanistic studies on the action of radiation at the subcellular level are few in number and there is a need for more research in this area if we are to gain a better understanding of how radiation affects living cells. The intent of this workshop was to bring together DOE contractors and grantees who are investigating the effects of radiation at the cellular and molecular levels. The aims were to foster the exchange of information on research projects and experimental results, promote collaborative research efforts, and obtain an overview of research currently supported by the Health Effects Research Division of the Office of Health and Environmental Research. The latter is needed by the Office for program planning purposes. This report on the workshop which took place in Albuquerque, New Mexico on March 10-11, 1987, includes an overview with future research recommendations, extended abstracts of the plenary presentations, shorter abstracts of each poster presentation, a workshop agenda and the names and addresses of the attendees.

  8. Testicular disorders induced by plant growth regulators: cellular protection with proanthocyanidins grape seeds extract.

    PubMed

    Hassan, Hanaa A; Isa, Ahmed M; El-Kholy, Wafaa M; Nour, Samar E

    2013-10-01

    The present study aims to investigate the adverse effects of plant growth regulators : gibberellic acid (GA3) and indoleacetic acid (IAA) on testicular functions in rats, and extends to investigate the possible protective role of grape seed extract, proanthocyanidin (PAC). Male rats were divided into six groups; control group, PAC, GA3, IAA, GA3 + PAC and IAA + PAC groups. The data showed that GA3 and IAA caused significant increase in total lipids, total cholesterol, triglycerides, phospholipids and low-density-lipoprotein cholesterol in the serum, concomitant with a significant decrease in high-density-lipoprotein cholesterol, total protein, and testosterone levels. In addition, there was significant decrease in the activity of alkaline phosphatase, acid phosphatase, and gamma-glutamyl transferase. A significant decrease was detected also in epididymyal fructose along with a significant reduction in sperm count. Testicular lipid peroxidation product and hydrogen peroxide (H2O2) levels were significantly increased. Meanwhile, the total antioxidant capacity, glutathione, sulphahydryl group content, as well as superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activity were significantly decreased. Moreover, there were a number of histopathological testicular changes including Leydig's cell degeneration, reduction in seminiferous tubule and necrotic symptoms and sperm degeneration in both GA3- and IAA-treated rats. However, an obvious recovery of all the above biochemical and histological testicular disorders was detected when PAC seed extract was supplemented to rats administered with GA3 or IAA indicating its protective effect. Therefore it was concluded that supplementation with PAC had ameliorative effects on those adverse effects of the mentioned plant growth regulators through its natural antioxidant properties.

  9. Testicular disorders induced by plant growth regulators: cellular protection with proanthocyanidins grape seeds extract.

    PubMed

    Hassan, Hanaa A; Isa, Ahmed M; El-Kholy, Wafaa M; Nour, Samar E

    2013-10-01

    The present study aims to investigate the adverse effects of plant growth regulators : gibberellic acid (GA3) and indoleacetic acid (IAA) on testicular functions in rats, and extends to investigate the possible protective role of grape seed extract, proanthocyanidin (PAC). Male rats were divided into six groups; control group, PAC, GA3, IAA, GA3 + PAC and IAA + PAC groups. The data showed that GA3 and IAA caused significant increase in total lipids, total cholesterol, triglycerides, phospholipids and low-density-lipoprotein cholesterol in the serum, concomitant with a significant decrease in high-density-lipoprotein cholesterol, total protein, and testosterone levels. In addition, there was significant decrease in the activity of alkaline phosphatase, acid phosphatase, and gamma-glutamyl transferase. A significant decrease was detected also in epididymyal fructose along with a significant reduction in sperm count. Testicular lipid peroxidation product and hydrogen peroxide (H2O2) levels were significantly increased. Meanwhile, the total antioxidant capacity, glutathione, sulphahydryl group content, as well as superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activity were significantly decreased. Moreover, there were a number of histopathological testicular changes including Leydig's cell degeneration, reduction in seminiferous tubule and necrotic symptoms and sperm degeneration in both GA3- and IAA-treated rats. However, an obvious recovery of all the above biochemical and histological testicular disorders was detected when PAC seed extract was supplemented to rats administered with GA3 or IAA indicating its protective effect. Therefore it was concluded that supplementation with PAC had ameliorative effects on those adverse effects of the mentioned plant growth regulators through its natural antioxidant properties. PMID:23292365

  10. Carotenoids exclusively synthesized in red pepper (capsanthin and capsorubin) protect human dermal fibroblasts against UVB induced DNA damage.

    PubMed

    Fernández-García, Elisabet; Carvajal-Lérida, Irene; Pérez-Gálvez, Antonio

    Photoprotection by dietary carotenoids has been linked to their antioxidant properties, in particular quenching of singlet molecular oxygen and scavenging of peroxyl radicals. Here, we compared the DNA-protection and antioxidant effects of selected carotenoids exclusively synthesized in red pepper (capsanthin and capsorubin) to the xanthophyll lutein. Preincubation of human dermal fibroblasts (hdf) with capsanthin and capsorubin significantly counteracted UVB induced cytotoxicity at doses between 0 and 300 mJ cm(-2). Pretreatment of hdf with capsanthin, capsorubin or lutein (1 μM) significantly decreased the formation of DNA strand breaks following irradiation with UVB light. All carotenoids studied decreased caspase-3 cleavage (a marker for UVB-induced apoptosis), however, caspase dependent PARP-1 cleavage was not affected suggesting that the remaining caspase activity is sufficient to promote UVB-induced apoptosis. It is conceivable that carotenoids selectively interfere with cellular responses activated by UVB-mediated damage. Our findings indicate that capsanthin and capsorubin exhibit similar properties to lutein and could be used as a dietary supplement to improve natural photoprotection. PMID:27537377

  11. An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping

    PubMed Central

    Gordon, Paul M. K.; Khan, Aneal; Sajid, Umair; Chang, Nicholas; Suresh, Varun; Dimnik, Leo; Lamont, Ryan E.; Parboosingh, Jillian S.; Martin, Steven R.; Pon, Richard T.; Weatherhead, Jene; Wegener, Shelly; Isaac, Debra; Greenway, Steven C.

    2016-01-01

    Cell-free DNA (cfDNA) has significant potential in the diagnosis and monitoring of clinical conditions. However, accurately and easily distinguishing the relative proportion of DNA molecules in a mixture derived from two different sources (i.e., donor and recipient tissues after transplantation) is challenging. In human cellular transplantation, there is currently no useable method to detect in vivo engraftment, and blood-based non-invasive tests for allograft rejection in solid organ transplantation are either non-specific or absent. Elevated levels of donor cfDNA have been shown to correlate with solid organ rejection, but complex methodology limits implementation of this promising biomarker. We describe a cost-effective method to quantify donor cfDNA in recipient plasma using a panel of high-frequency single nucleotide polymorphisms, next-generation (semiconductor) sequencing, and a novel mixture model algorithm. In vitro, our method accurately and rapidly determined donor:recipient DNA admixture. For in vivo testing, donor cfDNA was serially quantified in an infant with a urea cycle disorder after receiving six daily infusions of donor liver cells. Donor cfDNA isolated from 1 to 2 ml of recipient plasma was detected as late as 24 weeks after infusion suggesting engraftment. The percentage of circulating donor cfDNA was also assessed in pediatric and adult heart transplant recipients undergoing routine endomyocardial biopsy with levels observed to be stable over time and generally measuring <1% in cases without moderate or severe cellular rejection. Unlike existing non-invasive methods used to define the proportion of donor cfDNA in solid organ transplant patients, our assay does not require sex mismatch, donor genotyping, or whole-genome sequencing and potentially has broad application to detect cellular engraftment or allograft injury after transplantation. PMID:27713880

  12. Curcumin protects DNA damage in a chronically arsenic-exposed population of West Bengal.

    PubMed

    Biswas, Jaydip; Sinha, Dona; Mukherjee, Sutapa; Roy, Soumi; Siddiqi, Maqsood; Roy, Madhumita

    2010-06-01

    Groundwater arsenic contamination has been a health hazard for West Bengal, India. Oxidative stress to DNA is recognized as an underlying mechanism of arsenic carcinogenicity. A phytochemical, curcumin, from turmeric appears to be potent antioxidant and antimutagenic agent. DNA damage prevention with curcumin could be an effective strategy to combat arsenic toxicity. This field trial in Chakdah block of West Bengal evaluated the role of curcumin against the genotoxic effects of arsenic. DNA damage in human lymphocytes was assessed by comet assay and fluorescence-activated DNA unwinding assay. Curcumin was analyzed in blood by high performance liquid chromatography (HPLC). Arsenic induced oxidative stress and elucidation of the antagonistic role of curcumin was done by observation on reactive oxygen species (ROS) generation, lipid peroxidation and protein carbonyl. Antioxidant enzymes like catalase, superoxide dismutase, glutathione reductase, glutathioneS-transferase, glutathione peroxidase and non-enzymatic glutathione were also analyzed. The blood samples of the endemic regions showed severe DNA damage with increased levels of ROS and lipid peroxidation. The antioxidants were found with depleted activity. Three months curcumin intervention reduced the DNA damage, retarded ROS generation and lipid peroxidation and raised the level of antioxidant activity. Thus curcumin may have some protective role against the DNA damage caused by arsenic.

  13. Curcumin protects DNA damage in a chronically arsenic-exposed population of West Bengal.

    PubMed

    Biswas, Jaydip; Sinha, Dona; Mukherjee, Sutapa; Roy, Soumi; Siddiqi, Maqsood; Roy, Madhumita

    2010-06-01

    Groundwater arsenic contamination has been a health hazard for West Bengal, India. Oxidative stress to DNA is recognized as an underlying mechanism of arsenic carcinogenicity. A phytochemical, curcumin, from turmeric appears to be potent antioxidant and antimutagenic agent. DNA damage prevention with curcumin could be an effective strategy to combat arsenic toxicity. This field trial in Chakdah block of West Bengal evaluated the role of curcumin against the genotoxic effects of arsenic. DNA damage in human lymphocytes was assessed by comet assay and fluorescence-activated DNA unwinding assay. Curcumin was analyzed in blood by high performance liquid chromatography (HPLC). Arsenic induced oxidative stress and elucidation of the antagonistic role of curcumin was done by observation on reactive oxygen species (ROS) generation, lipid peroxidation and protein carbonyl. Antioxidant enzymes like catalase, superoxide dismutase, glutathione reductase, glutathioneS-transferase, glutathione peroxidase and non-enzymatic glutathione were also analyzed. The blood samples of the endemic regions showed severe DNA damage with increased levels of ROS and lipid peroxidation. The antioxidants were found with depleted activity. Three months curcumin intervention reduced the DNA damage, retarded ROS generation and lipid peroxidation and raised the level of antioxidant activity. Thus curcumin may have some protective role against the DNA damage caused by arsenic. PMID:20056736

  14. DNA methyltransferase inhibition increases efficacy of adoptive cellular immunotherapy of murine breast cancer.

    PubMed

    Terracina, Krista P; Graham, Laura J; Payne, Kyle K; Manjili, Masoud H; Baek, Annabel; Damle, Sheela R; Bear, Harry D

    2016-09-01

    Adoptive T cell immunotherapy is a promising approach to cancer treatment that currently has limited clinical applications. DNA methyltransferase inhibitors (DNAMTi) have known potential to affect the immune system through multiple mechanisms that could enhance the cytotoxic T cell responses, including: upregulation of tumor antigen expression, increased MHC class I expression, and blunting of myeloid derived suppressor cells (MDSCs) expansion. In this study, we have investigated the effect of combining the DNAMTi, decitabine, with adoptive T cell immunotherapy in the murine 4T1 mammary carcinoma model. We found that expression of neu, MHC class I molecules, and several murine cancer testis antigens (CTA) was increased by decitabine treatment of 4T1 cells in vitro. Decitabine also increased expression of multiple CTA in two human breast cancer cell lines. Decitabine-treated 4T1 cells stimulated greater IFN-gamma release from tumor-sensitized lymphocytes, implying increased immunogenicity. Expansion of CD11b + Gr1 + MDSC in 4T1 tumor-bearing mice was significantly diminished by decitabine treatment. Decitabine treatment improved the efficacy of adoptive T cell immunotherapy in mice with established 4T1 tumors, with greater inhibition of tumor growth and an increased cure rate. Decitabine may have a role in combination with existing and emerging immunotherapies for breast cancer. PMID:27416831

  15. Detection of cis- and trans-acting Factors in DNA Structure-Induced Genetic Instability Using In silico and Cellular Approaches

    PubMed Central

    Wang, Guliang; Zhao, Junhua; Vasquez, Karen M.

    2016-01-01

    Sequences that can adopt alternative DNA structures (i.e., non-B DNA) are very abundant in mammalian genomes, and recent studies have revealed many important biological functions of non-B DNA structures in chromatin remodeling, DNA replication, transcription, and genetic instability. Here, we provide results from an in silico web-based search engine coupled with cell-based experiments to characterize the roles of non-B DNA conformations in genetic instability in eukaryotes. The purpose of this article is to illustrate strategies that can be used to identify and interrogate the biological roles of non-B DNA structures, particularly on genetic instability. We have included unpublished data using a short H-DNA-forming sequence from the human c-MYC promoter region as an example, and identified two different mechanisms of H-DNA-induced genetic instability in yeast and mammalian cells: a DNA replication-related model of mutagenesis; and a replication-independent cleavage model. Further, we identified candidate proteins involved in H-DNA-induced genetic instability by using a yeast genetic screen. A combination of in silico and cellular methods, as described here, should provide further insight into the contributions of non-B DNA structures in biological functions, genetic evolution, and disease development. PMID:27532010

  16. Detection of cis- and trans-acting Factors in DNA Structure-Induced Genetic Instability Using In silico and Cellular Approaches.

    PubMed

    Wang, Guliang; Zhao, Junhua; Vasquez, Karen M

    2016-01-01

    Sequences that can adopt alternative DNA structures (i.e., non-B DNA) are very abundant in mammalian genomes, and recent studies have revealed many important biological functions of non-B DNA structures in chromatin remodeling, DNA replication, transcription, and genetic instability. Here, we provide results from an in silico web-based search engine coupled with cell-based experiments to characterize the roles of non-B DNA conformations in genetic instability in eukaryotes. The purpose of this article is to illustrate strategies that can be used to identify and interrogate the biological roles of non-B DNA structures, particularly on genetic instability. We have included unpublished data using a short H-DNA-forming sequence from the human c-MYC promoter region as an example, and identified two different mechanisms of H-DNA-induced genetic instability in yeast and mammalian cells: a DNA replication-related model of mutagenesis; and a replication-independent cleavage model. Further, we identified candidate proteins involved in H-DNA-induced genetic instability by using a yeast genetic screen. A combination of in silico and cellular methods, as described here, should provide further insight into the contributions of non-B DNA structures in biological functions, genetic evolution, and disease development. PMID:27532010

  17. Detection of cis- and trans-acting Factors in DNA Structure-Induced Genetic Instability Using In silico and Cellular Approaches.

    PubMed

    Wang, Guliang; Zhao, Junhua; Vasquez, Karen M

    2016-01-01

    Sequences that can adopt alternative DNA structures (i.e., non-B DNA) are very abundant in mammalian genomes, and recent studies have revealed many important biological functions of non-B DNA structures in chromatin remodeling, DNA replication, transcription, and genetic instability. Here, we provide results from an in silico web-based search engine coupled with cell-based experiments to characterize the roles of non-B DNA conformations in genetic instability in eukaryotes. The purpose of this article is to illustrate strategies that can be used to identify and interrogate the biological roles of non-B DNA structures, particularly on genetic instability. We have included unpublished data using a short H-DNA-forming sequence from the human c-MYC promoter region as an example, and identified two different mechanisms of H-DNA-induced genetic instability in yeast and mammalian cells: a DNA replication-related model of mutagenesis; and a replication-independent cleavage model. Further, we identified candidate proteins involved in H-DNA-induced genetic instability by using a yeast genetic screen. A combination of in silico and cellular methods, as described here, should provide further insight into the contributions of non-B DNA structures in biological functions, genetic evolution, and disease development.

  18. Rising Cellular Immune Response after Injection of pVax/iutA: A Genetic DNA Cassette as Candidate Vaccine against Urinary Tract Infection

    PubMed Central

    BAKHTIARI, Ronak; AHMADIAN, Shahin; FALLAH MEHRABADI, Jalil

    2016-01-01

    Background: Uropathogenic Escherichia coli (UPEC) are major bacterial agent of Urinary Tract Infection (UTI). This infection is more prevalent among women because approximately half of all women will experience a UTI in their life-time and near a quarter of them will have a recurrent infection within 6–12 months. IutA protein has a major role during UPEC pathogenesis and consequently infection. Therefore, the aim of current study was assessment of IutA protein roles as a potential candidate antigen based for vaccine designing. Methods: This survey was conducted during 2014–2015 at the University of Tehran, Iran. Chromosomal DNA extracted from E. coli 35218 and iutA gene amplified by PCR. The amplicon cloned to pVax.1 eukaryotic expression vector and recombinant vector confirmed by sequencing. The iutA gene expression in genetic cassette of pVax/iutA was evaluated in COS7 cell line by RT-PCR. Then, injected to mouse model, which divided to three groups: injected with pVax vector, PBS and pVax/iutA cassette respectively in two stages (d 1 and 14). One week after the second injection, bleeding from immunized mouse was performed and IFN-gamma was measured. Results: The mice immunized with pVax/iutA showed increased interferon-γ responses significantly higher than two non-immunized groups (P<0.05). Conclusion: Cellular immune response has a main protective role against UTI. Raising this kind of immune response is important to preventing of recurrent infection. Moreover, the current DNA cassette will be valuable for more trying to prepare a new vaccine against UTI. PMID:27516995

  19. Antioxidant, DNA protective efficacy and HPLC analysis of Annona muricata (soursop) extracts.

    PubMed

    George, V Cijo; Kumar, D R Naveen; Suresh, P K; Kumar, R Ashok

    2015-04-01

    Annona muricata is a naturally occurring edible plant with wide array of therapeutic potentials. In India, it has a long history of traditional use in treating various ailments. The present investigation was carried out to characterize the phytochemicals present in the methanolic and aqueous leaf extracts of A. muricata, followed by validation of its radical scavenging and DNA protection activities. The extracts were also analyzed for its total phenolic contents and subjected to HPLC analysis to determine its active metabolites. The radical scavenging activities were premeditated by various complementary assays (DRSA, FRAP and HRSA). Further, its DNA protection efficacy against H2O2 induced toxicity was evaluated using pBR322 plasmid DNA. The results revealed that the extracts were highly rich in various phytochemicals including luteolin, homoorientin, tangeretin, quercetin, daidzein, epicatechin gallate, emodin and coumaric acid. Both the extracts showed significant (p < 0.05) radical scavenging activities, while methanolic extract demonstrated improved protection against H2O2-induced DNA damage when compared to aqueous extract. A strong positive correlation was observed for the estimated total phenolic contents and radical scavenging potentials of the extracts. Further HPLC analysis of the phyto-constituents of the extracts provides a sound scientific basis for compound isolation.

  20. Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

    PubMed Central

    Sepúlveda, Dagoberto; Lorenzen, Niels

    2016-01-01

    DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms. PMID:27054895

  1. Can VHS Virus Bypass the Protective Immunity Induced by DNA Vaccination in Rainbow Trout?

    PubMed

    Sepúlveda, Dagoberto; Lorenzen, Niels

    2016-01-01

    DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms.

  2. Chronic administration of troxerutin protects mouse kidney against D-galactose-induced oxidative DNA damage.

    PubMed

    Liu, Chan-Min; Ma, Jie-Qiong; Lou, Yao

    2010-10-01

    Troxerutin, a natural bioflavonoid, has been reported to have many benefits and medicinal properties. In this study, we evaluated the protective effect of troxerutin against D-gal-induced oxidative DNA damage in mouse kidney, and explored the potential mechanism of its action. Our data showed that troxerutin significantly decreased levels of urea, uric acid and creatinine in serum and the renal histological injury in D-gal-treated mice. Troxerutin markedly restored Cu/Zn-SOD, CAT and GPx activities in the kidney of D-gal-treated mouse. Furthermore, the increase of 8-hydroxydeoxyguanosine (a marker of oxidative DNA damage) induced by d-gal was effectively suppressed by troxerutin. Internucleosomal DNA ladder fragmentation and the number of terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labeling (TUNEL)-positive cells in D-gal-treated mice were inhibited by troxerutin, which might be attributed to its antioxidant property by decreasing activities of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) and levels of reactive oxygen species (ROS). In conclusion, these results suggested that troxerutin could protect the mouse kidney against D-gal-induced injury by improving renal function, attenuating histopathologic changes, reducing ROS production, renewing the activities of antioxidant enzymes and decreasing DNA oxidative damage. This study provided novel insights into the protective mechanisms of troxerutin in D-gal-induced kidney injury.

  3. Protective Efficacy and Immunogenicity of a Combinatory DNA Vaccine against Influenza A Virus and the Respiratory Syncytial Virus

    PubMed Central

    Stab, Viktoria; Nitsche, Sandra; Niezold, Thomas; Storcksdieck genannt Bonsmann, Michael; Wiechers, Andrea; Tippler, Bettina; Hannaman, Drew; Ehrhardt, Christina; Überla, Klaus

    2013-01-01

    The Respiratory Syncytial Virus (RSV) and Influenza A Virus (IAV) are both two major causative agents of severe respiratory tract infections in humans leading to hospitalization and thousands of deaths each year. In this study, we evaluated the immunogenicity and efficacy of a combinatory DNA vaccine in comparison to the single component vaccines against both diseases in a mouse model. Intramuscular electroporation with plasmids expressing the hemagglutinin (HA) of IAV and the F protein of RSV induced strong humoral immune responses regardless if they were delivered in combination or alone. In consequence, high neutralizing antibody titers were detected, which conferred protection against a lethal challenge with IAV. Furthermore, the viral load in the lungs after a RSV infection could be dramatically reduced in vaccinated mice. Concurrently, substantial amounts of antigen-specific, polyfunctional CD8+ T-cells were measured after vaccination. Interestingly, the cellular response to the hemagglutinin was significantly reduced in the presence of the RSV-F encoding plasmid, but not vice versa. Although these results indicate a suppressive effect of the RSV-F protein, the protective efficacy of the combinatory vaccine was comparable to the efficacy of both single-component vaccines. In conclusion, the novel combinatory vaccine against RSV and IAV may have great potential to reduce the rate of severe respiratory tract infections in humans without increasing the number of necessary vaccinations. PMID:23967287

  4. The antioxidant activity of sulphurous thermal water protects against oxidative DNA damage: a comet assay investigation.

    PubMed

    Braga, P C; Ceci, C; Marabini, L; Nappi, G

    2013-04-01

    Various studies have recently shown that sulphurous waters acts against the oxidants released during respiratory bursts of human neutrophils, and free radicals such as HO•, O2¯•, Tempol and Fremy's salt. However, there is still a lack of data concerning their direct protection of DNA. The aim of this study was to investigate the antigenotoxicity effects of sulphurous water, which has never been previously investigated for this purpose, using the alkaline single cell gel electrophoresis (SCGE) approach (comet assay). The comet assay is a sensitive method for assessing DNA fragmentation in individual cells in genotoxicity studies but can also be used to investigate the activity of agents that protect against DNA damage. The extent of migration was measured by means of SCGE, and DNA damage was expressed as tail moment. All of these assays were made using natural sulphurous water, degassed sulphurous water (no detectable HS), and reconstituted sulphurous water (degassed plus NaHS). DNA damages was significantly inhibited by natural water with HS concentrations of 5.0 and 2.5 μg/mL. The use of degassed water did not lead to any significant differences from baseline values, whereas the reconstituted water led to significant results overlapping those obtained using natural water. These findings confirm the importance of the presence of an HS group (reductive activity) and indicate that, in addition to their known mucolytic activity and trophic effects on respiratory mucosa, HS groups in sulphurous water also protect against oxidative DNA damage and contribute to the water's therapeutic effects on upper and lower airway inflammatory diseases.

  5. Hydroxyl-radical-induced oxidation of 5-methylcytosine in isolated and cellular DNA

    PubMed Central

    Madugundu, Guru S.; Cadet, Jean; Wagner, J. Richard

    2014-01-01

    The methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the regulation of genes during cell differentiation, embryogenesis and carcinogenesis. Despite its low abundance, 5-methylcytosine (5mC) is a hotspot for mutations in mammalian cells. Here, we measured five oxidation products of 5mC together with the analogous products of cytosine and thymine in DNA exposed to ionizing radiation in oxygenated aqueous solution. The products can be divided into those that arise from hydroxyl radical (•OH) addition at the 5,6-double bond of 5mC (glycol, hydantoin and imidazolidine products) and those that arise from H-atom abstraction from the methyl group of 5mC including 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC). Based on the analysis of these products, we show that the total damage at 5mC is about 2-fold greater than that at C in identical sequences. The formation of hydantoin products of 5mC is favored, compared to analogous reactions of thymine and cytosine, which favor the formation of glycol products. The distribution of oxidation products is sequence dependent in specific ODN duplexes. In the case of 5mC, the formation of 5hmC and 5fC represents about half of the total of •OH-induced oxidation products of 5mC. Several products of thymine, cytosine, 5mC, as well as 8-oxo-7,8-dihydroguanine (8oxoG), were also estimated in irradiated cells. PMID:24852253

  6. Hydroxyl-radical-induced oxidation of 5-methylcytosine in isolated and cellular DNA.

    PubMed

    Madugundu, Guru S; Cadet, Jean; Wagner, J Richard

    2014-06-01

    The methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the regulation of genes during cell differentiation, embryogenesis and carcinogenesis. Despite its low abundance, 5-methylcytosine (5mC) is a hotspot for mutations in mammalian cells. Here, we measured five oxidation products of 5mC together with the analogous products of cytosine and thymine in DNA exposed to ionizing radiation in oxygenated aqueous solution. The products can be divided into those that arise from hydroxyl radical (•OH) addition at the 5,6-double bond of 5mC (glycol, hydantoin and imidazolidine products) and those that arise from H-atom abstraction from the methyl group of 5mC including 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC). Based on the analysis of these products, we show that the total damage at 5mC is about 2-fold greater than that at C in identical sequences. The formation of hydantoin products of 5mC is favored, compared to analogous reactions of thymine and cytosine, which favor the formation of glycol products. The distribution of oxidation products is sequence dependent in specific ODN duplexes. In the case of 5mC, the formation of 5hmC and 5fC represents about half of the total of •OH-induced oxidation products of 5mC. Several products of thymine, cytosine, 5mC, as well as 8-oxo-7,8-dihydroguanine (8oxoG), were also estimated in irradiated cells.

  7. Oxidation of guanine in cellular DNA by solar UV radiation: biological role.

    PubMed

    Douki, T; Perdiz, D; Gróf, P; Kuluncsics, Z; Moustacchi, E; Cadet, J; Sage, E

    1999-08-01

    The formation of cyclobutane pyrimidine dimers (CPD) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) was investigated in Chinese hamster ovary cells upon exposure to either UVC, UVB, UVA or simulated sunlight (SSL). Two cell lines were used, namely AT3-2 and UVL9, the latter being deficient in nucleotide excision repair and consequently UV sensitive. For all types of radiation, including UVA, CPD were found to be the predominant lesions quantitatively. At the biologically relevant doses used, UVC, UVB and SSL irradiation yielded 8-oxodGuo at a rather low level, whereas UVA radiation produced relatively higher amounts. The formation of CPD was 10(2) and 10(5) more effective upon UVC than UVB and UVA exposure. These yields of formation followed DNA absorption, even in the UVA range. The calculated relative spectral effectiveness in the production of the two lesions showed that efficient induction of 8-oxodGuo upon UVA irradiation was shifted toward longer wavelengths, in comparison with those for CPD formation, in agreement with a photosensitization mechanism. In addition, after exposure to SSL, about 19% and 20% of 8-oxodGuo were produced between 290-320 nm and 320-340 nm, respectively, whereas CPD were essentially (90%) induced in the UVB region. However, the ratio of CPD to 8-oxodGuo greatly differed from one source of light to the other: it was over 100 for UVB but only a few units for UVA source. The extent of 8-oxodGuo and CPD was also compared to the lethality for the different types of radiation. The involvement of 8-oxodGuo in cell killing by solar UV radiation was clearly ruled out. In addition, our previously reported mutation spectra demonstrated that the contribution of 8-oxodGuo in the overall solar UV mutagenic process is very minor.

  8. Protection of rainbow trout against infectious hematopoietic necrosis virus four days after specific or semi-specific DNA vaccination

    USGS Publications Warehouse

    LaPatra, S.E.; Corbeil, S.; Jones, G.R.; Shewmaker, W.D.; Lorenzen, N.; Anderson, E.D.; Kurath, G.

    2001-01-01

    A DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was shown to provide significant protection as soon as 4 d after intramuscular vaccination in 2 g rainbow trout (Oncorhynchus mykiss) held at 15??C. Nearly complete protection was also observed at later time points (7, 14, and 28 d) using a standardized waterborne challenge model. In a test of the specificity of this early protection, immunization of rainbow trout with a DNA vaccine against another fish rhabdovirus, viral hemorrhagic septicemia virus, provided a significant level of cross-protection against IHNV challenge for a transient period of time, whereas a rabies virus DNA vaccine was not protective. This indication of distinct early and late protective mechanisms was not dependent on DNA vaccine doses from 0.1 to 2.5 ??g. ?? 2001 Elsevier Science Ltd.

  9. Chemokine Adjuvanted Electroporated-DNA Vaccine Induces Substantial Protection from Simian Immunodeficiency Virus Vaginal Challenge

    PubMed Central

    Hutnick, N A; Moldoveanu, Z; Hunter, M; Reuter, M; Yuan, S; Yan, J; Ginsberg, A; Sylvester, A; Pahar, B; Carnathan, D; Kathuria, N; Khan, A S; Montefiori, D; Sardesai, N Y; Betts, M R; Mestecky, J; Marx, P; Weiner, D B

    2015-01-01

    There have been encouraging results for the development of an effective HIV vaccine. However, many questions remain regarding the quality of immune responses and the role of mucosal antibodies. We addressed some of these issues by using a simian immunodeficiency virus (SIV) DNA vaccine adjuvanted with plasmid-expressed mucosal chemokines combined with an intravaginal SIV challenge in rhesus macaque (RhM) model. We previously reported on the ability of CCR9 and CCR10 ligand (L) adjuvants to enhance mucosal and systemic IgA and IgG in small animals. In this study, RhMs were intramuscularly immunized five times with either DNA or DNA plus chemokine adjuvant delivered by electroporation followed by challenge with SIVsmE660. Sixty-eight percent of all vaccinated animals (P=0.0016) remained either uninfected or had aborted infection compared to only 14% in the vaccine naïve group. The highest protection was observed in the CCR10L chemokines group, where 6 of 9 animals had aborted infection and two remained uninfected, leading to 89% protection (P=0.0003). The induction of mucosal SIV-specific antibodies and neutralization titers correlated with trends in protection. These results indicate the need to further investigate the contribution of chemokine adjuvants to modulate immune responses and the role of mucosal antibodies in SIV/HIV protection. PMID:25943275

  10. Role of Mitochondria in Cerebral Vascular Function: Energy Production, Cellular Protection, and Regulation of Vascular Tone.

    PubMed

    Busija, David W; Rutkai, Ibolya; Dutta, Somhrita; Katakam, Prasad V

    2016-06-13

    Mitochondria not only produce energy in the form of ATP to support the activities of cells comprising the neurovascular unit, but mitochondrial events, such as depolarization and/or ROS release, also initiate signaling events which protect the endothelium and neurons against lethal stresses via pre-/postconditioning as well as promote changes in cerebral vascular tone. Mitochondrial depolarization in vascular smooth muscle (VSM), via pharmacological activation of the ATP-dependent potassium channels on the inner mitochondrial membrane (mitoKATP channels), leads to vasorelaxation through generation of calcium sparks by the sarcoplasmic reticulum and subsequent downstream signaling mechanisms. Increased release of ROS by mitochondria has similar effects. Relaxation of VSM can also be indirectly achieved via actions of nitric oxide (NO) and other vasoactive agents produced by endothelium, perivascular and parenchymal nerves, and astroglia following mitochondrial activation. Additionally, NO production following mitochondrial activation is involved in neuronal preconditioning. Cerebral arteries from female rats have greater mitochondrial mass and respiration and enhanced cerebral arterial dilation to mitochondrial activators. Preexisting chronic conditions such as insulin resistance and/or diabetes impair mitoKATP channel relaxation of cerebral arteries and preconditioning. Surprisingly, mitoKATP channel function after transient ischemia appears to be retained in the endothelium of large cerebral arteries despite generalized cerebral vascular dysfunction. Thus, mitochondrial mechanisms may represent the elusive signaling link between metabolic rate and blood flow as well as mediators of vascular change according to physiological status. Mitochondrial mechanisms are an important, but underutilized target for improving vascular function and decreasing brain injury in stroke patients. © 2016 American Physiological Society. Compr Physiol 6:1529-1548, 2016.

  11. Role of Mitochondria in Cerebral Vascular Function: Energy Production, Cellular Protection, and Regulation of Vascular Tone.

    PubMed

    Busija, David W; Rutkai, Ibolya; Dutta, Somhrita; Katakam, Prasad V

    2016-01-01

    Mitochondria not only produce energy in the form of ATP to support the activities of cells comprising the neurovascular unit, but mitochondrial events, such as depolarization and/or ROS release, also initiate signaling events which protect the endothelium and neurons against lethal stresses via pre-/postconditioning as well as promote changes in cerebral vascular tone. Mitochondrial depolarization in vascular smooth muscle (VSM), via pharmacological activation of the ATP-dependent potassium channels on the inner mitochondrial membrane (mitoKATP channels), leads to vasorelaxation through generation of calcium sparks by the sarcoplasmic reticulum and subsequent downstream signaling mechanisms. Increased release of ROS by mitochondria has similar effects. Relaxation of VSM can also be indirectly achieved via actions of nitric oxide (NO) and other vasoactive agents produced by endothelium, perivascular and parenchymal nerves, and astroglia following mitochondrial activation. Additionally, NO production following mitochondrial activation is involved in neuronal preconditioning. Cerebral arteries from female rats have greater mitochondrial mass and respiration and enhanced cerebral arterial dilation to mitochondrial activators. Preexisting chronic conditions such as insulin resistance and/or diabetes impair mitoKATP channel relaxation of cerebral arteries and preconditioning. Surprisingly, mitoKATP channel function after transient ischemia appears to be retained in the endothelium of large cerebral arteries despite generalized cerebral vascular dysfunction. Thus, mitochondrial mechanisms may represent the elusive signaling link between metabolic rate and blood flow as well as mediators of vascular change according to physiological status. Mitochondrial mechanisms are an important, but underutilized target for improving vascular function and decreasing brain injury in stroke patients. © 2016 American Physiological Society. Compr Physiol 6:1529-1548, 2016. PMID:27347901

  12. Grape seeds proanthocyanidin extract as a hepatic-reno-protective agent against gibberellic acid induced oxidative stress and cellular alterations.

    PubMed

    Hassan, Hanaa A; Al-Rawi, Maisaa M

    2013-08-01

    The present study aims to investigate the heptonephro-protective effect of grape seeds proanthocyanidin extract (GSPE) against the risks induced by gibberellic acid (GA3) in male rats. The results recorded that GA3 caused a significant increase in total lipids, total cholesterol, triglycerides and LDL-C levels in serum, concomitant with a significant decrease in serum HDL-C. A significant increase in serum AST, ALT, urea and creatinine, while, a significant decrease in total protein content in serum was observed in rats given GA3. Hepatic and renal lipid peroxidation product (MDA) was significantly increased, meanwhile, total antioxidant capacity (TAC), glutathione, and catalase levels were significantly decreased. In addition, there was a negative change in liver structure including dilatation in the central veins with degeneration of endothelium cells and cellular injury around the veins as well as in the kidney structure such as lesion in both glomeruli and tubules, detachment of the Malpighian corpuscles from the Bowman's capsule's epithelium, shrinkage in the glomerular capillary network. However, almost all of these adverse effects seemed to be ameliorated by oral administration of GSPE with GA3 to rats for 2 month indicating the protective effect of grape seeds GSPE on GA3 induced oxidative stress in rats. PMID:23135702

  13. Vaccination with Klebsiella pneumoniae-derived extracellular vesicles protects against bacteria-induced lethality via both humoral and cellular immunity.

    PubMed

    Lee, Won-Hee; Choi, Hyun-Il; Hong, Sung-Wook; Kim, Kwang-Sun; Gho, Yong Song; Jeon, Seong Gyu

    2015-01-01

    The emergence of multidrug-resistant Klebsiella pneumoniae highlights the need to develop preventive measures to ameliorate Klebsiella infections. Bacteria-derived extracellular vesicles (EVs) are spherical nanometer-sized proteolipids enriched with outer membrane proteins. Gram-negative bacteria-derived EVs have gained interest for use as nonliving complex vaccines. In the present study, we evaluated whether K. pneumoniae-derived EVs confer protection against bacteria-induced lethality. K. pneumoniae-derived EVs isolated from in vitro bacterial culture supernatants induced innate immunity, including the upregulation of co-stimulatory molecule expression and proinflammatory mediator production. EV vaccination via the intraperitoneal route elicited EV-reactive antibodies and interferon-gamma-producing T-cell responses. Three vaccinations with the EVs prevented bacteria-induced lethality. As verified by sera and splenocytes adoptive transfer, the protective effect of EV vaccination was dependent on both humoral and cellular immunity. Taken together, these findings suggest that K. pneumoniae-derived EVs are a novel vaccine candidate against K. pneumoniae infections. PMID:26358222

  14. Vaccination with Klebsiella pneumoniae-derived extracellular vesicles protects against bacteria-induced lethality via both humoral and cellular immunity

    PubMed Central

    Lee, Won-Hee; Choi, Hyun-Il; Hong, Sung-Wook; Kim, Kwang-sun; Gho, Yong Song; Jeon, Seong Gyu

    2015-01-01

    The emergence of multidrug-resistant Klebsiella pneumoniae highlights the need to develop preventive measures to ameliorate Klebsiella infections. Bacteria-derived extracellular vesicles (EVs) are spherical nanometer-sized proteolipids enriched with outer membrane proteins. Gram-negative bacteria-derived EVs have gained interest for use as nonliving complex vaccines. In the present study, we evaluated whether K. pneumoniae-derived EVs confer protection against bacteria-induced lethality. K. pneumoniae-derived EVs isolated from in vitro bacterial culture supernatants induced innate immunity, including the upregulation of co-stimulatory molecule expression and proinflammatory mediator production. EV vaccination via the intraperitoneal route elicited EV-reactive antibodies and interferon-gamma-producing T-cell responses. Three vaccinations with the EVs prevented bacteria-induced lethality. As verified by sera and splenocytes adoptive transfer, the protective effect of EV vaccination was dependent on both humoral and cellular immunity. Taken together, these findings suggest that K. pneumoniae-derived EVs are a novel vaccine candidate against K. pneumoniae infections. PMID:26358222

  15. Photoinduced conformational changes in DNA by poly(vinyl alcohol) carrying a malachite green moiety for protecting DNA against attack by nuclease.

    PubMed

    Uda, Ryoko M; Matsui, Takashi

    2015-11-14

    Light is a highly advantageous means of specific cell targeting. Though targeted gene delivery is an important characteristic of an ideal delivery vehicle, there has been little effort to develop a photoresponsive vector. Among nonviral vectors, cationic substances interact effectively with negatively charged DNA. With this property in mind, we designed copolymers of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG) with different molecular weights. Though PVAMG has no affinity for DNA in the absence of light, it undergoes photoionization in the presence of light to afford cationic DNA binding sites. The DNA-PVAMG complex was investigated with respect to DNA conformational changes and its protective nature, which are important properties for nonviral vectors. PVAMG irradiation promoted DNA conformational transitions from coils to partial globules to compacted globules. The complex had a protective effect against DNase I after PVAMG irradiation, while DNA was degraded under dark conditions. The effect on DNA transition and the protective nature were sensitive to the molecular weight of PVAMG. The data regarding binding constants and binding mode provided insight into the structure of the DNA-PVAMG complex. To withstand DNase I attacks, complexation results in the compaction of DNA, which is further covered with PVAMG.

  16. Photoinduced conformational changes in DNA by poly(vinyl alcohol) carrying a malachite green moiety for protecting DNA against attack by nuclease.

    PubMed

    Uda, Ryoko M; Matsui, Takashi

    2015-11-14

    Light is a highly advantageous means of specific cell targeting. Though targeted gene delivery is an important characteristic of an ideal delivery vehicle, there has been little effort to develop a photoresponsive vector. Among nonviral vectors, cationic substances interact effectively with negatively charged DNA. With this property in mind, we designed copolymers of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG) with different molecular weights. Though PVAMG has no affinity for DNA in the absence of light, it undergoes photoionization in the presence of light to afford cationic DNA binding sites. The DNA-PVAMG complex was investigated with respect to DNA conformational changes and its protective nature, which are important properties for nonviral vectors. PVAMG irradiation promoted DNA conformational transitions from coils to partial globules to compacted globules. The complex had a protective effect against DNase I after PVAMG irradiation, while DNA was degraded under dark conditions. The effect on DNA transition and the protective nature were sensitive to the molecular weight of PVAMG. The data regarding binding constants and binding mode provided insight into the structure of the DNA-PVAMG complex. To withstand DNase I attacks, complexation results in the compaction of DNA, which is further covered with PVAMG. PMID:26339777

  17. HTLV-I Tax-Mediated Inactivation of Cell Cycle Checkpoints and DNA Repair Pathways Contribute to Cellular Transformation: “A Random Mutagenesis Model”

    PubMed Central

    Nicot, Christophe

    2015-01-01

    To achieve cellular transformation, most oncogenic retroviruses use transduction by proto-oncogene capture or insertional mutagenesis, whereby provirus integration disrupts expression of tumor suppressors or proto-oncogenes. In contrast, the Human T-cell leukemia virus type 1 (HTLV-I) has been classified in a separate class referred to as “transactivating retroviruses”. Current views suggest that the viral encoded Tax protein transactivates expression of cellular genes leading to deregulated growth and transformation. However, if Tax-mediated transactivation was indeed sufficient for cellular transformation, a fairly high frequency of infected cells would eventually become transformed. In contrast, the frequency of transformation by HTLV-I is very low, likely less than 5%. This review will discuss the current understanding and recent discoveries highlighting critical functions of Tax in cellular transformation. HTLV-I Tax carries out essential functions in order to override cell cycle checkpoints and deregulate cellular division. In addition, Tax expression is associated with increased DNA damage and genome instability. Since Tax can inhibit multiple DNA repair pathways and stimulate unfaithful DNA repair or bypass checkpoints, these processes allow accumulation of genetic mutations in the host genome. Given this, a “Random Mutagenesis” transformation model seems more suitable to characterize the oncogenic activities of HTLV-I. PMID:26835512

  18. Immunization of DNA vaccine encoding C3d-VP1 fusion enhanced protective immune response against foot-and-mouth disease virus.

    PubMed

    Fan, Huiying; Tong, Tiezhu; Chen, Huanchun; Guo, Aizhen

    2007-10-01

    Because foot-and-mouth disease virus (FMDV) remains a great problem to many livestock of agricultural importance, safe, effective vaccines are in great need. DNA vaccine would be a promising candidate but the design remains to be optimized. VP1 gene of FMDV strain O/ES/2001 was linked to three copies of either porcine or murine C3d or four copies of a 28-aa fragment of murine C3d containing the CR2 receptor binding domain (M28). The resultant plasmids encoding C3d/M28-VP1 fusion or only VP1 as control were immunized guinea pigs. Both cellular and humoral immune responses were evaluated and protection was observed after virus challenge. As a result, although the plasmid encoding only VP1 could elicit virus-binding antibody detected by ELISA, splenocyte proliferation, IL-4 and IFN-gamma production, the levels were significantly less than C3d/M28-VP1 fusion. Furthermore, VP1 failed to induce neutralization antibody and protect animals against virus challenge, while murine C3d-VP1 fusion efficiently induced neutralization antibody response and provided 87.50% of the animals with complete protection and 12.50% with partial protection. Among murine C3d, M28, and porcine C3d, the adjuvant effect of murine C3d is strongest, followed by porcine C3d, and last murine M28. In conclusion, the fact that C3d genes, when coupled to VP1 gene, are able to greatly enhance the protective immune response of VP1 DNA in guinea pigs suggests that C3d-VP1 DNA chimera has a significant potential for use as a novel DNA vaccine against FMDV. PMID:17497212

  19. Intra-epithelial vaccination with COPV L1 DNA by particle-mediated DNA delivery protects against mucosal challenge with infectious COPV in beagle dogs.

    PubMed

    Stanley, M A; Moore, R A; Nicholls, P K; Santos, E B; Thomsen, L; Parry, N; Walcott, S; Gough, G

    2001-04-01

    Protection against viral challenge with canine oral papillomavirus (COPV) was achieved by immunisation via particle-mediated DNA delivery (PMDD) of a plasmid encoding the COPV L1 gene to cutaneous and oral mucosal sites in beagle dogs. The initial dose of approximately 9 microg of DNA was followed by two booster doses at 6 week intervals. A similar approach was used to vaccinate a control group of animals with plasmid DNA encoding the Hepatitis B virus S gene. Following challenge at the oral mucosa with COPV all animals vaccinated with the COPV L1 gene were protected against disease. However five of six animals in the control group developed COPV induced papillomas at the oral mucosa. Both cell-mediated lymphoproliferative and humoral antibody responses to the DNA vaccine were observed. Our data indicate that PMDD of plasmid DNA can protect against mucosal challenge with papillomavirus. PMID:11282188

  20. Ribonucleotide triggered DNA damage and RNA-DNA damage responses

    PubMed Central

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage. PMID:25692233

  1. Ubiquitin-specific Peptidase 10 (USP10) Deubiquitinates and Stabilizes MutS Homolog 2 (MSH2) to Regulate Cellular Sensitivity to DNA Damage.

    PubMed

    Zhang, Mu; Hu, Chen; Tong, Dan; Xiang, Shengyan; Williams, Kendra; Bai, Wenlong; Li, Guo-Min; Bepler, Gerold; Zhang, Xiaohong

    2016-05-13

    MSH2 is a key DNA mismatch repair protein, which plays an important role in genomic stability. In addition to its DNA repair function, MSH2 serves as a sensor for DNA base analogs-provoked DNA replication errors and binds to various DNA damage-induced adducts to trigger cell cycle arrest or apoptosis. Loss or depletion of MSH2 from cells renders resistance to certain DNA-damaging agents. Therefore, the level of MSH2 determines DNA damage response. Previous studies showed that the level of MSH2 protein is modulated by the ubiquitin-proteasome pathway, and histone deacetylase 6 (HDAC6) serves as an ubiquitin E3 ligase. However, the deubiquitinating enzymes, which regulate MSH2 remain unknown. Here we report that ubiquitin-specific peptidase 10 (USP10) interacts with and stabilizes MSH2. USP10 deubiquitinates MSH2 in vitro and in vivo Moreover, the protein level of MSH2 is positively correlated with the USP10 protein level in a panel of lung cancer cell lines. Knockdown of USP10 in lung cancer cells exhibits increased cell survival and decreased apoptosis upon the treatment of DNA-methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and antimetabolite 6-thioguanine (6-TG). The above phenotypes can be rescued by ectopic expression of MSH2. In addition, knockdown of MSH2 decreases the cellular mismatch repair activity. Overall, our results suggest a novel USP10-MSH2 pathway regulating DNA damage response and DNA mismatch repair.

  2. New insight into multifunctional role of peroxiredoxin family protein: Determination of DNA protection properties of bacterioferritin comigratory protein under hyperthermal and oxidative stresses.

    PubMed

    Lee, Sangmin; Chung, Jeong Min; Yun, Hyung Joong; Won, Jonghan; Jung, Hyun Suk

    2016-01-22

    Bacterioferritin comigratory protein (BCP) is a monomeric conformer acting as a putative thiol-dependent bacterial peroxidase, however molecular basis of DNA-protection via DNA-binding has not been clearly understood. In this study, we characterized the DNA binding properties of BCP using various lengths and differently shaped architectures of DNA. An electrophoretic mobility shift assay and electron microscopy analysis showed that recombinant TkBCP bound to DNA of a circular shape (double-stranded DNA and single-stranded DNA) and a linear shape (16-1000 bp) as well as various architectures of DNA. In addition, DNA protection experiments indicated that TkBCP can protect DNA against hyperthermal and oxidative stress by removing highly reactive oxygen species (ROS) or by protecting DNA from thermal degradation. Based on these results, we suggest that TkBCP is a multi-functional DNA-binding protein which has DNA chaperon and antioxidant functions.

  3. Latent infection of myeloid progenitors by human cytomegalovirus protects cells from FAS-mediated apoptosis through the cellular IL-10/PEA-15 pathway

    PubMed Central

    Lau, Jonathan C. H.; Sinclair, John

    2015-01-01

    Latent infection of primary CD34+ progenitor cells by human cytomegalovirus (HCMV) results in their increased survival in the face of pro-apoptotic signals. For instance, we have shown previously that primary myeloid cells are refractory to FAS-mediated killing and that cellular IL-10 (cIL-10) is an important survival factor for this effect. However, how cIL-10 mediates this protection is unclear. Here, we have shown that cIL-10 signalling leading to upregulation of the cellular factor PEA-15 mediates latency-associated protection of CD34+ progenitor cells from the extrinsic death pathway. PMID:25957098

  4. Cellular HIV type 1 DNA levels are equivalent among drug-sensitive and drug-resistant strains in newly diagnosed and antiretroviral naive patients.

    PubMed

    Antoniadou, Zoi-Anna; Hezka, Johana; Kousiappa, Ioanna; Mamais, Ioannis; Skoura, Lemonia; Pilalas, Dimitris; Metallidis, Simeon; Nicolaidis, Pavlos; Malisiovas, Nicolaos; Kostrikis, Leondios G

    2014-03-01

    The emergence of resistance against current antiretroviral drugs to human immunodeficiency virus type 1 (HIV-1) is an increasingly important concern to the continuous success of antiretroviral therapy to HIV-1-infected patients. In the past decade, a number of studies reported that the prevalence of transmitted drug resistance among newly diagnosed patients has reached an overall 9% prevalence worldwide. Also, a number of studies using longitudinal HIV-1 patient study cohorts demonstrated that the cellular HIV-1 DNA level in peripheral blood mononuclear cells (PBMCs) has a prognostic value for the progression of HIV-1 disease independently of plasma HIV-1 RNA load and CD4 count. Using a previously established molecular-beacon-based real-time PCR methodology, cellular HIV-1 DNA levels were quantified in newly diagnosed and antiretroviral-naive patients in Northern Greece recruited between 2009 and 2010 using a predefined enrolling strategy, in an effort to investigate whether there is any relationship between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. As part of the same study, DNA sequences encoding the env (C2-C5 region of gp120) were also amplified from PBMC-extracted DNA in order to determine the genotypic coreceptor tropism and genetic subtype. Cellular HIV-1 DNA levels had a median of 3.309 log10 HIV-1 copies per 10(6) PBMCs and demonstrated no correlation between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. An absence of association between cellular HIV-1 DNA levels with plasma viral HIV-1 RNA load and CD4 levels was also found reconfirming the previously published study. Genotypic analysis of coreceptor tropism indicated that 96% of samples, independently of the presence or not of genotypic drug resistance, were CCR5-tropic. Overall, the findings reconfirmed the previously proposed proposition that transmitted drug resistance does not have an impact on disease progression in HIV-1-infected individuals. Also, CCR5

  5. Identification of ALDH4 as a p53-inducible gene and its protective role in cellular stresses.

    PubMed

    Yoon, Kyong-Ah; Nakamura, Yusuke; Arakawa, Hirofumi

    2004-01-01

    To identify additional targets of p53, we used a cDNA microarray system to examine gene-expression patterns in response to enforced expression of exogenous p53 in p53-deficient cancer cells, and identified the aldehyde dehydrogenase 4 ( ALDH4) gene as a direct target of p53. ALDH4 is a mitochondrial-matrix NAD+-dependent enzyme catalyzing the second step of the proline degradation pathway. Expression of ALDH4 mRNA was induced in HCT116 cells in response to DNA damage caused by adriamycin treatment, in a p53-dependent manner. ALDH4 contains a potential p53 binding sequence in intron1 and the interaction of p53 with the site was shown by EMSA and ChIP assays. We confirmed p53-dependent transcriptional activity of the binding site by means of a reporter assay. Inhibition of ALDH4 expression by antisense oligonucleotides was able to enhance cell death induced by infection with Ad-p53. H1299 cells transformed to over-express ALDH4 showed significantly lower intracellular reactive oxygen species (ROS) levels than parental or control cells after treatment with hydrogen peroxide or UV. Those cells were also resistant to cell damage caused by hydrogen peroxide. These results suggest that p53 might play a protective role against cell damage induced by generation of intracellular ROS, through transcriptional activation of ALDH4.

  6. Mechanisms of radiation interaction with DNA: Potential implications for radiation protection

    SciTech Connect

    Sinclair, W.K.; Fry, R.J.M.

    1987-01-01

    An overview of presentations and discussions which took place at the US Department of Energy/Commission of European Communities (DOE/CEC) workshop on ''Mechanisms of Radiation Interaction with DNA: Potential Implications for Radiation Protection,'' held at San Diego, California, January 21-22, 1987, is provided. The Department has traditionally supported fundamental research on interactions of ionizing radiation with different biological systems and at all levels of biological organization. The aim of this workshop was to review the base of knowledge in the area of mechanisms of radiation action at the DNA level, and to explore ways in which this information can be applied to the development of scientifically sound concepts and procedures for use in the field of radiation protection.

  7. Dietary spices protect against hydrogen peroxide-induced DNA damage and inhibit nicotine-induced cancer cell migration.

    PubMed

    Jayakumar, R; Kanthimathi, M S

    2012-10-01

    Spices are rich sources of antioxidants due to the presence of phenols and flavonoids. In this study, the DNA protecting activity and inhibition of nicotine-induced cancer cell migration of 9 spices were analysed. Murine fibroblasts (3T3-L1) and human breast cancer (MCF-7) cells were pre-treated with spice extracts and then exposed to H₂O₂ and nicotine. The comet assay was used to analyse the DNA damage. Among the 9 spices, ginger, at 50 μg/ml protected against 68% of DNA damage in 3T3-L1 cells. Caraway, cumin and fennel showed statistically significant (p<0.05) DNA protecting activity. Treatment of MCF-7 cells with nicotine induced cell migration, whereas pre-treatment with spices reduced this migration. Pepper, long pepper and ginger exhibited a high rate of inhibition of cell migration. The results of this study prove that spices protect DNA and inhibit cancer cell migration. PMID:25005983

  8. A novel strategy of natural plant ferritin to protect DNA from oxidative damage during iron oxidation.

    PubMed

    Liao, Xiayun; Lv, Chenyan; Zhang, Xiuqing; Masuda, Taro; Li, Meiliang; Zhao, Guanghua

    2012-07-15

    Plant ferritin is a naturally occurring heteropolymer in plastids, where Fe(2+) is oxidatively deposited into the protein. However, the effect of this process on the coexistence of DNA and plant ferritin in the plastids is unknown. To investigate this effect, we built a system in which various plant ferritins and DNA coexist, followed by treatment with ferrous ions under aerobic conditions. Interestingly, naturally occurring soybean seed ferritin (SSF), a heteropolymer with an H-1/H-2 ratio of 1 to 1 in the apo form, completely protected DNA from oxidative damage during iron oxidative deposition into protein, and a similar result was obtained with its recombinant form, but not with its homopolymeric counterparts, apo rH-1 and apo rH-2. We demonstrate that the difference in DNA protection between heteropolymeric and homopolymeric plant ferritins stems from their different strategies to control iron chemistry during the above oxidative process. For example, the detoxification reaction occurs only in the presence of apo heteropolymeric SSF (hSSF), thereby preventing the production of hydroxyl radicals. In contrast, hydroxyl radicals are apparently generated via the Fenton reaction when apo rH-1 or rH-2 is used instead of apo hSSF. Thus, a combination of H-1 and H-2 subunits in hSSF seems to impart a unique DNA-protective function to the protein, which was previously unrecognized. This new finding advances our understanding of the structure and function of ferritin and of the widespread occurrence of heteropolymeric plant ferritin in nature. PMID:22580341

  9. DNA protective effect of ginseng and the antagonistic effect of Chinese turnip: A supplementation study

    PubMed Central

    Szeto, Yim Tong; Wong, Kam Shing; Han, Andrea; Pak, Sok Cheon; Kalle, Wouter

    2016-01-01

    Aim: The aim of this clinical study is to provide scientific evidence for supporting traditional Chinese application and usage to the patients. For this purpose, we tested the ability if Panax ginseng extract to lower oxidative damage to nuclear DNA in human lymphocytes by comparing the effect of cooked Chinese turnip on this effect. Materials and Methods: Seven healthy subjects (4 males and 3 females from 37 to 60 years) participated two occasions which were at least 2 weeks apart. About 2 mL of fasting blood sample for baseline measurement was taken on arrival. They were requested to ingest the content of 5 ginseng capsules in 200 mL water. The subject remained fasting for 2 h until the second blood sample taken. In the other occasion, the experiment was repeated except a piece of cooked turnip (10 g) was taken with the ginseng extract. The two occasions could be interchanged. Comet assay was performed on two specimens on the same day for the evaluation of lymphocytic DNA damage with or without oxidative stress. Results: For the group with ginseng supplementation, there was a significant decrease in comet score for hydrogen peroxide (H2O2) treatment over the 2-h period while no change in DNA damage for unstressed sample. For the group with ginseng together with turnip supplementation, there was no significant difference in comet score for both H2O2 treatment and phosphate-buffered saline treatment. Ginseng extract could reduce DNA damage mediated by H2O2 effectively, but this protection effect was antagonized by the ingestion of cooked turnip at the same time. Conclusion: In the current study, commercial ginseng extract was used for supplementing volunteers. Ginseng extract could protect DNA from oxidative stress in vivo while turnip diminished the protection. PMID:27757261

  10. Synthesis of novel MMT/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras

    PubMed Central

    Roviello, G. N.; Gröschel, S.; Pedone, C.

    2009-01-01

    Alanyl-peptide nucleic acid (alanyl-PNA)/DNA chimeras are oligomers envisaged to be beneficial in efficient DNA diagnostics based on an improved molecular beacon concept. A synthesis of alanyl-PNA/DNA chimera can be based on the solid phase assembly of the oligomer with mixed oligonucleotide/peptide backbone under DNA synthesis conditions, in which the nucleotides are introduced as phosphoramidites, whereas the nucleo amino acids make use of the acid labile monomethoxytrityl (MMT) group for temporary protection of the α-amino groups and acyl protecting groups for the exocyclic amino functions of the nucleobases. In this work, we realized for the first time the synthesis of all four MMT/acyl-protected nucleo alanines, achieved by deprotection/reprotection of the newly synthesized Boc/acyl intermediates, useful monomers for the obtainment of (alanyl-PNA)/DNA chimeras by conditions fully compatible with the standard phosphoramidite DNA synthesis strategy. PMID:19629638

  11. Synthesis of novel MMT/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras.

    PubMed

    Roviello, G N; Gröschel, S; Pedone, C; Diederichsen, U

    2010-05-01

    Alanyl-peptide nucleic acid (alanyl-PNA)/DNA chimeras are oligomers envisaged to be beneficial in efficient DNA diagnostics based on an improved molecular beacon concept. A synthesis of alanyl-PNA/DNA chimera can be based on the solid phase assembly of the oligomer with mixed oligonucleotide/peptide backbone under DNA synthesis conditions, in which the nucleotides are introduced as phosphoramidites, whereas the nucleo amino acids make use of the acid labile monomethoxytrityl (MMT) group for temporary protection of the alpha-amino groups and acyl protecting groups for the exocyclic amino functions of the nucleobases. In this work, we realized for the first time the synthesis of all four MMT/acyl-protected nucleo alanines, achieved by deprotection/reprotection of the newly synthesized Boc/acyl intermediates, useful monomers for the obtainment of (alanyl-PNA)/DNA chimeras by conditions fully compatible with the standard phosphoramidite DNA synthesis strategy.

  12. Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival.

    PubMed

    Tinkum, Kelsey L; Stemler, Kristina M; White, Lynn S; Loza, Andrew J; Jeter-Jones, Sabrina; Michalski, Basia M; Kuzmicki, Catherine; Pless, Robert; Stappenbeck, Thaddeus S; Piwnica-Worms, David; Piwnica-Worms, Helen

    2015-12-22

    Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.

  13. Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival

    PubMed Central

    Tinkum, Kelsey L.; Stemler, Kristina M.; White, Lynn S.; Loza, Andrew J.; Jeter-Jones, Sabrina; Michalski, Basia M.; Kuzmicki, Catherine; Pless, Robert; Stappenbeck, Thaddeus S.; Piwnica-Worms, David; Piwnica-Worms, Helen

    2015-01-01

    Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy. PMID:26644583

  14. Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish.

    PubMed

    de las Heras, Ana I; Rodríguez Saint-Jean, S; Pérez-Prieto, Sara I

    2010-04-01

    DNA vaccines and oral DNA-based immunotherapy against infectious pancreatic necrosis virus (IPNV) have scarcely been studied in salmonid fish. Here, a vector with the capsid VP2 gene inserted was encapsulated in alginate microspheres to avoid the aggressive gastrointestinal conditions experienced following oral administration. Alginate microspheres were effective to protect the pDNA encoding VP2, which was expressed early in different organs of the vaccinated trout and that persisted for at least 60 days. The vaccine induces innate immune responses, raising the expression of IFN more than 10-fold relative to the fish vaccinated with the empty plasmid, at 7 and 15 days post-vaccination. Likewise, maximal expression of the IFN-induced antiviral Mx protein was recorded 15 days post-vaccination and neutralizing antibodies were also detected after 15 days, although their titre rose further at 21 days post-vaccination. Protection was high in the immunized fish, which showed around an 80% relative survival when challenged 15 and 30 days after vaccine delivery. Very low viral load with respect to the control group was detected in the vaccinated fish that survived 45 days after challenge. Thus, this study demonstrates the potential of the encapsulation technique for IPNV-DNA vaccine delivery and the relevance of the IPNV-VP2 gene for future plasmid constructs.

  15. Mitochondrial aldehyde dehydrogenase 2 protects gastric mucosa cells against DNA damage caused by oxidative stress.

    PubMed

    Duan, Yantao; Gao, Yaohui; Zhang, Jun; Chen, Yinan; Jiang, Yannan; Ji, Jun; Zhang, Jianian; Chen, Xuehua; Yang, Qiumeng; Su, Liping; Zhang, Jun; Liu, Bingya; Zhu, Zhenggang; Wang, Lishun; Yu, Yingyan

    2016-04-01

    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a member of the aldehyde dehydrogenase superfamily and is involved with the metabolic processing of aldehydes. ALDH2 plays a cytoprotective role by removing aldehydes produced during normal metabolism. We examined the cytoprotective role of ALDH2 specifically in gastric mucosa cells. Overexpression of ALDH2 increased the viability of gastric mucosa cells treated with H2O2, while knockdown of ALDH2 had an opposite effect. Moreover, overexpression of ALDH2 protected gastric mucosa cells against oxidative stress-induced apoptosis as determined by flow cytometry, Hoechst 33342, and TUNEL assays. Consistently, ALDH2 knockdown had an opposite effect. Additionally, DNA damage was ameliorated in ALDH2-overexpressing gastric mucosa cells treated with H2O2. We further identified that this cytoprotective role of ALDH2 was mediated by metabolism of 4-hydroxynonenal (4-HNE). Consistently, 4-HNE mimicked the oxidative stress induced by H2O2 in gastric mucosa cells. Treatment with 4-HNE increased levels of DNA damage in ALDH2-knockdown GES-1 cells, while overexpression of ALDH2 decreased 4-HNE-induced DNA damage. These findings suggest that ALDH2 can protect gastric mucosa cells against DNA damage caused by oxidative stress by reducing levels of 4-HNE.

  16. Immunogenicity and protective efficacy of DNA vaccine against visceral leishmaniasis in BALB/c mice.

    PubMed

    Kaur, Sukhbir; Kaur, Tejinder; Joshi, Jyoti

    2016-07-01

    The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis. Inbred BALB/c mice were immunized subcutaneously twice at an interval of three weeks with pcDNA3.1(+) encoding T cell epitopes of gp63 and Hsp70 individually and in combination. Animals were challenged intracardially with 10(7) promastigotes of Leishmania donovani 10 days post immunization and sacrificed 1, 2 and 3 months post challenge. The immunized animals revealed a significant reduction (P < 0.05) in splenic and hepatic parasite burden as compared to the infected controls. Maximum reduction in parasite load (P < 0.05) was observed in animals treated with a combination of pcDNA/gp63 and pcDNA/Hsp70. These animals also showed heightened DTH response, increased IgG2a, elevated Th1 cytokines (IFN-γ and IL-2) and reduced IgG1 and IL-10 levels. Thus, mice immunized with the cocktail vaccine exhibited significantly greater protection in comparison to those immunized with individual antigens. PMID:27533939

  17. Immunogenicity and protective efficacy of DNA vaccine against visceral leishmaniasis in BALB/c mice

    PubMed Central

    Kaur, Sukhbir; Kaur, Tejinder; Joshi, Jyoti

    2016-01-01

    Abstract The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis. Inbred BALB/c mice were immunized subcutaneously twice at an interval of three weeks with pcDNA3.1(+) encoding T cell epitopes of gp63 and Hsp70 individually and in combination. Animals were challenged intracardially with 107 promastigotes of Leishmania donovani 10 days post immunization and sacrificed 1, 2 and 3 months post challenge. The immunized animals revealed a significant reduction (P < 0.05) in splenic and hepatic parasite burden as compared to the infected controls. Maximum reduction in parasite load (P < 0.05) was observed in animals treated with a combination of pcDNA/gp63 and pcDNA/Hsp70. These animals also showed heightened DTH response, increased IgG2a, elevated Th1 cytokines (IFN-γ and IL-2) and reduced IgG1 and IL-10 levels. Thus, mice immunized with the cocktail vaccine exhibited significantly greater protection in comparison to those immunized with individual antigens. PMID:27533939

  18. Effect of DNA Methylation in Various Diseases and the Probable Protective Role of Nutrition: A Mini-Review

    PubMed Central

    Vadakedath, Sabitha

    2015-01-01

    DNA methylation, a process of adding a methyl group to DNA done by a DNA methyltransferase is a heritable (epigenetic) alteration leading to cancer, atherosclerosis, nervous disorders (Imprinting disorders), and cardiovascular diseases. The role of nutrition in DNA methylation is revealed by identification of methyl variable positions (MVP) on DNA. These regions are more susceptible to DNA methylations. Nutritional supplementation of folic acid and methionine in utero and in adults decreased epigenetic modifications due to its role in DNA metabolism (one carbon metabolism). Thus, in utero and adult supplementation of folic acid and methionine may reduce DNA methylation. This review attempts to highlight the process of DNA methylation, its effect on various diseases, and the probable protective role of nutrition. PMID:26430583

  19. Immunotherapy with a posttranscriptionally modified DNA vaccine induces complete protection against metastatic neuroblastoma.

    PubMed

    Pertl, Ursula; Wodrich, Harald; Ruehlmann, J Michael; Gillies, Stephen D; Lode, Holger N; Reisfeld, Ralph A

    2003-01-15

    The successful induction of a T-cell-mediated tumor-protective immunity against poorly immunogenic malignancies remains a major challenge for cancer immunotherapy. We achieved this by immunization with a tyrosine hydroxylase (mTH)-based DNA vaccine, enhanced with the posttranscriptional regulatory acting RNA element (WPRE), derived from woodchuck hepatitis virus in combination with an antibody-cytokine fusion protein (ch14.18-IL-2) that targets interleukin-2 (IL-2) to the tumor microenvironment. This DNA vaccine mTH-WPRE was carried by attenuated Salmonella typhimurium and applied by oral gavage in a mouse model of neuroblastoma. Mice immunized with the mTH-WPRE vaccine, and which additionally received a boost with suboptimal doses of ch14.18-IL-2, were completely protected against hepatic neuroblastoma metastases. In contrast, all controls presented with disseminated metastases. Both T-cell and natural killer (NK) cell-dependent mechanisms were involved in the induction of a systemic tumor-protective immunity. Thus, up-regulation of interferon-gamma (IFN-gamma) expression in CD8(+) T cells occurred only in those animals that received the mTH-WPRE vaccine plus the ch14.18-IL-2 boost. Up-regulation of this proinflammatory cytokine was not observed in mice immunized with mTH-WPRE vaccine alone. A role for NK cells was indicated by the complete abrogation of systemic tumor-protective immunity in all animals that were depleted of NK cells in vivo. Taken together, these data demonstrate that immunization with a posttranscriptionally enhanced DNA vaccine encoding the WPRE sequence, combined with a boost of the ch14.18-IL-2 fusion protein, completely protects against hepatic metastases in a murine model of neuroblastoma and therefore may lead to a new strategy for immunotherapy and prevention of metastatic neuroblastoma.

  20. Aqueous extracts of the edible Gracilaria tenuistipitata are protective against H₂O₂-induced DNA damage, growth inhibition, and cell cycle arrest.

    PubMed

    Yang, Jing-Iong; Yeh, Chi-Chen; Lee, Jin-Ching; Yi, Szu-Cheng; Huang, Hurng-Wern; Tseng, Chao-Neng; Chang, Hsueh-Wei

    2012-06-13

    Potential antioxidant properties of an aqueous extract of the edible red seaweed Gracilaria tenuistipitata (AEGT) against oxidative DNA damage were evaluated. The AEGT revealed several antioxidant molecules, including phenolics, flavonoids and ascorbic acid. In a cell-free assay, the extract exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity that significantly reduced H₂O₂-induced plasmid DNA breaks in a dose-response manner (P < 0.001). The AEGT also suppressed H₂O₂-induced oxidative DNA damage in H1299 cells by reducing the percentage of damaged DNA in a dose-response manner (P < 0.001) as measured by a modified alkaline comet-nuclear extract (comet-NE) assay. The MTT assay results showed that AEGT confers significant protection against H₂O₂-induced cytotoxicity and that AEGT itself is not cytotoxic (P < 0.001). Moreover, H₂O₂-induced cell cycle G2/M arrest was significantly released when cells were co-treated with different concentrations of AEGT (P < 0.001). Taken together, these findings suggest that edible red algae Gracilaria water extract can prevent H₂O₂-induced oxidative DNA damage and its related cellular responses.

  1. Vector optimization and needle-free intradermal application of a broadly protective polyvalent influenza A DNA vaccine for pigs and humans.

    PubMed

    Borggren, Marie; Nielsen, Jens; Bragstad, Karoline; Karlsson, Ingrid; Krog, Jesper S; Williams, James A; Fomsgaard, Anders

    2015-01-01

    The threat posed by the 2009 pandemic H1N1 virus emphasized the need for new influenza A virus vaccines inducing a broad cross-protective immune response for use in both humans and pigs. An effective and broad influenza vaccine for pigs would greatly benefit the pork industry and contribute to public health by diminishing the risk of emerging highly pathogenic reassortants. Current inactivated protein vaccines against swine influenza produce only short-lived immunity and have no efficacy against heterologous strains. DNA vaccines are a potential alternative with advantages such as the induction of cellular and humoral immunity, inherent safety and rapid production time. We have previously developed a DNA vaccine encoding selected influenza proteins of pandemic origin and demonstrated broad protective immune responses in ferrets and pigs. In this study, we evaluated our DNA vaccine expressed by next-generation vectors. These new vectors can improve gene expression, but they are also efficiently produced on large scales and comply with regulatory guidelines by avoiding antibiotic resistance genes. In addition, a new needle-free delivery of the vaccine, convenient for mass vaccinations, was compared with intradermal needle injection followed by electroporation. We report that when our DNA vaccine is expressed by the new vectors and delivered to the skin with the needle-free device in the rabbit model, it can elicit an antibody response with the same titers as a conventional vector with intradermal electroporation. The needle-free delivery is already in use for traditional protein vaccines in pigs but should be considered as a practical alternative for the mass administration of broadly protective influenza DNA vaccines. PMID:25746201

  2. RecO acts with RecF and RecR to protect and maintain replication forks blocked by UV-induced DNA damage in Escherichia coli.

    PubMed

    Chow, Kin-Hoe; Courcelle, Justin

    2004-01-30

    In Escherichia coli, recF and recR are required to stabilize and maintain replication forks arrested by UV-induced DNA damage. In the absence of RecF, replication fails to recover, and the nascent lagging strand of the arrested replication fork is extensively degraded by the RecQ helicase and RecJ nuclease. recO mutants are epistatic with recF and recR with respect to recombination and survival assays after DNA damage. In this study, we show that RecO functions with RecF and RecR to protect the nascent lagging strand of arrested replication forks after UV-irradiation. In the absence of RecO, the nascent DNA at arrested replication forks is extensively degraded and replication fails to recover. The extent of nascent DNA degradation is equivalent in single, double, or triple mutants of recF, recO, or recR, and the degradation is dependent upon RecJ and RecQ functions. Because RecF has been shown to protect the nascent lagging strand from degradation, these observations indicate that RecR and RecO function with RecF to protect the same nascent strand of the arrested replication fork and are likely to act at a common point during the recovery process. We discuss these results in relation to the biochemical and cellular properties of RecF, RecO, and RecR and their potential role in loading RecA filaments to maintain the replication fork structure after the arrest of replication by UV-induced DNA damage. PMID:14625283

  3. Vector optimization and needle-free intradermal application of a broadly protective polyvalent influenza A DNA vaccine for pigs and humans.

    PubMed

    Borggren, Marie; Nielsen, Jens; Bragstad, Karoline; Karlsson, Ingrid; Krog, Jesper S; Williams, James A; Fomsgaard, Anders

    2015-01-01

    The threat posed by the 2009 pandemic H1N1 virus emphasized the need for new influenza A virus vaccines inducing a broad cross-protective immune response for use in both humans and pigs. An effective and broad influenza vaccine for pigs would greatly benefit the pork industry and contribute to public health by diminishing the risk of emerging highly pathogenic reassortants. Current inactivated protein vaccines against swine influenza produce only short-lived immunity and have no efficacy against heterologous strains. DNA vaccines are a potential alternative with advantages such as the induction of cellular and humoral immunity, inherent safety and rapid production time. We have previously developed a DNA vaccine encoding selected influenza proteins of pandemic origin and demonstrated broad protective immune responses in ferrets and pigs. In this study, we evaluated our DNA vaccine expressed by next-generation vectors. These new vectors can improve gene expression, but they are also efficiently produced on large scales and comply with regulatory guidelines by avoiding antibiotic resistance genes. In addition, a new needle-free delivery of the vaccine, convenient for mass vaccinations, was compared with intradermal needle injection followed by electroporation. We report that when our DNA vaccine is expressed by the new vectors and delivered to the skin with the needle-free device in the rabbit model, it can elicit an antibody response with the same titers as a conventional vector with intradermal electroporation. The needle-free delivery is already in use for traditional protein vaccines in pigs but should be considered as a practical alternative for the mass administration of broadly protective influenza DNA vaccines.

  4. Oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by dietary polyphenols: implications for a cancer preventive mechanism.

    PubMed

    Khan, Husain Y; Zubair, Haseeb; Ullah, Mohd F; Ahmad, Aamir; Hadi, Sheikh M

    2011-12-01

    To account for the observed anticancer properties of plant polyphenols, we have earlier proposed a mechanism which involves the mobilization of endogenous copper ions by polyphenols leading to the generation of reactive oxygen species (ROS) that serve as proximal DNA cleaving agents and lead to cell death. Over the last decade we have proceeded to validate our hypothesis with considerable success. As a further confirmation of our hypothesis, in this paper we first show that oral administration of copper to rats leads to elevated copper levels in lymphocytes. When such lymphocytes with a copper overload were isolated and treated with polyphenols EGCG, genistein and resveratrol, an increased level of DNA breakage was observed. Further, preincubation of lymphocytes having elevated copper levels with the membrane permeable copper chelator neocuproine, resulted in inhibition of polyphenol induced DNA degradation. However, membrane impermeable chelator of copper bathocuproine, as well as iron and zinc chelators were ineffective in causing such inhibition in DNA breakage, confirming the involvement of endogenous copper in polyphenol induced cellular DNA degradation. It is well established that serum and tissue concentrations of copper are greatly increased in various malignancies. In view of this fact, the present results further confirm our earlier findings and strengthen our hypothesis that an important anticancer mechanism of plant polyphenols could be the mobilization of intracellular copper leading to ROS-mediated cellular DNA breakage. In this context, it may be noted that cancer cells are under considerable oxidative stress and increasing such stress to cytotoxic levels could be a successful anticancer approach.

  5. Increased Cellular NAD+ Level through NQO1 Enzymatic Action Has Protective Effects on Bleomycin-Induced Lung Fibrosis in Mice

    PubMed Central

    Oh, Gi-Su; Lee, Su-Bin; Karna, Anjani; Kim, Hyung-Jin; Shen, AiHua; Pandit, Arpana; Lee, SeungHoon

    2016-01-01

    Background Idiopathic pulmonary fibrosis is a common interstitial lung disease; it is a chronic, progressive, and fatal lung disease of unknown etiology. Over the last two decades, knowledge about the underlying mechanisms of pulmonary fibrosis has improved markedly and facilitated the identification of potential targets for novel therapies. However, despite the large number of antifibrotic drugs being described in experimental pre-clinical studies, the translation of these findings into clinical practices has not been accomplished yet. NADH:quinone oxidoreductase 1 (NQO1) is a homodimeric enzyme that catalyzes the oxidation of NADH to NAD+ by various quinones and thereby elevates the intracellular NAD+ levels. In this study, we examined the effect of increase in cellular NAD+ levels on bleomycin-induced lung fibrosis in mice. Methods C57BL/6 mice were treated with intratracheal instillation of bleomycin. The mice were orally administered with β-lapachone from 3 days before exposure to bleomycin to 1-3 weeks after exposure to bleomycin. Bronchoalveolar lavage fluid (BALF) was collected for analyzing the infiltration of immune cells. In vitro, A549 cells were treated with transforming growth factor β1 (TGF-β1) and β-lapachone to analyze the extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT). Results β-Lapachone strongly attenuated bleomycin-induced lung inflammation and fibrosis, characterized by histological staining, infiltrated immune cells in BALF, inflammatory cytokines, fibrotic score, and TGF-β1, α-smooth muscle actin accumulation. In addition, β-lapachone showed a protective role in TGF-β1–induced ECM expression and EMT in A549 cells. Conclusion Our results suggest that β-lapachone can protect against bleomycin-induced lung inflammation and fibrosis in mice and TGF-β1–induced EMT in vitro, by elevating the NAD+/NADH ratio through NQO1 activation. PMID:27790277

  6. XPD localizes in mitochondria and protects the mitochondrial genome from oxidative DNA damage.

    PubMed

    Liu, Jing; Fang, Hongbo; Chi, Zhenfen; Wu, Zan; Wei, Di; Mo, Dongliang; Niu, Kaifeng; Balajee, Adayabalam S; Hei, Tom K; Nie, Linghu; Zhao, Yongliang

    2015-06-23

    Xeroderma pigmentosum group D (XPD/ERCC2) encodes an ATP-dependent helicase that plays essential roles in both transcription and nucleotide excision repair of nuclear DNA, however, whether or not XPD exerts similar functions in mitochondria remains elusive. In this study, we provide the first evidence that XPD is localized in the inner membrane of mitochondria, and cells under oxidative stress showed an enhanced recruitment of XPD into mitochondrial compartment. Furthermore, mitochondrial reactive oxygen species production and levels of oxidative stress-induced mitochondrial DNA (mtDNA) common deletion were significantly elevated, whereas capacity for oxidative damage repair of mtDNA was markedly reduced in both XPD-suppressed human osteosarcoma (U2OS) cells and XPD-deficient human fibroblasts. Immunoprecipitation-mass spectrometry analysis was used to identify interacting factor(s) with XPD and TUFM, a mitochondrial Tu translation elongation factor was detected to be physically interacted with XPD. Similar to the findings in XPD-deficient cells, mitochondrial common deletion and oxidative damage repair capacity in U2OS cells were found to be significantly altered after TUFM knock-down. Our findings clearly demonstrate that XPD plays crucial role(s) in protecting mitochondrial genome stability by facilitating an efficient repair of oxidative DNA damage in mitochondria.

  7. XPD localizes in mitochondria and protects the mitochondrial genome from oxidative DNA damage

    PubMed Central

    Liu, Jing; Fang, Hongbo; Chi, Zhenfen; Wu, Zan; Wei, Di; Mo, Dongliang; Niu, Kaifeng; Balajee, Adayabalam S.; Hei, Tom K.; Nie, Linghu; Zhao, Yongliang

    2015-01-01

    Xeroderma pigmentosum group D (XPD/ERCC2) encodes an ATP-dependent helicase that plays essential roles in both transcription and nucleotide excision repair of nuclear DNA, however, whether or not XPD exerts similar functions in mitochondria remains elusive. In this study, we provide the first evidence that XPD is localized in the inner membrane of mitochondria, and cells under oxidative stress showed an enhanced recruitment of XPD into mitochondrial compartment. Furthermore, mitochondrial reactive oxygen species production and levels of oxidative stress-induced mitochondrial DNA (mtDNA) common deletion were significantly elevated, whereas capacity for oxidative damage repair of mtDNA was markedly reduced in both XPD-suppressed human osteosarcoma (U2OS) cells and XPD-deficient human fibroblasts. Immunoprecipitation-mass spectrometry analysis was used to identify interacting factor(s) with XPD and TUFM, a mitochondrial Tu translation elongation factor was detected to be physically interacted with XPD. Similar to the findings in XPD-deficient cells, mitochondrial common deletion and oxidative damage repair capacity in U2OS cells were found to be significantly altered after TUFM knock-down. Our findings clearly demonstrate that XPD plays crucial role(s) in protecting mitochondrial genome stability by facilitating an efficient repair of oxidative DNA damage in mitochondria. PMID:25969448

  8. The tertiary structure of the four-way DNA junction affords protection against DNase I cleavage.

    PubMed Central

    Murchie, A I; Carter, W A; Portugal, J; Lilley, D M

    1990-01-01

    The accessibility of phosphodiester bonds in the DNA of four-way helical junctions has been probed with the nuclease DNase I. Regions of protection were observed on all four strands of the junctions, that tended to be longer on the strands that are exchanged between the coaxially stacked pairs of helices. The protected regions on the continuous strands of the stacked helices were not located exactly at the junction, but were displaced towards the 3' side of the strand. This is the region of backbone that becomes located in the major groove of the opposed helix in the non-crossed, right-handed structure for the junction, and might therefore be predicted to be protected against cleavage by an enzyme. However, the major grooves of the structure remain accessible to the much smaller probe dimethyl sulphate. Images PMID:2339051

  9. A DNA vaccine encoding VP22 of herpes simplex virus type I (HSV-1) and OprF confers enhanced protection from Pseudomonas aeruginosa in mice.

    PubMed

    Yu, Xian; Wang, Yan; Xia, Yifan; Zhang, Lijuan; Yang, Qin; Lei, Jun

    2016-08-17

    Pseudomonas aeruginosa antimicrobial resistance is a major therapeutic challenge. DNA vaccination is an attractive approach for antigen-specific immunotherapy against P. aeruginosa. We explored the feasibility of employing Herpes simplex virus type 1 tegument protein, VP22, as a molecular tool to enhance the immunogenicity of an OprF DNA vaccine against P. aeruginosa. Recombinant DNA vaccines, pVAX1-OprF, pVAX1-OprF-VP22 (encoding a n-OprF-VP22-c fusion protein) and pVAX1-VP22-OprF (encoding a n-VP22-OprF-c fusion protein) were constructed. The humoral and cellular immune responses and immune protective effects of these DNA vaccines in mice were evaluated. In this report, we showed that vaccination with pVAX1-OprF-VP22 induced higher levels of IgG titer, T cell proliferation rate. It also provided better immune protection against the P. aeruginosa challenge when compared to that induced by pVAX1-OprF or pVAX1-VP22-OprF DNA vaccines. Molecular mechanistic analyses indicated vaccination with pVAX1-OprF-VP22 triggered immune responses characterized by a preferential increase in antigen specific IgG2a and IFN-γ in mice, indicating Th1 polarization. We concluded that VP22 is a potent stimulatory molecular tool for DNA vaccination when fused to the carboxyl end of OprF gene. Our study provides a novel strategy for prevention and treatment of P. aeruginosa infection. PMID:27449680

  10. The Leishmania infantum Acidic Ribosomal Protein P0 Administered as a DNA Vaccine Confers Protective Immunity to Leishmania major Infection in BALB/c Mice

    PubMed Central

    Iborra, Salvador; Soto, Manuel; Carrión, Javier; Nieto, Ana; Fernández, Edgar; Alonso, Carlos; Requena, Jose M.

    2003-01-01

    In this study, we examined the immunogenic properties of the Leishmania infantum acidic ribosomal protein P0 (LiP0) in the BALB/c mouse model. The humoral and cellular responses induced by the administration of the LiP0 antigen, either as soluble recombinant LiP0 (rLiP0) or as a plasmid DNA formulation (pcDNA3-LiP0), were determined. Also, the immunological response associated with a prime-boost strategy, consisting of immunization with pcDNA3-LiP0 followed by a boost with rLiP0, was assayed. Immunization with rLiP0 induced a predominant Th2-like humoral response, but no anti-LiP0 antibodies were induced after immunization with pcDNA3-LiP0, whereas a strong humoral response consisting of a mixed immunoglobulin G2a (IgG2a)-IgG1 isotype profile was induced in mice immunized with the prime-boost regime. For all three immunization protocols, rLiP0-stimulated production of gamma interferon (IFN-γ) in both splenocytes and lymph node cells from immunized mice was observed. However, it was only when mice were immunized with pcDNA3-LiP0 that noticeable protection against L. major infection was achieved, as determined by both lesion development and parasite burden. Immunization of mice with LiP0-DNA primes both CD4+ and CD8+ T cells, which, with the L. major challenge, were boosted to produce significant levels of IL-12-dependent, antigen-specific IFN-γ. Taken together, these data indicate that genetic vaccination with LiP0 induces protective immunological effector mechanisms, yet the immunological response elicited by LiP0 is not sufficient to keep the infection from progressing. PMID:14573678

  11. Priming with two DNA vaccines expressing hepatitis C virus NS3 protein targeting dendritic cells elicits superior heterologous protective potential in mice.

    PubMed

    Guan, Jie; Deng, Yao; Chen, Hong; Yin, Xiao; Yang, Yang; Tan, Wenjie

    2015-10-01

    Development an effective vaccine may offer an alternative preventive and therapeutic strategy against HCV infection. DNA vaccination has been shown to induce robust humoral and cellular immunity and overcome many problems associated with conventional vaccines. In this study, mice were primed with either conventional pVRC-based or suicidal pSC-based DNA vaccines carrying DEC-205-targeted NS3 antigen (DEC-NS3) and boosted with type 5 adenoviral vectors encoding the partial NS3 and core antigens (C44P). The prime boost regimen induced a marked increase in antigen-specific humoral and T-cell responses in comparison with either rAd5-based vaccines or DEC-205-targeted DNA immunization in isolation. The protective effect against heterogeneous challenge was correlated with high levels of anti-NS3 IgG and T-cell-mediated immunity against NS3 peptides. Moreover, priming with a suicidal DNA vaccine (pSC-DEC-NS3), which elicited increased TNF-α-producing CD4+ and CD8+ T-cells against NS3-2 peptides (aa 1245-1461), after boosting, showed increased heterogeneous protective potential compared with priming with a conventional DNA vaccine (pVRC-DEC-NS3). In conclusion, a suicidal DNA vector (pSC-DEC-NS3) expressing DEC-205-targeted NS3 combined with boosting using an rAd5-based HCV vaccine (rAd5-C44P) is a good candidate for a safe and effective vaccine against HCV infection.

  12. YAP activation protects urothelial cell carcinoma from treatment-induced DNA damage

    PubMed Central

    Ciamporcero, Eric; Shen, He; Ramakrishnan, Swathi; Ku, Sheng Yu; Chintala, Sreenivasulu; Shen, Li; Adelaiye, Remi; Miles, Kiersten Marie; Ullio, Chiara; Pizzimenti, Stefania; Daga, Martina; Azabdaftari, Gissou; Attwood, Kris; Johnson, Candace; Zhang, Jianmin; Barrera, Giuseppina; Pili, Roberto

    2015-01-01

    Current standard of care for muscle-invasive urothelial cell carcinoma (UCC) is surgery along with perioperative platinum-based chemotherapy. UCC is sensitive to cisplatin-based regimens, but acquired resistance eventually occurs, and a subset of tumors is intrinsically resistant. Thus, there is an unmet need for new therapeutic approaches to target chemotherapy-resistant UCC. Yes-associated protein (YAP) is a transcriptional co-activator that has been associated with bladder cancer progression and cisplatin resistance in ovarian cancer. In contrast, YAP has been shown to induce DNA damage associated apoptosis in non-small cell lung carcinoma. However, no data have been reported on the YAP role in UCC chemo-resistance. Thus, we have investigated the potential dichotomous role of YAP in UCC response to chemotherapy utilizing two patient-derived xenograft models recently established. Constitutive expression and activation of YAP inversely correlated with in vitro and in vivo cisplatin sensitivity. YAP overexpression protected while YAP knock-down sensitized UCC cells to chemotherapy and radiation effects via increased accumulation of DNA damage and apoptosis. Furthermore, pharmacological YAP inhibition with verteporfin inhibited tumor cell proliferation and restored sensitivity to cisplatin. In addition, nuclear YAP expression was associated with poor outcome in UCC patients who received perioperative chemotherapy. In conclusion, these results suggest that YAP activation exerts a protective role and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of UCC. PMID:26119935

  13. Ganglioside GT1b protects human spermatozoa from hydrogen peroxide-induced DNA and membrane damage.

    PubMed

    Gavella, Mirjana; Garaj-Vrhovac, Verica; Lipovac, Vaskresenija; Antica, Mariastefania; Gajski, Goran; Car, Nikica

    2010-06-01

    We have reported previously that various gangliosides, the sialic acid containing glycosphingolipids, provide protection against sperm injury caused by reactive oxygen species (ROS). In this study, we investigated the effect of treatment of human spermatozoa with ganglioside GT1b on hydrogen peroxide (H(2)O(2))-induced DNA fragmentation and plasma membrane damage. Single-cell gel electrophoresis (Comet assay) used in the assessment of sperm DNA integrity showed that in vitro supplemented GT1b (100 microm) significantly reduced DNA damage induced by H(2)O(2) (200 microm) (p < 0.05). Measurements of Annexin V binding in combination with the propidium iodide vital dye labelling demonstrated that the spermatozoa pre-treated with GT1b exhibited a significant increase (p < 0.05) in the percentage of live cells with intact membrane and decreased phosphatidylserine translocation after exposure to H(2)O(2). Flow cytometry using the intracellular ROS-sensitive fluorescence dichlorodihydrofluorescein diacetate dye employed to investigate the transport of the extracellularly supplied H(2)O(2) into the cell interior revealed that ganglioside GT1b completely inhibited the passage of H(2)O(2) through the sperm membrane. These results suggest that ganglioside GT1b may protect human spermatozoa from H(2)O(2)-induced damage by rendering sperm membrane more hydrophobic, thus inhibiting the diffusion of H(2)O(2) across the membrane.

  14. Neisseria gonorrhoeae DNA recombination and repair enzymes protect against oxidative damage caused by hydrogen peroxide.

    PubMed

    Stohl, Elizabeth A; Seifert, H Steven

    2006-11-01

    The strict human pathogen Neisseria gonorrhoeae is exposed to oxidative damage during infection. N. gonorrhoeae has many defenses that have been demonstrated to counteract oxidative damage. However, recN is the only DNA repair and recombination gene upregulated in response to hydrogen peroxide (H(2)O(2)) by microarray analysis and subsequently shown to be important for oxidative damage protection. We therefore tested the importance of RecA and DNA recombination and repair enzymes in conferring resistance to H(2)O(2) damage. recA mutants, as well as RecBCD (recB, recC, and recD) and RecF-like pathway mutants (recJ, recO, and recQ), all showed decreased resistance to H(2)O(2). Holliday junction processing mutants (ruvA, ruvC, and recG) showed decreased resistance to H(2)O(2) resistance as well. Finally, we show that RecA protein levels did not increase as a result of H(2)O(2) treatment. We propose that RecA, recombinational DNA repair, and branch migration are all important for H(2)O(2) resistance in N. gonorrhoeae but that constitutive levels of these enzymes are sufficient for providing protection against oxidative damage by H(2)O(2). PMID:16936020

  15. Protective effect of carboxymethyl-glucan (CM-G) against DNA damage in patients with advanced prostate cancer.

    PubMed

    Magnani, Marciane; Castro-Gomez, Raul Jorge Hernan; Mori, Mateus Prates; Kuasne, Hellen; Gregório, Emerson Pereira; Libos, Farid; de Syllos Cólus, Ilce Mara

    2011-01-01

    Carboxymethyl-glucan (CM-G) is a soluble derivative from Saccharomyces cerevisiae (1 → 3)(1 → 6)-β-D-glucan. The protective efficiency of CM-G against DNA damage in cells from patients with advanced prostate cancer (PCa), and undergoing Androgen Deprivation Therapy (ADT), was evaluated. DNA damage scores were obtained by the comet assay, both before and after treatment with CM-G. The reduction in DNA damage, ranging from 18% to 87%, with an average of 59%, was not related to the increased number of leukocytes in peripheral blood. The results demonstrate for the first time the protective effect of CM-G against DNA damage in patients with advanced PCa. Among smokers, three presented the highest reduction in DNA damage after treatment with CM-G. There was no observable relationship between DNA damage scores before and after treatment, and age, alcoholism and radiotherapy.

  16. Protective effect of carboxymethyl-glucan (CM-G) against DNA damage in patients with advanced prostate cancer

    PubMed Central

    Magnani, Marciane; Castro-Gomez, Raul Jorge Hernan; Mori, Mateus Prates; Kuasne, Hellen; Gregório, Emerson Pereira; Libos, Farid; de Syllos Cólus, Ilce Mara

    2011-01-01

    Carboxymethyl-glucan (CM-G) is a soluble derivative from Saccharomyces cerevisiae (1 → 3)(1 → 6)-β-D-glucan. The protective efficiency of CM-G against DNA damage in cells from patients with advanced prostate cancer (PCa), and undergoing Androgen Deprivation Therapy (ADT), was evaluated. DNA damage scores were obtained by the comet assay, both before and after treatment with CM-G. The reduction in DNA damage, ranging from 18% to 87%, with an average of 59%, was not related to the increased number of leukocytes in peripheral blood. The results demonstrate for the first time the protective effect of CM-G against DNA damage in patients with advanced PCa. Among smokers, three presented the highest reduction in DNA damage after treatment with CM-G. There was no observable relationship between DNA damage scores before and after treatment, and age, alcoholism and radiotherapy. PMID:21637556

  17. Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.

    PubMed Central

    Balagúe, C; Kalla, M; Zhang, W W

    1997-01-01

    Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78. PMID:9060699

  18. A synthetic consensus anti–spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates

    PubMed Central

    Muthumani, Karuppiah; Falzarano, Darryl; Reuschel, Emma L.; Tingey, Colleen; Flingai, Seleeke; Villarreal, Daniel O.; Wise, Megan; Patel, Ami; Izmirly, Abdullah; Aljuaid, Abdulelah; Seliga, Alecia M.; Soule, Geoff; Morrow, Matthew; Kraynyak, Kimberly A.; Khan, Amir S.; Scott, Dana P.; Feldmann, Friederike; LaCasse, Rachel; Meade-White, Kimberly; Okumura, Atsushi; Ugen, Kenneth E.; Sardesai, Niranjan Y.; Kim, J. Joseph; Kobinger, Gary; Feldmann, Heinz; Weiner, David B.

    2015-01-01

    First identified in 2012, Middle East respiratory syndrome (MERS) is caused by an emerging human coronavirus, which is distinct from the severe acute respiratory syndrome coronavirus (SARS-CoV), and represents a novel member of the lineage C betacoronoviruses. Since its identification, MERS coronavirus (MERS-CoV) has been linked to more than 1372 infections manifesting with severe morbidity and, often, mortality (about 495 deaths) in the Arabian Peninsula, Europe, and, most recently, the United States. Human-to-human transmission has been documented, with nosocomial transmission appearing to be an important route of infection. The recent increase in cases of MERS in the Middle East coupled with the lack of approved antiviral therapies or vaccines to treat or prevent this infection are causes for concern. We report on the development of a synthetic DNA vaccine against MERS-CoV. An optimized DNA vaccine encoding the MERS spike protein induced potent cellular immunity and antigen-specific neutralizing antibodies in mice, macaques, and camels. Vaccinated rhesus macaques seroconverted rapidly and exhibited high levels of virus-neutralizing activity. Upon MERS viral challenge, all of the monkeys in the control-vaccinated group developed characteristic disease, including pneumonia. Vaccinated macaques were protected and failed to demonstrate any clinical or radiographic signs of pneumonia. These studies demonstrate that a consensus MERS spike protein synthetic DNA vaccine can induce protective responses against viral challenge, indicating that this strategy may have value as a possible vaccine modality against this emerging pathogen. PMID:26290414

  19. P53 Binding Protein 1 (53bp1) Is an Early Participant in the Cellular Response to DNA Double-Strand Breaks

    PubMed Central

    Schultz, Linda B.; Chehab, Nabil H.; Malikzay, Asra; Halazonetis, Thanos D.

    2000-01-01

    p53 binding protein 1 (53BP1), a protein proposed to function as a transcriptional coactivator of the p53 tumor suppressor, has BRCT domains with high homology to the Saccharomyces cerevisiae Rad9p DNA damage checkpoint protein. To examine whether 53BP1 has a role in the cellular response to DNA damage, we probed its intracellular localization by immunofluorescence. In untreated primary cells and U2OS osteosarcoma cells, 53BP1 exhibited diffuse nuclear staining; whereas, within 5–15 min after exposure to ionizing radiation (IR), 53BP1 localized at discreet nuclear foci. We propose that these foci represent sites of processing of DNA double-strand breaks (DSBs), because they were induced by IR and chemicals that cause DSBs, but not by ultraviolet light; their peak number approximated the number of DSBs induced by IR and decreased over time with kinetics that parallel the rate of DNA repair; and they colocalized with IR-induced Mre11/NBS and γ-H2AX foci, which have been previously shown to localize at sites of DSBs. Formation of 53BP1 foci after irradiation was not dependent on ataxia-telangiectasia mutated (ATM), Nijmegen breakage syndrome (NBS1), or wild-type p53. Thus, the fast kinetics of 53BP1 focus formation after irradiation and the lack of dependency on ATM and NBS1 suggest that 53BP1 functions early in the cellular response to DNA DSBs. PMID:11134068

  20. Induction of Broad Cytotoxic T Cells by Protective DNA Vaccination Against Marburg and Ebola

    PubMed Central

    Shedlock, Devon J; Aviles, Jenna; Talbott, Kendra T; Wong, Gary; Wu, Stephan J; Villarreal, Daniel O; Myles, Devin JF; Croyle, Maria A; Yan, Jian; Kobinger, Gary P; Weiner, David B

    2013-01-01

    Marburg and Ebola hemorrhagic fevers have been described as the most virulent viral diseases known to man due to associative lethality rates of up to 90%. Death can occur within days to weeks of exposure and there is currently no licensed vaccine or therapeutic. Recent evidence suggests an important role for antiviral T cells in conferring protection, but little detailed analysis of this response as driven by a protective vaccine has been reported. We developed a synthetic polyvalent-filovirus DNA vaccine against Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV). Preclinical efficacy studies were performed in guinea pigs and mice using rodent-adapted viruses, whereas murine T-cell responses were extensively analyzed using a novel modified assay described herein. Vaccination was highly potent, elicited robust neutralizing antibodies, and completely protected against MARV and ZEBOV challenge. Comprehensive T-cell analysis revealed cytotoxic T lymphocytes (CTLs) of great magnitude, epitopic breadth, and Th1-type marker expression. This model provides an important preclinical tool for studying protective immune correlates that could be applied to existing platforms. Data herein support further evaluation of this enhanced gene-based approach in nonhuman primate studies for in depth analyses of T-cell epitopes in understanding protective efficacy. PMID:23670573

  1. MPT-51/CpG DNA vaccine protects mice against Mycobacterium tuberculosis.

    PubMed

    Silva, Bruna Daniella de Souza; da Silva, Ediane Batista; do Nascimento, Ivan Pereira; Dos Reis, Michelle Cristina Guerreiro; Kipnis, André; Junqueira-Kipnis, Ana Paula

    2009-07-16

    Tuberculosis (TB) is a severe infectious disease that kills approximately two million people worldwide every year. Because BCG protection is variable and does not protects adults, there is a great need for a new vaccine against TB that does not represent a risk for immunocompromised patients and that is also capable of protecting adult individuals. MPT-51 is a protein found in the genome of mycobacteria and binds to the fibronectin of the extracellular matrix, which may have a role in host tissue attachment and virulence. In order to test the usefulness of MPT-51 as a subunit vaccine, BALB/c were vaccinated and challenged with Mycobacterium tuberculosis. The infection of BALB/c with M. tuberculosis increased the number of IFN-gamma(+) T lymphocytes specific to MPT-51 in the spleen and lungs. Inoculation with rMPT-51/FIA and with rMPT-51/CpG DNA in non-infected BALB/c increased the amounts of IFN-gamma(+) T lymphocytes. Inoculation with rMPT-51/FIA also induced a humoral response specific to MPT-51. CFU counts of lung tissues done 60 days after infection showed a reduction of about 2 log in the bacteria load in the group of animals inoculated with rMPT-51/CpG DNA. These results make MPT-51 a valuable component to be further evaluated in the development of other subunit vaccines.

  2. Short-step chemical synthesis of DNA by use of MMTrS group for protection of 5'-hydroxyl group.

    PubMed

    Shiraishi, Miyuki; Utagawa, Eri; Ohkubo, Akihiro; Sekine, Mitsuo; Seio, Kohji

    2007-01-01

    4-methoxytrithylthio (MMTrS) group was applied for the appropriately protected four canonical nucleosides. We prepared the phosphoroamidite units by use of these nucleosides and developed the synthesis of oligodeoxynucleotides without any acidic treatment. Moreover, the new DNA synthesis protocol was applied to an automated DNA synthesizer for the synthesis of longer oligodeoxynucleotides. PMID:18029620

  3. Human REV3 DNA Polymerase Zeta Localizes to Mitochondria and Protects the Mitochondrial Genome.

    PubMed

    Singh, Bhupendra; Li, Xiurong; Owens, Kjerstin M; Vanniarajan, Ayyasamy; Liang, Ping; Singh, Keshav K

    2015-01-01

    To date, mitochondrial DNA polymerase γ (POLG) is the only polymerase known to be present in mammalian mitochondria. A dogma in the mitochondria field is that there is no other polymerase present in the mitochondria of mammalian cells. Here we demonstrate localization of REV3 DNA polymerase in the mammalian mitochondria. We demonstrate localization of REV3 in the mitochondria of mammalian tissue as well as cell lines. REV3 associates with POLG and mitochondrial DNA and protects the mitochondrial genome from DNA damage. Inactivation of Rev3 leads to reduced mitochondrial membrane potential, reduced OXPHOS activity, and increased glucose consumption. Conversely, inhibition of the OXPHOS increases expression of Rev3. Rev3 expression is increased in human primary breast tumors and breast cancer cell lines. Inactivation of Rev3 decreases cell migration and invasion, and localization of Rev3 in mitochondria increases survival and the invasive potential of cancer cells. Taken together, we demonstrate that REV3 functions in mammalian mitochondria and that mitochondrial REV3 is associated with the tumorigenic potential of cells.

  4. Dormant Bacillus spores protect their DNA in crystalline nucleoids against environmental stress.

    PubMed

    Dittmann, Christin; Han, Hong-Mei; Grabenbauer, Markus; Laue, Michael

    2015-08-01

    Bacterial spores of the genera Bacillus and Clostridium are extremely resistant against desiccation, heat and radiation and involved in the spread and pathogenicity of health relevant species such as Bacillus anthracis (anthrax) or Clostridium botulinum. While the resistance of spores is very well documented, underlying mechanisms are not fully understood. In this study we show, by cryo-electron microscopy of vitreous sections and particular resin thin section electron microscopy, that dormant Bacillus spores possess highly ordered crystalline core structures, which contain the DNA, but only if small acid soluble proteins (SASPs) are present. We found those core structures in spores of all Bacillus species investigated, including spores of anthrax. Similar core structures were detected in Geobacillus and Clostridium species which suggest that highly ordered, at least partially crystalline core regions represent a general feature of bacterial endospores. The crystalline core structures disintegrate in a period during spore germination, when resistance against most stresses is lost. Our results suggest that the DNA is tightly packed into a crystalline nucleoid by binding SASPs, which stabilizes DNA fibrils and protects them against modification. Thus, the crystalline nucleoid seems to be the structural and functional correlate for the remarkable stability of the DNA in bacterial endospores. PMID:26094877

  5. Chromosome 11 protects against DNA damage by reactive oxygen species (ROS)

    SciTech Connect

    Hofseth, L.J.; Rosin, M.P.

    1994-12-31

    Recent evidence suggests that chromosome 11 plays an important role in determining a cell`s sensitivity to DNA damage by ROS. The induction of micronuclei (MN) by treatment with ROS was found to be reduced in a bladder carcinoma cell line ({open_quotes}parent{close_quotes}) when a normal chromosome 11 was inserted ({open_quotes}hybrid{close_quotes}). These studies have been extended to include a third cell line ({open_quotes}revertant{close_quotes}) derived from the {open_quotes}hybrid{close_quotes} by spontaneous loss of the chromosome (11) insert. In this study, MN induction and nuclear division indices (NDI) were determined in all 3 lines after exposure to either hydrogen peroxide (H{sub 2}O{sub 2}) (0-32.3 {mu}M) or to {gamma}-radiation (0-3 Gy). For both agents, there was a significant protection against induction of MN in the hybrid cell line (p<0.0001). This protection was lost in the revertant line. There was significantly more protection against H{sub 2}O{sub 2}-induced damage than against damage by irradiation. At the highest dose for H{sub 2}O{sub 2} (32.3{mu}M), induced MN frequencies for hybrid cells (3.7%) were approximately 5-fold lower than frequencies in the parent (17.1%) or revertant (18.6%) cells. In contrast, after 1 Gy of irradiation, induced MN frequencies in hybrid cells (18.7%) were 1.4-fold less than either the parent (26.2%) or revertant (27.6%) cells (ratio at 3 Gy was 1.4-fold also). The 2 treatments also had a different effect on the NDI (an index of toxicity and/or growth inhibition). Both treatments induced a decrease in NDI. The chromosome 11 insert protected hybrid cells from this effect but only after exposure to H{sub 2}O{sub 2} and not to irradiation. These data suggest a protective effect against ROS. Since only a portion of DNA damage from {gamma}-ray treatment is due to ROS, this may account for the reduced ability of the chromosome 11 to provide a protective effect against DNA damage due to {gamma}-ray exposure.

  6. A New Blood Collection Device Minimizes Cellular DNA Release During Sample Storage and Shipping When Compared to a Standard Device

    PubMed Central

    Norton, Sheila E; Luna, Kristin K; Lechner, Joel M; Qin, Jianbing; Fernando, M Rohan

    2013-01-01

    Background Cell-free DNA (cfDNA) circulating in blood is currently used for noninvasive diagnostic and prognostic tests. Minimizing background DNA is vital for detection of low abundance cfDNA. We investigated whether a new blood collection device could reduce background levels of genomic DNA (gDNA) in plasma compared to K3EDTA tubes, when subjected to conditions that may occur during sample storage and shipping. Methods Blood samples were drawn from healthy donors into K3EDTA and Cell-Free DNA™ BCT (BCT). To simulate shipping, samples were shaken or left unshaken. In a shipping study, samples were shipped or not shipped. To assess temperature variations, samples were incubated at 6°C, 22°C, and 37°C. In all cases, plasma was harvested by centrifugation and total plasma DNA (pDNA) assayed by quantitative real-time polymerase chain reaction (qPCR). Results Shaking and shipping blood in K3EDTA tubes showed significant increases in pDNA, whereas no change was seen in BCTs. Blood in K3EDTA tubes incubated at 6°C, 22°C, and 37°C showed increases in pDNA while pDNA from BCTs remained stable. Conclusions BCTs prevent increases in gDNA levels that can occur during sample storage and shipping. This new device permits low abundance DNA target detection and allows accurate cfDNA concentrations. PMID:23852790

  7. Simultaneous disruption of two DNA polymerases, Polη and Polζ, in Avian DT40 cells unmasks the role of Polη in cellular response to various DNA lesions.

    PubMed

    Hirota, Kouji; Sonoda, Eiichiro; Kawamoto, Takuo; Motegi, Akira; Masutani, Chikahide; Hanaoka, Fumio; Szüts, Dávid; Iwai, Shigenori; Sale, Julian E; Lehmann, Alan; Takeda, Shunichi

    2010-10-07

    Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ζ by generating POLη(-/-)/POLζ(-/-) cells from the chicken DT40 cell line. POLζ(-/-) cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Polη plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLη(-/-)/POLζ(-/-) cells shows that, unexpectedly, the loss of Polη significantly rescued all mutant phenotypes of POLζ(-/-) cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Polη contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells.

  8. DNA sensor's selectivity enhancement and protection from contaminating nucleases due to a hydrated ionic liquid.

    PubMed

    Tateishi-Karimata, Hisae; Pramanik, Smritimoy; Sugimoto, Naoki

    2015-07-01

    The thermodynamic stability of certain mismatched base pairs has made the development of DNA sequence sensing systems challenging. Thus, the stability of fully matched and mismatched DNA oligonucleotides in the hydrated ionic liquid choline dihydrogen phosphate (choline dhp) was investigated. Mismatched base pairs were significantly destabilized in choline dhp relative to those in aqueous buffer. A molecular beacon that forms a triplex with a conserved HIV-1 sequence was then designed and tested in choline dhp. The molecular beacon specifically detected the target duplex via triplex formation at concentrations as low as 1 pmol per 10 μL with 10,000-fold sequence selectivity. Moreover, the molecular beacon was protected from a contaminating nuclease in choline dhp, and DNAs in aqueous solutions were not sufficiently stable for practical use.

  9. DNA Binding and Photocleavage Properties, Cellular Uptake and Localization, and in-Vitro Cytotoxicity of Dinuclear Ruthenium(II) Complexes with Varying Lengths in Bridging Alkyl Linkers.

    PubMed

    Liu, Ping; Wu, Bao-Yan; Liu, Jin; Dai, Yong-Cheng; Wang, You-Jun; Wang, Ke-Zhi

    2016-02-15

    Two new dinuclear Ru(II) polypyridyl complexes containing three and ten methylene chains in their bridging linkers are synthesized and characterized. Their calf thymus DNA-binding and plasmid DNA photocleavage behaviors are comparatively studied with a previously reported, six-methylene-containing analog by absorption and luminescence spectroscopy, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, DNA viscosity measurements, DNA thermal denaturation, and agarose gel electrophoresis analyses. Theoretical calculations applying the density functional theory (DFT) method for the three complexes are also performed to understand experimentally observed DNA binding properties. The results show that the two complexes partially intercalate between the base pairs of DNA. Cellular uptake and colocalization studies have demonstrated that the complexes could enter HeLa cells efficiently and localize within lysosomes. The in-vitro antitumor activity against HeLa and MCF-7 tumor cells of the complexes are studied by MTT cytotoxic analysis. A new method, high-content analysis (HCA), is also used to assess cytotoxicity, apoptosis and cell cycle arrest of the three complexes. The results show that the lengths of the alkyl linkers could effectively tune their biological properties and that HCA is suitable for rapidly identifying cytotoxicity and can be substituted for MTT assays to evaluate the cell cytotoxicity of chemotherapeutic agents.

  10. pH effects on binding between the anthrax protective antigen and the host cellular receptor CMG2

    PubMed Central

    Rajapaksha, Maheshinie; Lovell, Scott; Janowiak, Blythe E; Andra, Kiran K; Battaile, Kevin P; Bann, James G

    2012-01-01

    The anthrax protective antigen (PA) binds to the host cellular receptor capillary morphogenesis protein 2 (CMG2) with high affinity. To gain a better understanding of how pH may affect binding to the receptor, we have investigated the kinetics of binding as a function of pH to the full-length monomeric PA and to two variants: a 2-fluorohistidine-labeled PA (2-FHisPA), which is ∼1 pH unit more stable to variations in pH than WT, and an ∼1 pH unit less stable variant in which Trp346 in the domain 2β3-2β4 loop is substituted with a Phe (W346F). We show using stopped-flow fluorescence that the binding rate increases as the pH is lowered for all proteins, with little influence on the rate of dissociation. In addition, we have crystallized PA and the two variants and examine the influence of pH on structure. In contrast to previous X-ray studies, the domain 2β3-2β4 loop undergoes little change in structure from pH ∼8 to 5.5 for the WT protein, but for the 2-FHis labeled and W346F mutant there are changes in structure consistent with previous X-ray studies. In accord with pH stability studies, we find that the average B-factor values increase by ∼20–30% for all three proteins at low pH. Our results suggest that for the full-length PA, low pH increases the binding affinity, likely through a change in structure that favors a more “bound-like” conformation. PMID:22855243

  11. Protective effect of naringin against ischemic reperfusion cerebral injury: possible neurobehavioral, biochemical and cellular alterations in rat brain.

    PubMed

    Gaur, Vaibhav; Aggarwal, Aditi; Kumar, Anil

    2009-08-15

    The present study was conducted with an aim to explore the possible role of naringin against ischemia reperfusion induced-neurobehavioral alterations, oxidative damage, cellular and histopathological alterations in cortex, striatum, hippocampus areas of brain. Male Wistar rats (200-220 g) were subjected to bilateral carotid artery occlusion for 30 min followed by reperfusion for 24 h to induce reperfusion (I/R) cerebral injury. Naringin (50, 100 mg/kg, i.p.) was administered for 7 days continuously before animals were subjected to ischemia reperfusion injury. Various behavioral tests [locomotor activity, neurological score (inclined beam test), transfer latency, resistance to lateral push] and biochemical parameters (lipid peroxidation, nitrite level, reduced glutathione, superoxide dismutase and catalase activity), mitochondrial enzyme dysfunctions (Complex I, II, III and IV) in cortex, striatum, hippocampus of brain and histopathological alterations were assessed subsequently. Seven days naringin (50 and 100 mg/kg) treatment significantly improved neurobehavioral alterations (improved locomotor activity, inclined beam walking and reduced resistance to lateral push, transfer latency) as compared to control ischemia reperfusion. Naringin (50 mg/kg and 100 mg/kg) treatment significantly attenuated oxidative damage as indicated by reduced lipid peroxidation, nitrite concentration, restored reduced glutathione and catalase activity and mitochondrial enzyme activities in cortex, striatum, cerebellum as compared to control (ischemia reperfusion) animals. In addition, naringin treatment significantly reversed histopathological alterations in cortex, striatum, hippocampus areas as compared to control (ischemia reperfusion). Present study suggests the protective effect of naringin and its therapeutic potential against ischemia reperfusion induced and related behavioral alterations in rats. PMID:19577560

  12. Alpha/Beta Interferon Protects against Lethal West Nile Virus Infection by Restricting Cellular Tropism and Enhancing Neuronal Survival

    PubMed Central

    Samuel, Melanie A.; Diamond, Michael S.

    2005-01-01

    West Nile virus (WNV) is a mosquito-borne flavivirus that is neurotropic in humans, birds, and other animals. While adaptive immunity plays an important role in preventing WNV spread to the central nervous system (CNS), little is known about how alpha/beta interferon (IFN-α/β) protects against peripheral and CNS infection. In this study, we examine the virulence and tropism of WNV in IFN-α/β receptor-deficient (IFN- α/βR−/−) mice and primary neuronal cultures. IFN-α/βR−/− mice were acutely susceptible to WNV infection through subcutaneous inoculation, with 100% mortality and a mean time to death (MTD) of 4.6 ± 0.7 and 3.8± 0.5 days after infection with 100 and 102 PFU, respectively. In contrast, congenic wild-type 129Sv/Ev mice infected with 102 PFU showed 62% mortality and a MTD of 11.9 ± 1.9 days. IFN-α/βR−/− mice developed high viral loads by day 3 after infection in nearly all tissues assayed, including many that were not infected in wild-type mice. IFN-α/βR−/− mice also demonstrated altered cellular tropism, with increased infection in macrophages, B cells, and T cells in the spleen. Additionally, treatment of primary wild-type neurons in vitro with IFN-β either before or after infection increased neuronal survival independent of its effect on WNV replication. Collectively, our data suggest that IFN-α/β controls WNV infection by restricting tropism and viral burden and by preventing death of infected neurons. PMID:16227257

  13. Oral azathioprine leads to higher incorporation of 6-thioguanine in DNA of skin than liver: the protective role of the Keap1/Nrf2/ARE pathway.

    PubMed

    Kalra, Sukirti; Zhang, Ying; Knatko, Elena V; Finlayson, Stewart; Yamamoto, Masayuki; Dinkova-Kostova, Albena T

    2011-10-01

    Azathioprine is a widely used anti-inflammatory, immunosuppressive, and anticancer agent. However, chronic treatment with this drug is associated with a profoundly increased risk (in certain cases by more than 100-fold) of developing squamous cell carcinoma of the skin. Incorporation of its ultimate metabolite, thio-dGTP, in DNA results in partial substitution of guanine with 6-thioguanine which, combined with exposure to UVA radiation, creates a source of synergistic mutagenic damage to DNA. We now report that oral treatment with azathioprine leads to a much greater incorporation of 6-thioguanine in DNA of mouse skin than liver. These higher levels of 6-thioguanine, together with the fact that the skin is constantly exposed to UV radiation from the sun, may be responsible, at least in part, for the increased susceptibility of this organ to tumor development. Genetic upregulation of the Keap1/Nrf2/ARE pathway, a major cellular regulator of the expression of a network of cytoprotective genes, reduces the incorporation of 6-thioguanine in DNA of both skin and liver following treatment with azathioprine. Similarly, pharmacologic activation of the pathway by the potent inducer sulforaphane results in lower 6-thioguanine incorporation in DNA and protects 6-thioguanine-treated cells against oxidative stress following exposure to UVA radiation. Protection is accompanied by increased levels of glutathione and induction of multidrug resistance-associated protein 4, an organic anion efflux pump that also exports nucleoside monophosphate analogues. Our findings suggest that activation of the Keap1/Nrf2/ARE pathway could reduce the risk for skin cancer in patients receiving long-term azathioprine therapy. PMID:21803983

  14. AP39, a Mitochondria-Targeted Hydrogen Sulfide Donor, Supports Cellular Bioenergetics and Protects against Alzheimer's Disease by Preserving Mitochondrial Function in APP/PS1 Mice and Neurons

    PubMed Central

    Zhao, Feng-li; Fang, Fang; Qiao, Pei-feng; Yan, Ning; Gao, Dan; Yan, Yong

    2016-01-01

    Increasing evidence suggests that mitochondrial functions are altered in AD and play an important role in AD pathogenesis. It has been established that H2S homeostasis is balanced in AD. The emerging mitochondrial roles of H2S include antioxidation, antiapoptosis, and the modulation of cellular bioenergetics. Here, using primary neurons from the well-characterized APP/PS1 transgenic mouse model, we studied the effects of AP39 (a newly synthesized mitochondrially targeted H2S donor) on mitochondrial function. AP39 increased intracellular H2S levels, mainly in mitochondrial regions. AP39 exerted dose-dependent effects on mitochondrial activity in APP/PS1 neurons, including increased cellular bioenergy metabolism and cell viability at low concentrations (25–100 nM) and decreased energy production and cell viability at a high concentration (250 nM). Furthermore, AP39 (100 nM) increased ATP levels, protected mitochondrial DNA, and decreased ROS generation. AP39 regulated mitochondrial dynamics, shifting from fission toward fusion. After 6 weeks, AP39 administration to APP/PS1 mice significantly ameliorated their spatial memory deficits in the Morris water maze and NORT and reduced Aβ deposition in their brains. Additionally, AP39 inhibited brain atrophy in APP/PS1 mice. Based on these results, AP39 was proposed as a promising drug candidate for AD treatment, and its anti-AD mechanism may involve protection against mitochondrial damage. PMID:27057285

  15. Zingerone protects against stannous chloride-induced and hydrogen peroxide-induced oxidative DNA damage in vitro.

    PubMed

    Rajan, Iyappan; Narayanan, Nithya; Rabindran, Remitha; Jayasree, P R; Manish Kumar, P R

    2013-12-01

    In this paper, we report the dose-dependent antioxidant activity and DNA protective effects of zingerone. At 500 μg/mL, the DPPH radical scavenging activity of zingerone and ascorbic acid as a standard was found to be 86.7 and 94.2 % respectively. At the same concentration, zingerone also showed significant reducing power (absorbance 0.471) compared to that of ascorbic acid (absorbance 0.394). The in vitro toxicity of stannous chloride (SnCl2) was evaluated using genomic and plasmid DNA. SnCl2-induced degradation of genomic DNA was found to occur at a concentration of 0.8 mM onwards with complete degradation at 1.02 mM and above. In the case of plasmid DNA, conversion of supercoiled DNA into the open circular form indicative of DNA nicking activity was observed at a concentration of 0.2 mM onwards; complete conversion was observed at a concentration of 1.02 mM and above. Zingerone was found to confer protection against SnCl2-induced oxidative damage to genomic and plasmid DNA at concentrations of 500 and 750 μg/mL onwards, respectively. This protective effect was further confirmed in the presence of UV/H2O2-a known reactive oxygen species (ROS) generating system-wherein protection by zingerone against ROS-mediated DNA damage was observed at a concentration of 250 μg/mL onwards in a dose-dependent manner. This study clearly indicated the in vitro DNA protective property of zingerone against SnCl2-induced, ROS-mediated DNA damage.

  16. Tc52 amino-terminal-domain DNA carried by attenuated Salmonella enterica serovar Typhimurium induces protection against a Trypanosoma cruzi lethal challenge.

    PubMed

    Matos, Marina N; Cazorla, Silvia I; Bivona, Augusto E; Morales, Celina; Guzmán, Carlos A; Malchiodi, Emilio L

    2014-10-01

    In this work we immunized mice with DNA encoding full-length Tc52 or its amino- or carboxy-terminal (N- and C-term, respectively) domain carried by attenuated Salmonella as a DNA delivery system. As expected, Salmonella-mediated DNA delivery resulted in low antibody titers and a predominantly Th1 response, as shown by the ratio of IgG2a/IgG1-specific antibodies. Despite modest expression of Tc52 in trypomastigotes, the antibodies elicited by vaccination were able to mediate lysis of the trypomastigotes in the presence of complement and inhibit their invasion of mammal cells in vitro. The strongest functional activity was observed with sera from mice immunized with Salmonella carrying the N-term domain (SN-term), followed by Tc52 (STc52), and the C-term domain (SC-term). All immunized groups developed strong cellular responses, with predominant activation of Th1 cells. However, mice immunized with SN-term showed higher levels of interleukin-10 (IL-10), counterbalancing the inflammatory reaction, and also strong activation of Tc52-specific gamma interferon-positive (IFN-γ(+)) CD8(+) T cells. In agreement with this, although all prototypes conferred protection against infection, immunization with SN-term promoted greater protection than that with SC-term for all parameters tested and slightly better protection than that with STc52, especially in the acute stage of infection. We conclude that the N-terminal domain of Tc52 is the section of the protein that confers maximal protection against infection and propose it as a promising candidate for vaccine development. PMID:25069980

  17. Tc52 Amino-Terminal-Domain DNA Carried by Attenuated Salmonella enterica Serovar Typhimurium Induces Protection against a Trypanosoma cruzi Lethal Challenge

    PubMed Central

    Matos, Marina N.; Cazorla, Silvia I.; Bivona, Augusto E.; Morales, Celina; Guzmán, Carlos A.

    2014-01-01

    In this work we immunized mice with DNA encoding full-length Tc52 or its amino- or carboxy-terminal (N- and C-term, respectively) domain carried by attenuated Salmonella as a DNA delivery system. As expected, Salmonella-mediated DNA delivery resulted in low antibody titers and a predominantly Th1 response, as shown by the ratio of IgG2a/IgG1-specific antibodies. Despite modest expression of Tc52 in trypomastigotes, the antibodies elicited by vaccination were able to mediate lysis of the trypomastigotes in the presence of complement and inhibit their invasion of mammal cells in vitro. The strongest functional activity was observed with sera from mice immunized with Salmonella carrying the N-term domain (SN-term), followed by Tc52 (STc52), and the C-term domain (SC-term). All immunized groups developed strong cellular responses, with predominant activation of Th1 cells. However, mice immunized with SN-term showed higher levels of interleukin-10 (IL-10), counterbalancing the inflammatory reaction, and also strong activation of Tc52-specific gamma interferon-positive (IFN-γ+) CD8+ T cells. In agreement with this, although all prototypes conferred protection against infection, immunization with SN-term promoted greater protection than that with SC-term for all parameters tested and slightly better protection than that with STc52, especially in the acute stage of infection. We conclude that the N-terminal domain of Tc52 is the section of the protein that confers maximal protection against infection and propose it as a promising candidate for vaccine development. PMID:25069980

  18. Apurinic/Apyrimidinic Endonuclease 1 Upregulation Reduces Oxidative DNA Damage and Protects Hippocampal Neurons from Ischemic Injury

    PubMed Central

    Leak, Rehana K.; Li, Peiying; Zhang, Feng; Sulaiman, Hassan H.; Weng, Zhongfang; Wang, Guohua; Stetler, R. Anne; Shi, Yejie; Cao, Guodong

    2015-01-01

    Abstract Aims: Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme that participates in base-excision repair of oxidative DNA damage and in the redox activation of transcription factors. We tested the hypothesis that APE1 upregulation protects neuronal structure and function against transient global cerebral ischemia (tGCI). Results: Upregulation of APE1 by low-dose proton irradiation (PI) or by transgene overexpression protected hippocampal CA1 neurons against tGCI-induced cell loss and reduced apurinic/apyrimidinic sites and DNA fragmentation. Conversely, APE1 knockdown attenuated the protection afforded by PI and ischemic preconditioning. APE1 overexpression inhibited the DNA damage response, as evidenced by lower phospho-histone H2A and p53-upregulated modulator of apoptosis levels. APE1 overexpression also partially rescued dendritic spines and attenuated the decrease in field excitatory postsynaptic potentials in hippocampal CA1. Presynaptic and postsynaptic markers were reduced after tGCI, and this effect was blunted in APE1 transgenics. The Morris water maze test revealed that APE1 protected against learning and memory deficits for at least 27 days post-injury. Animals expressing DNA repair-disabled mutant APE1 (D210A) exhibited more DNA damage than wild-type controls and were not protected against tGCI-induced cell loss. Innovation: This is the first study that thoroughly characterizes structural and functional protection against ischemia after APE1 upregulation by measuring synaptic markers, electrophysiological function, and long-term neurological deficits in vivo. Furthermore, disabling the DNA repair activity of APE1 was found to abrogate its protective impact. Conclusion: APE1 upregulation, either endogenously or through transgene overexpression, protects DNA, neuronal structures, synaptic function, and behavioral output from ischemic injury. Antioxid. Redox Signal. 22, 135–148. PMID:24180454

  19. Introduction of translation stop codons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

    USGS Publications Warehouse

    Garver, K.A.; Conway, C.M.; Kurath, G.

    2006-01-01

    A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine. ?? Springer Science+Business Media, Inc. 2006.

  20. Protective Immunity to H7N9 Influenza viruses elicited by synthetic DNA Vaccine

    PubMed Central

    Yan, Jian; Villarreal, Daniel O.; Racine, Trina; Chu, Jaemi S.; Walters, Jewell N.; Morrow, Matthew P.; Khan, Amir S.; Sardesai, Niranjan Y.; Kim, J. Joseph; Kobinger, Gary P.; Weiner, David B.

    2014-01-01

    Despite an intensive vaccine program influenza infections remain a major health problem, due to the viruses’ ability to change its envelope glycoprotein hemagglutinin (HA), through shift and drift, permitting influenza to escape protection induced by current vaccines or natural immunity. Recently a new variant, H7N9, has emerged in China causing global concern. First, there have been more than 130 laboratory-confirmed human infections resulting in an alarmingly high death rate (32.3%). Second, genetic changes found in H7N9 appear to be associated with enabling avian influenza viruses to spread more effectively in mammals, thus transmitting infections on a larger scale. Currently, no vaccines or drugs are effectively able to target H7N9. Here, we report the rapid development of a synthetic consensus DNA vaccine (pH7HA) to elicit potent protective immunity against the H7N9 viruses. We show that pH7HA induces broad antibody responses that bind to divergent HAs from multiple new members of the H7N9 family. These antibody responses result in high-titer HAI against H7N9. Simultaneously, this vaccine induces potent polyfunctional effector CD4 and CD8 T cell memory responses. Animals vaccinated with pH7HA are completely protected from H7N9 virus infection and any morbidity associated with lethal challenge. This study establishes that this synthetic consensus DNA vaccine represents a new tool for targeting emerging infection, and more importantly, its design, testing and development into seed stock for vaccine production in a few days in the pandemic setting has significant implications for the rapid deployment of vaccines protecting against emerging infectious diseases. PMID:24631084

  1. Protective immunity to H7N9 influenza viruses elicited by synthetic DNA vaccine.

    PubMed

    Yan, Jian; Villarreal, Daniel O; Racine, Trina; Chu, Jaemi S; Walters, Jewell N; Morrow, Matthew P; Khan, Amir S; Sardesai, Niranjan Y; Kim, J Joseph; Kobinger, Gary P; Weiner, David B

    2014-05-19

    Despite an intensive vaccine program influenza infections remain a major health problem, due to the viruses' ability to change its envelope glycoprotein hemagglutinin (HA), through shift and drift, permitting influenza to escape protection induced by current vaccines or natural immunity. Recently a new variant, H7N9, has emerged in China causing global concern. First, there have been more than 130 laboratory-confirmed human infections resulting in an alarmingly high death rate (32.3%). Second, genetic changes found in H7N9 appear to be associated with enabling avian influenza viruses to spread more effectively in mammals, thus transmitting infections on a larger scale. Currently, no vaccines or drugs are effectively able to target H7N9. Here, we report the rapid development of a synthetic consensus DNA vaccine (pH7HA) to elicit potent protective immunity against the H7N9 viruses. We show that pH7HA induces broad antibody responses that bind to divergent HAs from multiple new members of the H7N9 family. These antibody responses result in high-titer HAI against H7N9. Simultaneously, this vaccine induces potent polyfunctional effector CD4 and CD8T cell memory responses. Animals vaccinated with pH7HA are completely protected from H7N9 virus infection and any morbidity associated with lethal challenge. This study establishes that this synthetic consensus DNA vaccine represents a new tool for targeting emerging infection, and more importantly, its design, testing and development into seed stock for vaccine production in a few days in the pandemic setting has significant implications for the rapid deployment of vaccines protecting against emerging infectious diseases.

  2. Energy, ageing, fidelity and sex: oocyte mitochondrial DNA as a protected genetic template

    PubMed Central

    de Paula, Wilson B. M.; Lucas, Cathy H.; Agip, Ahmed-Noor A.; Vizcay-Barrena, Gema; Allen, John F.

    2013-01-01

    Oxidative phosphorylation couples ATP synthesis to respiratory electron transport. In eukaryotes, this coupling occurs in mitochondria, which carry DNA. Respiratory electron transport in the presence of molecular oxygen generates free radicals, reactive oxygen species (ROS), which are mutagenic. In animals, mutational damage to mitochondrial DNA therefore accumulates within the lifespan of the individual. Fertilization generally requires motility of one gamete, and motility requires ATP. It has been proposed that oxidative phosphorylation is nevertheless absent in the special case of quiescent, template mitochondria, that these remain sequestered in oocytes and female germ lines and that oocyte mitochondrial DNA is thus protected from damage, but evidence to support that view has hitherto been lacking. Here we show that female gametes of Aurelia aurita, the common jellyfish, do not transcribe mitochondrial DNA, lack electron transport, and produce no free radicals. In contrast, male gametes actively transcribe mitochondrial genes for respiratory chain components and produce ROS. Electron microscopy shows that this functional division of labour between sperm and egg is accompanied by contrasting mitochondrial morphology. We suggest that mitochondrial anisogamy underlies division of any animal species into two sexes with complementary roles in sexual reproduction. We predict that quiescent oocyte mitochondria contain DNA as an unexpressed template that avoids mutational accumulation by being transmitted through the female germ line. The active descendants of oocyte mitochondria perform oxidative phosphorylation in somatic cells and in male gametes of each new generation, and the mutations that they accumulated are not inherited. We propose that the avoidance of ROS-dependent mutation is the evolutionary pressure underlying maternal mitochondrial inheritance and the developmental origin of the female germ line. PMID:23754815

  3. Energy, ageing, fidelity and sex: oocyte mitochondrial DNA as a protected genetic template.

    PubMed

    de Paula, Wilson B M; Lucas, Cathy H; Agip, Ahmed-Noor A; Vizcay-Barrena, Gema; Allen, John F

    2013-07-19

    Oxidative phosphorylation couples ATP synthesis to respiratory electron transport. In eukaryotes, this coupling occurs in mitochondria, which carry DNA. Respiratory electron transport in the presence of molecular oxygen generates free radicals, reactive oxygen species (ROS), which are mutagenic. In animals, mutational damage to mitochondrial DNA therefore accumulates within the lifespan of the individual. Fertilization generally requires motility of one gamete, and motility requires ATP. It has been proposed that oxidative phosphorylation is nevertheless absent in the special case of quiescent, template mitochondria, that these remain sequestered in oocytes and female germ lines and that oocyte mitochondrial DNA is thus protected from damage, but evidence to support that view has hitherto been lacking. Here we show that female gametes of Aurelia aurita, the common jellyfish, do not transcribe mitochondrial DNA, lack electron transport, and produce no free radicals. In contrast, male gametes actively transcribe mitochondrial genes for respiratory chain components and produce ROS. Electron microscopy shows that this functional division of labour between sperm and egg is accompanied by contrasting mitochondrial morphology. We suggest that mitochondrial anisogamy underlies division of any animal species into two sexes with complementary roles in sexual reproduction. We predict that quiescent oocyte mitochondria contain DNA as an unexpressed template that avoids mutational accumulation by being transmitted through the female germ line. The active descendants of oocyte mitochondria perform oxidative phosphorylation in somatic cells and in male gametes of each new generation, and the mutations that they accumulated are not inherited. We propose that the avoidance of ROS-dependent mutation is the evolutionary pressure underlying maternal mitochondrial inheritance and the developmental origin of the female germ line.

  4. Protection of mice against challenge with Bacillus anthracis STI spores after DNA vaccination.

    PubMed

    Hahn, Ulrike K; Alex, Michaela; Czerny, Claus-Peter; Böhm, Reinhard; Beyer, Wolfgang

    2004-07-01

    Immune responses against the protective antigen (PA) of Bacillus anthracis are known to confer immunity against anthrax. We evaluated the efficacy of genetic vaccination with plasmid vectors encoding PA, in protecting mice from a lethal challenge with B. anthracis STI spores. BALB/c and A/J mice were immunized via gene gun inoculation, using eukaryotic expression vectors with different cellular targeting signals for the encoded antigen. The vector pSecTag PA83, encoding the full-length PA protein, has a signal sequence for secretion of the expressed protein. The plasmids pCMV/ER PA83 and pCMV/ER PA63, encoding the full-length and the physiologically active form of PA, respectively, target and retain the expressed antigen in the endoplasmic reticulum of transfected cells. All three plasmids induced PA-specific humoral immune responses, predominantly IgG1 antibodies, in mice. Spleen cells collected from plasmid-vaccinated BALB/c mice produced PA-specific interleukin-4, interleukin-5, and interferon-gamma in vitro. Vaccination with either pSecTag PA83 or pCMV/ER PA83 showed significant protection of A/J mice against infection with B. anthracis STI spores. PMID:15293452

  5. Characterization of coal fly ash nanoparticles and their induced in vitro cellular toxicity and oxidative DNA damage in different cell lines.

    PubMed

    Sambandam, Bharathi; Devasena, Thiyagarajan; Islam, Villianur Ibrahim Hairul; Prakhya, Balkrishna Murthy

    2015-09-01

    Coal combustion generates considerable amount of ultrafine particles and exposure to such particulate matter is a major health concern in the developing countries. In this study, we collected nano sized coal fly ash (CFA) and characterized them by scanning electron microscope-energy dispersive X-ray analysis (SEM-EDX), particle size analyzer (PSA) and transmission electron microscope (TEM), and investigated its toxicity in vitro using different cell lines. The imaging techniques showed that the coal fly ash nanoparticles (CFA-NPs) are predominately spherical shaped. The analyses have revealed that the CFA-NPs are 7-50 nm in diameter and contain several heavy metals associated with CFA particles. The studies showed significant amount of toxicity in all cell lines on treatment with CFA-NPs. The cytotoxicity and oxidative DNA damage caused by CFA-NPs were determined by inhibition of cellular metabolism (MTT), total intracellular glutathione (GSH), reactive oxygen species (ROS) and DNA fragmentation in cultured cell lines (Chang liver, HS294T and LL29). The cellular metabolism was inhibited in a dose-dependent manner in CFA-NPs treated cell lines. The CFA-NPs induced ROS and decreased the total intracellular glutathione with increased dose. Further, the CFA-NPs treated cells showed severe DNA laddering as a result of DNA fragmentation.

  6. [Study of blue light induced DNA damage of retinal pigment epithelium(RPE) cells and the protection of vitamin C].

    PubMed

    Zhou, Jian Wei; Ren, Guo Liang; Zhang, Xiao Ming; Zhu, Xi; Lin, Hai Yan; Zhou, Ji Lin

    2003-10-01

    To evaluate protection of vitamin C on blue light-induced DNA damage of human retinal pigment epithelium (RPE) cells. The cultured RPE cells were divided into 3 groups: Control group (no blue light exposure), blue light exposure group (blue light exposure for 20 minutes) and blue light exposure + vitamin C group (blue light exposure + 100 mumol/L vitamin C). Travigen's comet assay kit and Euclid comet assay software were used to assay the DNA damage levels. The DNA percentage in the tail of electrophoretogram in the three groups were 18.44%, 54.42% and 32.43% respectively (p < 0.01). Tail moments were 8.2, 48.3, and 18.4 respectively (p < 0.01). Blue light could induce DNA damage to RPE cells but vitamin C could protect the RPE cells from the blue light-induced DNA damage.

  7. Structural, molecular and cellular functions of MSH2 and MSH6 during DNA mismatch repair, damage signaling and other noncanonical activities

    PubMed Central

    Edelbrock, Michael A.; Kaliyaperumal, Saravanan; Williams, Kandace J.

    2013-01-01

    The field of DNA mismatch repair (MMR) has rapidly expanded after the discovery of the MutHLS repair system in bacteria. By the mid 1990s yeast and human homologues to bacterial MutL and MutS had been identified and their contribution to hereditary non-polyposis colorectal cancer (HNPCC; Lynch Syndrome) was under intense investigation. The human MutS homologue 6 protein (hMSH6), was first reported in 1995 as a G:T binding partner (GTBP) of hMSH2, forming the hMutSα mismatch-binding complex. Signal transduction from each DNA-bound hMutSα complex is accomplished by the hMutLα heterodimer (hMLH1 and hPMS2). Molecular mechanisms and cellular regulation of individual MMR proteins are now areas of intensive research. This review will focus on molecular mechanisms associated with mismatch binding, as well as emerging evidence that MutSα and in particular, MSH6, is a key protein in MMR-dependent DNA damage response and communication with other DNA repair pathways within the cell. MSH6 is unstable in the absence of MSH2, however it is the DNA lesion-binding partner of this heterodimer. MSH6, but not MSH2, has a conserved Phe-X-Glu motif that recognizes and binds several different DNA structural distortions, initiating different cellular responses. hMSH6 also contains the nuclear localization sequences required to shuttle hMutSα into the nucleus. For example, upon binding to O6meG:T, MSH6 triggers a DNA damage response that involves altered phosphorylation within the N-terminal disordered domain of this unique protein. While many investigations have focused on MMR as a post-replication DNA repair mechanism, MMR proteins are expressed and active in all phases of the cell cycle. There is much more to be discovered about regulatory cellular roles that require the presence of MutSα and, in particular, MSH6. PMID:23391514

  8. Low levels of methylmercury induce DNA damage in rats: protective effects of selenium.

    PubMed

    Grotto, Denise; Barcelos, Gustavo R M; Valentini, Juliana; Antunes, Lusânia M G; Angeli, José Pedro F; Garcia, Solange C; Barbosa, Fernando

    2009-03-01

    In this study we examined the possible antigenotoxic effect of selenium (Se) in rats chronically exposed to low levels of methylmercury (MeHg) and the association between glutathione peroxidase (GSH-Px) activity and DNA lesions (via comet assay) in the same exposed animals. Rats were divided into six groups as follows: (Group I) received water; (Group II) received MeHg (100 mug/day); (Group III) received Se (2 mg/L drinking water); (Group IV) received Se (6 mg/L drinking water); (Group V) received MeHg (100 mug/day) and Se (2 mg/L drinking water); (Group VI) received MeHg (100 mug/day) and Se (6 mg/L drinking water). Total treatment time was 100 days. GSH-Px activity was determined spectrophotometrically and DNA damage was determined by comet assay. Mean GSH-Px activity in groups I, II, III, IV, V and VI were, respectively: 40.19 +/- 17.21; 23.63 +/- 6.04; 42.64 +/- 5.70; 38.50 +/- 7.15; 34.54 +/- 6.18 and 41.39 +/- 11.67 nmolNADPH/min/gHb. DNA damage was represented by a mean score from 0 to 300; the results for groups I, II, III, IV, V and VI were, respectively: 6.87 +/- 3.27; 124.12 +/- 13.74; 10.62 +/- 3.81; 13.25 +/- 1.76; 86.87 +/- 11.95 and 76.25 +/- 7.48. There was a significant inhibition of GSH-Px activity in group II compared with group I (P < 0.05). Groups V and VI did not show a difference in enzyme activity compared with groups III and IV, showing the possible protective action of Se. Comet assay presented a significant difference in DNA migration between group II and group I (P < 0.0001). Groups V and VI showed a significant reduction in MeHg-induced genotoxicity (P < 0.001) when compared with group II. A negative correlation (r = -0.559, P < 0.05) was found between GSH-Px activity and DNA lesion, showing that the greater the DNA damage, the lower the GSH-Px activity. Our findings demonstrated the oxidative and genotoxic properties of MeHg, even at low doses. Moreover, Se co-administration reestablished GSH-Px activity and reduced DNA damage.

  9. RSV fusion (F) protein DNA vaccine provides partial protection against viral infection.

    PubMed

    Wu, Hongzhuan; Dennis, Vida A; Pillai, Shreekumar R; Singh, Shree R

    2009-10-01

    The present study was conducted to investigate the feasibility and efficacy of a RSV F DNA vaccine incorporated with a mucosal adjuvant. Two DNA vaccine vectors (DRF-412 and DRF-412-P) were developed containing residues 412-524 of the RSV F gene. These antigenic regions were cloned into the phCMV1 DNA vaccine vector. One of the DNA vaccine vectors, DRF-412, contained the ctxA(2)B region of the cholera toxin gene as a mucosal adjuvant. The in vitro expressions of these DNA vectors were confirmed in Cos-7 cells by indirect immunofluorescence and Western blot analyses. In vivo expression of the cloned gene was further confirmed in mouse muscle tissue by immunohistological analysis. The active transcription of the RSV F gene in mouse muscle cells was confirmed by RT-PCR. The purified DRF-412 and DRF-412-P DNA vectors were used to immunize mice by intramuscular injections. Our results indicated that DRF-412 and DRF-412-P vaccine vectors were as effective as live RSV in inducing neutralization antibody, systemic Ab (IgG, IgG1, IgG2a, and IgG2b) responses, and mucosal antibody responses (Ig A). The Th1 (TNF-alpha, IL-12p70, IFN-gamma, IL-2) and Th2 (IL-10, IL-6) cytokine profiles were analyzed after stimulation of spleen cells from mice immunized with purified RF-412 protein. We observed that mice inoculated with vector DRF-412 induced a higher mixed Th1/Th2 cytokine immune response than DRF-412-P. Reverse transcriptase and quantitative real-time PCR (qRT-PCR) revealed that mice immunized with the DRF-412 vector contained less viral RNA in lung tissue and the lung immunohistology study confirmed that mice immunized with DRF-412 had better protection than those immunized with the DRF-412-P vector. These results indicate that the RSV DRF-412 vaccine vector, which contains the cholera toxin subunit ctxA2B as a mucosal adjuvant may provide a better DNA vaccination strategy against RSV. PMID:19540885

  10. Positive impact of sucrose supplementation during slow freezing of cat ovarian tissues on cellular viability, follicle morphology, and DNA integrity.

    PubMed

    Tanpradit, Nae; Comizzoli, Pierre; Srisuwatanasagul, Sayamon; Chatdarong, Kaywalee

    2015-06-01

    The objectives of the study were to (1) examine and optimize the impact of sucrose during slow freezing and (2) compare the results of two freezing methods (slow freezing and vitrification) on cellular viability (germinal and stromal cells), follicle morphology, DNA integrity, and gap junction protein expression (connexin 43 [Cx 43]). Different sucrose supplementations (0, 0.1, and 0.3 M) in standard freezing medium were compared before and after slow freezing. Ovarian tissue slow frozen using 0.1- (4.0 ± 0.4) or 0.3-M sucrose (3.9 ± 0.5) yielded better follicular viability (number of positive follicles per 0.0625 mm(2)) than the group without sucrose (1.9 ± 0.2; P < 0.05). Morphologically normal primordial follicles were higher in the sucrose-treated groups (0.1 M, 47.4% and 0.3 M, 43.5%) than the group without sucrose (0 M, 33.8%; P < 0.05). Moreover, less apoptotic primordial follicles were found in both sucrose groups (0.1 M, 1.2% and 0.3 M, 1.9%) than the group without sucrose (7.7%; P < 0.05). However, their Cx 43 expression showed no difference among the groups of different sucrose concentrations. In terms of the freezing methods used, vitrified ovarian tissues had fewer viable follicles (3.2 ± 0.6) than the slow-freezing method (4.6 ± 0.6; P < 0.05). In addition, the slow freezing resulted in more postthawed morphologically normal primordial follicles (38.8% vs. 28.3%, P < 0.05) and less apoptotic primordial follicles (3.8% vs. 8.9%, P < 0.05) than vitrification. The Cx 43 expression showed no difference between slow freezing and vitrification. The present study reported the positive effects of sucrose supplementation and slow-freezing method on the follicular viability, follicular histologic appearances of follicles, and apoptosis of the follicles and stromal cells in cat ovarian tissues.

  11. Chi sequence protects against RecBCD degradation of DNA in vivo.

    PubMed Central

    Dabert, P; Ehrlich, S D; Gruss, A

    1992-01-01

    RecBCD is a multifunctional enzyme involved in DNA degradation and homologous recombination. It also produces an endonucleolytic cleavage near properly oriented chi sites (5'-GCTGGTGG-3'). Plasmids are not known to be affected by either RecBCD enzyme or the presence of a chi site. We report here that plasmids that replicate by a rolling circle mechanism accumulate large amounts of high molecular weight linear multimers (HMW), either if they contain a chi site or if RecBCD is absent. An in vivo inducible system for rolling circle replication was constructed to study RecBCD and its interactions with chi. Results show that (i) HMW accumulation is chi orientation dependent, and (ii) a succession of chi sites prevents degradation of HMW by RecBCD enzyme. These results demonstrate chi activity in plasmids. The rolling circle mechanism produces a sigma structure during plasmid replication; we propose that the double-stranded DNA tail of this sigma form allows RecBCD entry; the tail is degraded unless it is protected by a chi site. By analogy, a principal role of chi in the survival of lambda red-gam- mutants in wild-type strains may be to protect rolling circle concatemers (in late replication) from degradation by RecBCD. Images PMID:1465442

  12. DNA Protection against Oxidative Damage Using the Hydroalcoholic Extract of Garcinia mangostana and Alpha-Mangostin.

    PubMed

    Carvalho-Silva, Ronaldo; Pereira, Alanna Cibelle Fernandes; Dos Santos Alves, Rúbens Prince; Guecheva, Temenouga N; Henriques, João A P; Brendel, Martin; Pungartnik, Cristina; Rios-Santos, Fabrício

    2016-01-01

    Garcinia mangostana, popularly known as "mangosteen fruit," originates from Southeast Asia and came to Brazil about 80 years ago where it mainly grows in the states of Pará and Bahia. Although mangosteen or its extracts have been used for ages in Asian folk medicine, data on its potential genotoxicity is missing. We, therefore, evaluated genotoxicity/mutagenicity of hydroethanolic mangosteen extract [HEGM, 10 to 640 μg/mL] in established test assays (Comet assay, micronucleus test, and Salmonella/microsome test). In the Comet assay, HEGM-exposed human leukocytes showed no DNA damage. No significant HEGM-induced mutation in TA98 and TA100 strains of Salmonella typhimurium (with or without metabolic activation) was observed and HEGM-exposed human lymphocytes had no increase of micronuclei. However, HEGM suggested exposure concentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeast Saccharomyces cerevisiae. HEGM preloading effectively protected against H2O2-induced DNA damage in leukocytes (Comet assay). Preloading of yeast with HEGM for up to 4 h significantly protected the cells from lethality of chronic H2O2-exposure, as expressed in better survival. Absence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substances contained in it may hold promise for pharmaceutical or nutraceutical application.

  13. DNA Protection against Oxidative Damage Using the Hydroalcoholic Extract of Garcinia mangostana and Alpha-Mangostin

    PubMed Central

    Carvalho-Silva, Ronaldo; Pereira, Alanna Cibelle Fernandes; dos Santos Alves, Rúbens Prince; Guecheva, Temenouga N.; Henriques, João A. P.; Brendel, Martin; Rios-Santos, Fabrício

    2016-01-01

    Garcinia mangostana, popularly known as “mangosteen fruit,” originates from Southeast Asia and came to Brazil about 80 years ago where it mainly grows in the states of Pará and Bahia. Although mangosteen or its extracts have been used for ages in Asian folk medicine, data on its potential genotoxicity is missing. We, therefore, evaluated genotoxicity/mutagenicity of hydroethanolic mangosteen extract [HEGM, 10 to 640 μg/mL] in established test assays (Comet assay, micronucleus test, and Salmonella/microsome test). In the Comet assay, HEGM-exposed human leukocytes showed no DNA damage. No significant HEGM-induced mutation in TA98 and TA100 strains of Salmonella typhimurium (with or without metabolic activation) was observed and HEGM-exposed human lymphocytes had no increase of micronuclei. However, HEGM suggested exposure concentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeast Saccharomyces cerevisiae. HEGM preloading effectively protected against H2O2-induced DNA damage in leukocytes (Comet assay). Preloading of yeast with HEGM for up to 4 h significantly protected the cells from lethality of chronic H2O2-exposure, as expressed in better survival. Absence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substances contained in it may hold promise for pharmaceutical or nutraceutical application. PMID:27042187

  14. Phenolic profile, antioxidant potential and DNA damage protecting activity of sugarcane (Saccharum officinarum).

    PubMed

    Abbas, Syed Rizwan; Sabir, Syed Mubashar; Ahmad, Syed Dilnawaz; Boligon, Aline Augusti; Athayde, Margareth Linde

    2014-03-15

    The present study investigated the antioxidant and phenolic composition of sugarcane. The leaves and juices of thirteen varieties of sugarcane were studied for their antioxidant activity and protective effect on DNA damage. 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) assay was used to determine the radical scavenging activities in leaves and juices. Different varieties of sugarcane showed good antioxidant properties, IC50 values ranged from 20.82 to 27.47 μg/ml for leaves and from 63.95 to higher than 200 μg/ml for juice. The leaves and juice possess strong ability to protect against DNA damage induced by hydroxyl radical generated in Fenton reaction. The major phenolic acids, some flavonoid aglycone and glycosides were identified in leaves by high performance liquid chromatography. Ferulic acid (14.63 ± 0.03 mg/g), cumaric acid (11.65 ± 0.03 mg/g), quercetrin (10.96 ± 0.02 mg/g), caffeic acid (9.16 ± 0.01 mg/g) and ellagic acid (9.03 ± 0.02 mg/g) were prédominant in infusion of sugarcane.

  15. Antioxidant and cyto/DNA protective properties of apple pomace enriched bakery products.

    PubMed

    Sudha, M L; Dharmesh, Shylaja M; Pynam, Hasitha; Bhimangouder, Shivaleela V; Eipson, Sushma W; Somasundaram, Rajarathnam; Nanjarajurs, Shashirekha M

    2016-04-01

    Apple pomace (AP), the residue that remains after the extraction of juice from apple accounts for ~25 % of total apple weight. Current study is aimed at identification of phytochemicals and utilization of Dehydrated apple pomace (DAP) in the preparation of bakery products with potential health benefits. DAP was prepared by drying the pomace obtained by crushing peeled apple fruits. DAP was incorporated into bakery products such as bun, muffin and cookies for value addition. Bioactivity such as free radical scavenging, cyto/DNA protectivity was evaluated in these products. DAP contained 17 g/100 g starch, 49.86 g/100 g fructose and 37 g/100 g dietary fibre. The phenolics and flavonoids content was 1.5 mg/g and 3.92 mg/g, respectively. Increase in DAP resulted in decreased volume and enhanced firmness of buns and muffins. DAP at 15 % in buns, 30 % in muffins and 20 % in cookies were found to be acceptable. DAP blended products exhibited better free radical scavenging as well as cyto/DNA protective properties suggesting the retention of bioactivity after baking. Addition of DAP potentially enhanced the bioactivity of the products evaluated. PMID:27413217

  16. Antioxidant and cyto/DNA protective properties of apple pomace enriched bakery products.

    PubMed

    Sudha, M L; Dharmesh, Shylaja M; Pynam, Hasitha; Bhimangouder, Shivaleela V; Eipson, Sushma W; Somasundaram, Rajarathnam; Nanjarajurs, Shashirekha M

    2016-04-01

    Apple pomace (AP), the residue that remains after the extraction of juice from apple accounts for ~25 % of total apple weight. Current study is aimed at identification of phytochemicals and utilization of Dehydrated apple pomace (DAP) in the preparation of bakery products with potential health benefits. DAP was prepared by drying the pomace obtained by crushing peeled apple fruits. DAP was incorporated into bakery products such as bun, muffin and cookies for value addition. Bioactivity such as free radical scavenging, cyto/DNA protectivity was evaluated in these products. DAP contained 17 g/100 g starch, 49.86 g/100 g fructose and 37 g/100 g dietary fibre. The phenolics and flavonoids content was 1.5 mg/g and 3.92 mg/g, respectively. Increase in DAP resulted in decreased volume and enhanced firmness of buns and muffins. DAP at 15 % in buns, 30 % in muffins and 20 % in cookies were found to be acceptable. DAP blended products exhibited better free radical scavenging as well as cyto/DNA protective properties suggesting the retention of bioactivity after baking. Addition of DAP potentially enhanced the bioactivity of the products evaluated.

  17. DNA Protection against Oxidative Damage Using the Hydroalcoholic Extract of Garcinia mangostana and Alpha-Mangostin.

    PubMed

    Carvalho-Silva, Ronaldo; Pereira, Alanna Cibelle Fernandes; Dos Santos Alves, Rúbens Prince; Guecheva, Temenouga N; Henriques, João A P; Brendel, Martin; Pungartnik, Cristina; Rios-Santos, Fabrício

    2016-01-01

    Garcinia mangostana, popularly known as "mangosteen fruit," originates from Southeast Asia and came to Brazil about 80 years ago where it mainly grows in the states of Pará and Bahia. Although mangosteen or its extracts have been used for ages in Asian folk medicine, data on its potential genotoxicity is missing. We, therefore, evaluated genotoxicity/mutagenicity of hydroethanolic mangosteen extract [HEGM, 10 to 640 μg/mL] in established test assays (Comet assay, micronucleus test, and Salmonella/microsome test). In the Comet assay, HEGM-exposed human leukocytes showed no DNA damage. No significant HEGM-induced mutation in TA98 and TA100 strains of Salmonella typhimurium (with or without metabolic activation) was observed and HEGM-exposed human lymphocytes had no increase of micronuclei. However, HEGM suggested exposure concentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeast Saccharomyces cerevisiae. HEGM preloading effectively protected against H2O2-induced DNA damage in leukocytes (Comet assay). Preloading of yeast with HEGM for up to 4 h significantly protected the cells from lethality of chronic H2O2-exposure, as expressed in better survival. Absence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substances contained in it may hold promise for pharmaceutical or nutraceutical application. PMID:27042187

  18. Illegal trade of regulated and protected aquatic species in the Philippines detected by DNA barcoding.

    PubMed

    Asis, Angelli Marie Jacynth M; Lacsamana, Joanne Krisha M; Santos, Mudjekeewis D

    2016-01-01

    Illegal trade has greatly affected marine fish stocks, decreasing fish populations worldwide. Despite having a number of aquatic species being regulated, illegal trade still persists through the transport of dried or processed products and juvenile species trafficking. In this regard, accurate species identification of illegally traded marine fish stocks by DNA barcoding is deemed to be a more efficient method in regulating and monitoring trade than by morphological means which is very difficult due to the absence of key morphological characters in juveniles and processed products. Here, live juvenile eels (elvers) and dried products of sharks and rays confiscated for illegal trade were identified. Twenty out of 23 (87%) randomly selected "elvers" were identified as Anguilla bicolor pacifica and 3 (13%) samples as Anguilla marmorata. On the other hand, 4 out of 11 (36%) of the randomly selected dried samples of sharks and rays were Manta birostris. The rest of the samples were identified as Alopias pelagicus, Taeniura meyeni, Carcharhinus falciformis, Himantura fai and Mobula japonica. These results confirm that wild juvenile eels and species of manta rays are still being caught in the country regardless of its protected status under Philippine and international laws. It is evident that the illegal trade of protected aquatic species is happening in the guise of dried or processed products thus the need to put emphasis on strengthening conservation measures. This study aims to underscore the importance of accurate species identification in such cases of illegal trade and the effectivity of DNA barcoding as a tool to do this.

  19. DNA damage protective effect of honey-sweetened cashew apple nectar in Drosophila melanogaster.

    PubMed

    Silva, Robson Alves da; Dihl, Rafael Rodrigues; Dias, Lucas Pinheiro; Costa, Maiane Papke; Abreu, Bianca Regina Ribas de; Cunha, Kênya Silva; Lehmann, Mauricio

    2016-01-01

    Fruits and derivatives, such as juices, are complex mixtures of chemicals, some of which may have mutagenic and/or carcinogenic potential, while others may have antimutagenic and/or anticancer activities. The modulating effects of honey-sweetened cashew apple nectar (HSCAN), on somatic mutation and recombination induced by ethyl methanesulfonate (EMS) and mitomycin C (MMC) were evaluated with the wing spot test in Drosophila melanogaster using co- and post-treatment protocols. Additionally, the antimutagenic activity of two HSCAN components, cashew apple pulp and honey, in MMC-induced DNA damage was also investigated. HSCAN reduced the mutagenic activity of both EMS and MMC in the co-treatment protocol, but had a co-mutagenic effect when post-administered. Similar results were also observed with honey on MMC mutagenic activity. Cashew apple pulp was effective in exerting protective or enhancing effects on the MMC mutagenicity, depending on the administration protocol and concentration used. Overall, these results indicate that HSCAN, cashew apple and honey seem capable of modulating not only the events that precede the induced DNA damages, but also the Drosophila DNA repair processes involved in the correction of EMS and MMC-induced damages. PMID:27560988

  20. DNA damage protective effect of honey-sweetened cashew apple nectar in Drosophila melanogaster

    PubMed Central

    da Silva, Robson Alves; Dihl, Rafael Rodrigues; Dias, Lucas Pinheiro; Costa, Maiane Papke; de Abreu, Bianca Regina Ribas; Cunha, Kênya Silva; Lehmann, Mauricio

    2016-01-01

    Abstract Fruits and derivatives, such as juices, are complex mixtures of chemicals, some of which may have mutagenic and/or carcinogenic potential, while others may have antimutagenic and/or anticancer activities. The modulating effects of honey-sweetened cashew apple nectar (HSCAN), on somatic mutation and recombination induced by ethyl methanesulfonate (EMS) and mitomycin C (MMC) were evaluated with the wing spot test in Drosophila melanogaster using co- and post-treatment protocols. Additionally, the antimutagenic activity of two HSCAN components, cashew apple pulp and honey, in MMC-induced DNA damage was also investigated. HSCAN reduced the mutagenic activity of both EMS and MMC in the co-treatment protocol, but had a co-mutagenic effect when post-administered. Similar results were also observed with honey on MMC mutagenic activity. Cashew apple pulp was effective in exerting protective or enhancing effects on the MMC mutagenicity, depending on the administration protocol and concentration used. Overall, these results indicate that HSCAN, cashew apple and honey seem capable of modulating not only the events that precede the induced DNA damages, but also the Drosophila DNA repair processes involved in the correction of EMS and MMC-induced damages. PMID:27560988

  1. Ancylostoma ceylanicum metalloprotease 6 DNA vaccination induces partial protection against hookworm challenge infection.

    PubMed

    Wiśniewski, Marcin; Jaros, Sławomir; Bąska, Piotr; Cappello, Michael; Wędrychowicz, Halina

    2013-09-01

    Hookworms are blood feeding intestinal nematodes that infect more than 500 million people and cause iron deficiency anemia. Infected children suffer from physical and cognitive growth retardation. Because of potential anthelminthic drug resistance, the need for vaccine development is urgent. Numerous antigens have been tested in animal models as vaccines against hookworm infection, but there is no effective human vaccine. We cloned a cDNA encoding Ancylostoma ceylanicum metalloprotease 6 (Acemep-6). Ace-MEP-6 is a protein with a predicted molecular mass of 101.87 kDa and based on computational analysis it is very likely to be engaged in food processing via hemoglobin digestion. Groups of hamsters were immunized with an Ace-mep-6 cDNA vaccine, either once or three times. Animals that were administered one dose developed high resistance (80%, p < 0.01) against challenge infection, whereas triple immunization resulted in no worm burden reduction. These results suggest that DNA vaccines can be powerful tools in ancylostomiasis control, although the mechanisms through which protection is conferred remain unclear.

  2. Hepatitis B: progress in understanding chronicity, the innate immune response, and cccDNA protection

    PubMed Central

    Shimazaki, Tomoe; Takeda, Rei; Izumi, Takaaki; Umumura, Machiko; Sakamoto, Naoya

    2016-01-01

    Hepatitis B virus (HBV) infection is a serious health threat around the world. Despite the availability of an effective hepatitis B vaccine, the number of HBV carriers is estimated to be as high as 240 million worldwide. Global mortality due to HBV-related liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) may be as high as 1 million deaths per year. HBV is transmitted via blood and body fluids, and is much more infectious than both human immunodeficiency virus (HIV) and hepatitis C virus. While HBV infection exhibits a variety of clinical presentations, even asymptomatic carriers can develop HCC without liver fibrosis. Current therapeutic options against HBV include pegylated interferon (Peg-IFN) and nucleos(t)ide reverse transcriptase inhibitors (NRTIs), with clinical studies showing a significant association between loss of HBV DNA and a decrease in cancer risk. However, the ultimate goal of HBV therapy is a complete cure of HBV—including the elimination of covalently closed circular DNA (cccDNA)—in order to further decrease the risk of developing HCC. The development of hepatitis B is associated with the host immune response to virus-infected hepatocytes, as HBV is understood to lack direct cytotoxicity. While HBV-specific CD8+ T cells are thus involved in hepatitis development, they also play an important role in eliminating HBV infection. Indeed, the innate immune response during the initial phase of HBV infection is essential to the induction of acquired immunity. However, the innate immune response to HBV infection, including the roles of specific immunocompetent cells and associated molecules, is not well understood. In this review, we focus on the current understanding of the mechanisms underlying hepatitis development by HBV infection. We also address the mechanisms by which HBV protects cccDNA. PMID:27761441

  3. Synthesis, DNA binding and photocleavage, and cellular uptake of an alkyl chain-linked dinuclear ruthenium(II) complex.

    PubMed

    Liu, Ping; Liu, Jin; Zhang, Yu-Qi; Wu, Bao-Yan; Wang, Ke-Zhi

    2015-02-01

    A dinuclear ruthenium(II) complex [(bpy)2Ru(L(1))Ru(bpy)2]Cl4 {bpy=2,2'-bipyridine, L(1)=1,6-bis(3-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-9H-carbazol-9-yl)hexane} was synthesised and characterized. The calf thymus DNA (ct-DNA) binding properties of the complex were investigated by means of UV-Visible absorption and emission spectrophotometric titrations, ethidium bromide competitive binding, steady-state emission quenching with ferrocyanide, DNA viscosity measurements, and DNA thermal denaturation. The results indicated that the complex avidly binds to ct-DNA most probably through a threading bis-intercalative mode. The complex was also evidenced to act as an efficient DNA photocleaver, and an effective luminescent stain for cytoplasmic of HeLa cells with low cytotoxicity.

  4. Interaction of Iron II Complexes with B-DNA. Insights from Molecular Modeling, Spectroscopy and Cellular Biology.

    NASA Astrophysics Data System (ADS)

    Gattuso, Hugo; Duchanois, Thibaut; Besancenot, Vanessa; Barbieux, Claire; Assfeld, Xavier; Becuwe, Philippe; Gros, Philippe; Grandemange, Stephanie; Monari, Antonio

    2015-12-01

    We report the characterization of the interaction between B-DNA and three terpyridin iron II complexes. Relatively long time-scale molecular dynamics is used in order to characterize the stable interaction modes. By means of molecular modeling and UV-vis spectroscopy, we prove that they may lead to stable interactions with the DNA duplex. Furthermore, the presence of larger π-conjugated moieties also leads to the appearance of intercalation binding mode. Non-covalent stabilizing interactions between the iron complexes and the DNA are also characterized and evidenced by the analysis of the gradient of the electronic density. Finally, the structural deformations induced on the DNA in the different binding modes are also evidenced. The synthesis and chemical characterization of the three complexes is reported, as well as their absorption spectra in presence of DNA duplexes to prove the interaction with DNA. Finally, their effects on human cell cultures have also been evidenced to further enlighten their biological effects.

  5. Protection against Vibrio alginolyticus in crimson snapper Lutjanus erythropterus immunized with a DNA vaccine containing the ompW gene.

    PubMed

    Cai, Shuang-Hu; Lu, Yi-Shan; Jian, Ji-Chang; Wang, Bei; Huang, Yu-Cong; Tang, Ju-Fen; Ding, Yu; Wu, Zao-He

    2013-09-24

    The outer membrane proteins of Vibrio alginolyticus play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In the present study, the ompW gene was cloned, expressed and purified. A DNA vaccine was constructed by inserting the ompW gene into a pcDNA plasmid. Crimson snapper Lutjanus erythropterus (Bloch) were injected intramuscularly with the recombinant plasmid pcDNA-ompW. The expression of the DNA vaccine was detected in gill, head kidney, heart, liver, spleen and injection site muscle of crimson snapper by RT-PCR 7 and 28 d post-vaccination. The ELISA results demonstrated that the DNA vaccine produced an observable antibody response in all sera of the vaccinated fish. In addition, crimson snapper immunized with the DNA vaccine showed a relative percentage survival (RPS) of 92.53%, indicating effective protection against V. alginolyticus infection.

  6. Nanogram quantities of a DNA vaccine protect rainbow trout fry against heterologous strains of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Corbeil, S.; LaPatra, S.E.; Anderson, E.D.; Kurath, G.

    2000-01-01

    The efficacy of a DNA vaccine containing the glycoprotein gene of infectious hematopoietic necrosis virus (IHNV), a rhabdovirus affecting trout and salmon, was investigated. The minimal dose of vaccine required, the protection against heterologous strains, and the titers of neutralizing antibodies produced were used to evaluate the potential of the vaccine as a control pharmaceutical. Results indicated that a single dose of as little as 1–10 ng of vaccine protected rainbow trout fry against waterborne challenge by IHNV. An optimal dose of 100 ng per fish was selected to assure strong protection under various conditions. Neutralizing antibody titers were detected in fish vaccinated with concentrations of DNA ranging from 5 to 0.01 μg. Furthermore, the DNA vaccine protected fish against a broad range of viral strains from different geographic locations, including isolates from France and Japan, suggesting that the vaccine could be used worldwide. A single dose of this DNA vaccine induced protection in fish at a lower dose than is usually reported in mammalian DNA vaccine studies.

  7. Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage▿

    PubMed Central

    Alekseev, Sergey; Luijsterburg, Martijn S.; Pines, Alex; Geverts, Bart; Mari, Pierre-Olivier; Giglia-Mari, Giuseppina; Lans, Hannes; Houtsmuller, Adriaan B.; Mullenders, Leon H. F.; Hoeijmakers, Jan H. J.; Vermeulen, Wim

    2008-01-01

    Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions. PMID:18936169

  8. The DNA-induced protective immunity with chicken interferon gamma against poultry coccidiosis.

    PubMed

    Shah, Muhammad Ali A; Song, Xiaokai; Xu, Lixin; Yan, Roufeng; Song, Hongyan; Ruirui, Zhang; Chengyu, Lei; Li, Xiangrui

    2010-08-01

    The immunogenicity of Eimeria acervulina cSZ-2 and chicken interferon gamma was observed against Eimeria tenella challenge. The chickens were randomly divided into six groups of 24 chicks each. Three groups of chickens were injected with DNA vaccines pVAX1-cSZ2, pVAX1-chIFN-gamma and pVAX1-cSZ2-chIFN-gamma two times (at days 14 and 21) at a dose of 100 microg intramuscularly. Three other groups were kept as control and injected with TE buffer (10 mM Tris-HCl pH 7.6 and 1 mM EDTA). One week following the booster dose, all chickens except the non-infected, non-vaccinated control group were inoculated orally with 5 x 10(4) sporulated oocysts of E. tenella. Seven days post challenge, all chickens were weighted and slaughtered for cecal lesion scoring and oocyst counts. The results demonstrated that cSZ-2 in combination with interferon gamma can protect chickens from coccidiosis by significantly decreasing body weight loss and oocyst excretion reflecting partial protection against E. tenella infection, and further studies are necessary to test for protection against other Eimeria species.

  9. Rectal single dose immunization of mice with Escherichia coli O157:H7 bacterial ghosts induces efficient humoral and cellular immune responses and protects against the lethal heterologous challenge

    PubMed Central

    Mayr, Ulrike Beate; Kudela, Pavol; Atrasheuskaya, Alena; Bukin, Eugenij; Ignatyev, Georgy; Lubitz, Werner

    2012-01-01

    Summary Bacterial ghosts (BGs) have been applied through oral, aerogenic, intraocular or intranasal routes for mucosal immunization using a wide range of experimental animals. All these applications required a booster after primary immunization to achieve protective immunity against the lethal challenge. Here we report for the first time that a single rectal dose of BGs produced from enterohaemorrhagic Escherichia coli (EHEC) O157:H7 fully protects mice against a 50% lethal challenge with a heterologous EHEC strain given at day 55. BGs from EHEC O157:H7 were prepared by a combination of protein E‐mediated cell lysis and expression of staphylococcal nuclease A guaranteeing the complete degradation of pathogen residual DNA. The lack of genetic material in the EHEC BGs vaccine abolished any potential hazard for horizontal gene transfer of plasmid encoded antibiotic resistance genes or pathogenic islands to the recipient's gut flora. Single rectal immunization using EHEC O157:H7 BGs without any addition of adjuvant significantly stimulated efficient humoral and cellular immune responses, and was equally protective as two immunizations, which indicates the possibility to develop a novel efficacious single dose mucosal EHEC O157:H7 BGs vaccine using a simplified immunization regimen. PMID:22103353

  10. Thermal stress and cellular signaling processes in hemocytes of native (Mytilus californianus) and invasive (M. galloprovincialis) mussels: cell cycle regulation and DNA repair.

    PubMed

    Yao, Cui-Luan; Somero, George N

    2013-06-01

    In a previous study using hemocytes from native and invasive congeners of Mytilus (Mytilus californianus and Mytilus galloprovincialis, respectively) we showed that DNA damage and cell signaling transduction processes related to the cellular stress response and apoptosis were induced by acute temperature stress. The present study extends this work by examining effects of acute heat- and cold stress on total hemocyte counts (THCs) and expression of key regulatory molecules involved in responding to stress: tumor suppressor factor (p53), cell cycle arrest activator (p21), and a DNA base excision repair enzyme (apurinic/apyrimidinic endonuclease (APE)). Hyperthermia (28 °C, 32 °C) led to significant decreases of THCs in both species. The extent of decrease in THC was temperature-, time-, and species-dependent; lower THC values were found in M. californianus, the more cold-adapted species. Western blot analyses of hemocyte extracts with antibodies specific for p53 protein, several site-specific phosphorylation states of p53, p21 protein, and APE indicated that heat- and cold (2 °C) stress induced a time-dependent activation of stress-related proteins in response to DNA damage; these stress-induced changes could govern cell cycle arrest or DNA damage repair. Our results show that the downstream regulatory response to temperature-induced cell damage may play an important role in deciding cellular fate following heat- and cold stress. Compared to M. californianus, the more warm-adapted M. galloprovincialis appears to have a higher temperature tolerance due to a lesser reduction in THC, faster signaling activation and transduction, and stronger DNA repair ability following heat stress.

  11. SWI/SNF factors required for cellular resistance to DNA damage include ARID1A and ARID1B and show interdependent protein stability.

    PubMed

    Watanabe, Reiko; Ui, Ayako; Kanno, Shin-Ichiro; Ogiwara, Hideaki; Nagase, Takahiro; Kohno, Takashi; Yasui, Akira

    2014-05-01

    The SWI/SNF chromatin-remodeling family contains various protein complexes, which regulate gene expression during cellular development and influence DNA damage response in an ATP- and complex-dependent manner, of which details remain elusive. Recent human genome sequencing of various cancer cells revealed frequent mutations in SWI/SNF factors, especially ARID1A, a variant subunit in the BRG1-associated factor (BAF) complex of the SWI/SNF family. We combined live-cell analysis and gene-suppression experiments to show that suppression of either ARID1A or its paralog ARID1B led to reduced nonhomologous end joining activity of DNA double-strand breaks (DSB), decreased accumulation of KU70/KU80 proteins at DSB, and sensitivity to ionizing radiation, as well as to cisplatin and UV. Thus, in contrast to transcriptional regulation, both ARID1 proteins are required for cellular resistance to various types of DNA damage, including DSB. The suppression of other SWI/SNF factors, namely SNF5, BAF60a, BAF60c, BAF155, or BAF170, exhibits a similar phenotype. Of these factors, ARID1A, ARID1B, SNF5, and BAF60c are necessary for the immediate recruitment of the ATPase subunit of the SWI/SNF complex to DSB, arguing that both ARID1 proteins facilitate the damage response of the complex. Finally, we found interdependent protein stability among the SWI/SNF factors, suggesting their direct interaction within the complex and the reason why multiple factors are frequently lost in parallel in cancer cells. Taken together, we show that cancer cells lacking in the expression of certain SWI/SNF factors, including ARID1A, are deficient in DNA repair and potentially vulnerable to DNA damage.

  12. XRCC4 and XLF form long helical protein filaments suitable for DNA end protection and alignment to facilitate DNA double strand break repair

    PubMed Central

    Mahaney, Brandi L.; Hammel, Michal; Meek, Katheryn; Tainer, John A.; Lees-Miller, Susan P.

    2013-01-01

    DNA double strand breaks (DSBs), induced by ionizing radiation (IR) and endogenous stress including replication failure, are the most cytotoxic form of DNA damage. In human cells, most IR-induced DSBs are repaired by the non-homologous end joining (NHEJ) pathway. One of the most critical steps in NHEJ is ligation of DNA ends by DNA ligase IV (LIG4), which interacts with, and is stabilized by, the scaffolding protein X-ray cross-complementing gene 4 (XRCC4). XRCC4 also interacts with XRCC4-like factor (XLF, also called Cernunnos); yet, XLF has been one of the least mechanistically understood proteins and precisely how XLF functions in NHEJ has been enigmatic. Here, we examine current combined structural and mutational findings that uncover integrated functions of XRCC4 and XLF and reveal their interactions to form long, helical protein filaments suitable to protect and align DSB ends. XLF-XRCC4 provides a global structural scaffold for ligating DSBs without requiring long complementary DNA ends, thus ensuring accurate and efficient ligation and repair. The assembly of these XRCC4-XLF filaments, providing both DNA end protection and alignment, may commit cells to NHEJ with general biological implications for NHEJ and DSB repair processes and their links to cancer predispositions and interventions. PMID:23442139

  13. Ran Binding Protein 9 (RanBP9) is a novel mediator of cellular DNA damage response in lung cancer cells

    PubMed Central

    Palmieri, Dario; Scarpa, Mario; Tessari, Anna; Uka, Rexhep; Amari, Foued; Lee, Cindy; Richmond, Timothy; Foray, Claudia; Sheetz, Tyler; Braddom, Ashley; Burd, Christin E.; Parvin, Jeffrey D.; Ludwig, Thomas; Croce, Carlo M.; Coppola, Vincenzo

    2016-01-01

    Ran Binding Protein 9 (RanBP9, also known as RanBPM) is an evolutionary conserved scaffold protein present both in the nucleus and the cytoplasm of cells whose biological functions remain elusive. We show that active ATM phosphorylates RanBP9 on at least two different residues (S181 and S603). In response to IR, RanBP9 rapidly accumulates into the nucleus of lung cancer cells, but this nuclear accumulation is prevented by ATM inhibition. RanBP9 stable silencing in three different lung cancer cell lines significantly affects the DNA Damage Response (DDR), resulting in delayed activation of key components of the cellular response to IR such as ATM itself, Chk2, γH2AX, and p53. Accordingly, abrogation of RanBP9 expression reduces homologous recombination-dependent DNA repair efficiency, causing an abnormal activation of IR-induced senescence and apoptosis. In summary, here we report that RanBP9 is a novel mediator of the cellular DDR, whose accumulation into the nucleus upon IR is dependent on ATM kinase activity. RanBP9 absence hampers the molecular mechanisms leading to efficient repair of damaged DNA, resulting in enhanced sensitivity to genotoxic stress. These findings suggest that targeting RanBP9 might enhance lung cancer cell sensitivity to genotoxic anti-neoplastic treatment. PMID:26943034

  14. Clinical, cellular, and molecular features of an Israeli xeroderma pigmentosum family with a frameshift mutation in the XPC gene: sun protection prolongs life.

    PubMed

    Slor, H; Batko, S; Khan, S G; Sobe, T; Emmert, S; Khadavi, A; Frumkin, A; Busch, D B; Albert, R B; Kraemer, K H

    2000-12-01

    An Ashkenazi Jewish Israeli family with two children affected with severe xeroderma pigmentosum was investigated. A son, XP12TA, developed skin cancer at 2 y and died at 10 y. A daughter, XP25TA, now 24 y old, was sun protected and began developing skin cancers at 10 y. Their cultured skin fibroblasts showed reductions in post-ultraviolet survival (11% of normal), unscheduled DNA synthesis (10% of normal), global genome DNA repair (15% of normal), and plasmid host cell reactivation (5% of normal). Transcription-coupled DNA repair was normal, however. Northern blot analysis revealed greatly reduced xeroderma pigmentosum complementation group C mRNA. A plasmid host cell reactivation assay assigned the cells to xeroderma pigmentosum complementation group C. Cells from both parents and an unaffected child exhibited normal post-ultraviolet-C survival and normal DNA repair. Sequencing the xeroderma pigmentosum complementation group C cDNA of XP12TA and XP25TA revealed a homozygous deletion of two bases (del AT 669-670) in exon 5 with a new termination site 10 codons downstream that is expected to encode a truncated xeroderma pigmentosum complementation group C protein. Sequence analysis of the xeroderma pigmentosum complementation group C cDNA in cells from the parents found identical heterozygous mutations: one allele carries both the exon 5 frameshift and an exon 15 polymorphism and the other allele carries neither alteration. Cells from the unaffected brother had two normal xeroderma pigmentosum complementation group C alleles. This frameshift mutation in the xeroderma pigmentosum complementation group C gene led to reduced DNA repair with multiple skin cancers and early death. Sun protection delayed the onset of skin cancer and prolonged life in a sibling with the same mutation. PMID:11121128

  15. Different sets of translesion synthesis DNA polymerases protect from genome instability induced by distinct food-derived genotoxins.

    PubMed

    Temviriyanukul, Piya; Meijers, Matty; van Hees-Stuivenberg, Sandrine; Boei, Jan J W A; Delbos, Frédéric; Ohmori, Haruo; de Wind, Niels; Jansen, Jacob G

    2012-05-01

    DNA lesions, induced by genotoxic compounds, block the processive replication fork but can be bypassed by specialized translesion synthesis (TLS) DNA polymerases (Pols). TLS safeguards the completion of replication, albeit at the expense of nucleotide substitution mutations. We studied the in vivo role of individual TLS Pols in cellular responses to benzo[a]pyrene diolepoxide (BPDE), a polycyclic aromatic hydrocarbon, and 4-hydroxynonenal (4-HNE), a product of lipid peroxidation. To this aim, we used mouse embryonic fibroblasts with targeted disruptions in the TLS-associated Pols η, ι, κ, and Rev1 as well as in Rev3, the catalytic subunit of TLS Polζ. After exposure, cellular survival, replication fork progression, DNA damage responses (DDR), and the induction of micronuclei were investigated. The results demonstrate that Rev1, Rev3, and, to a lesser extent, Polη are involved in TLS and the prevention of DDR and of DNA breaks, in response to both agents. Conversely, Polκ and the N-terminal BRCT domain of Rev1 are specifically involved in TLS of BPDE-induced DNA damage. We furthermore describe a novel role of Polι in TLS of 4-HNE-induced DNA damage in vivo. We hypothesize that different sets of TLS polymerases act on structurally different genotoxic DNA lesions in vivo, thereby suppressing genomic instability associated with cancer. Our experimental approach may provide a significant contribution in delineating the molecular bases of the genotoxicity in vivo of different classes of DNA-damaging agents. PMID:22331492

  16. Pregnane X receptor protects HepG2 cells from BaP-induced DNA damage.

    PubMed

    Naspinski, Christine; Gu, Xinsheng; Zhou, Guo-Dong; Mertens-Talcott, Susanne U; Donnelly, Kirby C; Tian, Yanan

    2008-07-01

    Pregnane X receptor (PXR) is a nuclear receptor that coordinately regulates transcriptional expression of both phase I and phase II metabolizing enzymes. PXR plays an important role in the pharmacokinetics of a broad spectrum of endogenous and xenobiotic compounds and appears to have evolved in part to protect organisms from toxic xenobiotics. Metabolism of benzo[a]pyrene (BaP), a well-established carcinogen and ubiquitous environmental contaminant, can result in either detoxification or bioactivation to its genotoxic forms. Therefore, PXR could modulate the genotoxicity of BaP by changing the balance of the metabolic pathways in favor of BaP detoxification. To examine the role of PXR in BaP genotoxicity, BaP-DNA adduct formation was measured by 32P-postlabeling in BaP-treated parental HepG2 cells and human PXR-transfected HepG2 cells. The presence of transfected PXR significantly reduced the level of adducts relative to parental cells by 50-65% (p < 0.001), demonstrating that PXR protects liver cells from genotoxicity induced by exposure to BaP. To analyze potential PXR-regulated detoxification pathways in liver cells, a panel of genes involved in phase I and phase II metabolism and excretion was surveyed with real-time quantitative reverse transcription PCR. The messenger RNA levels of CYP1A2, GSTA1, GSTA2, GSTM1, UGT1A6, and BCRP (ABCG2) were significantly higher in cells overexpressing PXR, independent of exposure to BaP. In addition, the total GST enzymatic activity, which favors the metabolic detoxification of BaP, was significantly increased by the presence of PXR (p < 0.001), independent of BaP exposure. Taken together, these results suggest that PXR plays an important role in protection against DNA damage by polycyclic aromatic hydrocarbons (PAHs) such as BaP, and that these protective effects may be through a coordinated regulation of genes involved in xenobiotic metabolism.

  17. Control of Trx1 redox state modulates protection against methyl methanesulfonate-induced DNA damage via stabilization of p21.

    PubMed

    Gu, Li; Gao, Wei; Yang, Hui Min; Wang, Bei Bei; Wang, Xiao Na; Xu, Jianguo; Zhang, Hong

    2016-01-01

    Thioredoxin 1 (Trx1) is known to play an important role in protecting against cell death. However, the mechanism for control of Trx1 in cell death resulting from DNA damage has not been fully investigated. In this study, we used the DNA-damaging agent methyl methanesulfonate (MMS) to investigate the protective effects of Trx1 against DNA damage and cell death in HEK293 cells. We found that MMS application caused dose-dependent changes in the Trx1 redox state determined by redox western blotting. At lower concentrations, both reduced and oxidized Trx1 were observed, whereas the reduced band was fully oxidized at the higher concentration. Trx1 overexpression and small interfering RNA knockdown in cells revealed that reduced Trx1 after exposure to lower doses of MMS attenuated DNA damage, assessed by comet assay, and level of the DNA-damage marker histone γ-H2AX, possibly through scavenging intracellular ROS and an increase in p21 protein level via enhancing its stability. However, oxidized Trx1 lost its protective ability to DNA damage in response to higher concentration of MMS. Corresponding to the redox state control of Trx1, cell death induced by different dose of MMS was also found, by inhibiting phosphorylations of p38 and 4E-BP1. These results indicate that reduced Trx1 plays important protective roles against MMS-induced DNA damage and cell death, suggesting that cell protection is regulated by the intracellular redox state. Control of the redox state of Trx1 and its regulating proteins may offer a novel therapeutic strategy for the control of cancer.

  18. Use of a Sindbis virus DNA-based expression vector for induction of protective immunity against pseudorabies virus in pigs.

    PubMed

    Dufour, Vinciane; De Boisséson, Claire

    2003-06-20

    Injection of plasmid DNA encoding pseudorabies virus (PRV) glycoproteins into pig muscle has been shown to result in protective immunity against lethal infection. Nevertheless, such DNA vaccines are still less efficient than some attenuated or killed live vaccines. One way to increase DNA vaccine efficacy is to improve the vectorisation system at the molecular level, thereby enhancing the rate of in vivo-produced immunogen protein and consequently specific acquired immunity. The present study compared the effectiveness of the protein expression system depending on Sindbis virus (SIN) replicase [J. Virol. 70 (1996) 508] with that of more classical pcDNA3 plasmid. Pigs were vaccinated twice at 3-week interval with a mixture of three pcDNA3 plasmids expressing gB, gC and gD (designated as PRV-pcDNA3) or a mixture of three SIN plasmids expressing the same glycoproteins (PRV-pSINCP), and were challenged with a highly virulent PRV strain. The two DNA vaccines induced PRV-specific T cell-mediated immune response characterized by very low levels of IFN-gamma mRNA in PBMC after in vitro antigen-specific stimulation. Very low levels of neutralizing antibodies (NAb) were also obtained in sera following DNA injection(s). A second DNA injection did not boost immune responses. After a lethal challenge, high levels of IFN-gamma mRNA and high NAb response were induced in all DNA-vaccinated pigs, regardless of the vector used. Therefore, the two eukaryotic expression systems showed comparable efficacy in inducing antiviral immunity and clinical protection against PRV in pigs. This suggests that SIN DNA-based vector immunizing potential may differ according to antigen and/or host. PMID:12814698

  19. Heterologous Protection Against Influenza by Injection of DNA Encoding a Viral Protein

    NASA Astrophysics Data System (ADS)

    Ulmer, Jeffrey B.; Donnelly, John J.; Parker, Suezanne E.; Rhodes, Gary H.; Felgner, Philip L.; Dwarki, V. J.; Gromkowski, Stanislaw H.; Deck, R. Randall; Dewitt, Corrille M.; Friedman, Arthur; Hawe, Linda A.; Leander, Karen R.; Martinez, Douglas; Perry, Helen C.; Shiver, John W.; Montgomery, Donna L.; Liu, Margaret A.

    1993-03-01

    Cytotoxic T lymphocytes (CTLs) specific for conserved viral antigens can respond to different strains of virus, in contrast to antibodies, which are generally strain-specific. The generation of such CTLs in vivo usually requires endogenous expression of the antigen, as occurs in the case of virus infection. To generate a viral antigen for presentation to the immune system without the limitations of direct peptide delivery or viral vectors, plasmid DNA encoding influenza A nucleoprotein was injected into the quadriceps of BALB/c mice. This resulted in the generation of nucleoprotein-specific CTLs and protection from a subsequent challenge with a heterologous strain of influenza A virus, as measured by decreased viral lung titers, inhibition of mass loss, and increased survival.

  20. Protective immunity against Taenia crassiceps murine cysticercosis induced by DNA vaccination with a Taenia saginata tegument antigen.

    PubMed

    Rosas, Gabriela; Fragoso, Gladis; Garate, Teresa; Hernández, Beatriz; Ferrero, Patricia; Foster-Cuevas, Mildred; Parkhouse, R Michael E; Harrison, Leslie J S; Briones, Sergio López; González, Luis Miguel; Sciutto, Edda

    2002-11-01

    This study investigated the protective capacity of the recombinant Taenia saginata Tso18 antigen administered as a DNA vaccine in the Taenia crassiceps murine model of cysticercosis. This Tso18 DNA sequence, isolated from a T. saginata oncosphere cDNA library, has homologies with Taenia solium and Echinococcus sp. It was cloned in the pcDNA3.1 plasmid and injected once intramuscularly into mice. Compared to saline-vaccinated control mice, immunization reduced the parasite burden by 57.3-81.4%, while lower levels of non-specific protection were induced in control mice injected with the plasmid pcDNA3.1 (18.8-33.1%) or a plasmid with irrelevant construct, pcDNA3.1/3D15 (33.4-38.8%). Importantly, significant levels of protection were observed between the pcDNA3.1/Tso18 plasmid and pcDNA3.1/3D15 plasmid immunized mice. Mice immunized with pTso18 synthesized low levels of, primarily IgG1 sub-class, antibodies. These antibodies were shown to recognize a 66 kDa antigen fraction of T. crassiceps and T. solium. Splenocytes enriched in both CD4+CD8- and CD4-CD8+ T cells from these vaccinated mice proliferated in vitro when exposed to antigens from both T. solium and T. crassiceps cestodes. Immunolocalization studies revealed the Tso18 antigen in oncospheres of T. saginata and T. solium, in the adult tapeworm and in the tegument of T. solium cysticerci. The protective capacity of this antigen and its extensive distribution in different stages, species and genera of cestodes points to the potential of Tso18 antigen for the possible design of a vaccine against cestodes.

  1. Illegal trade of regulated and protected aquatic species in the Philippines detected by DNA barcoding.

    PubMed

    Asis, Angelli Marie Jacynth M; Lacsamana, Joanne Krisha M; Santos, Mudjekeewis D

    2016-01-01

    Illegal trade has greatly affected marine fish stocks, decreasing fish populations worldwide. Despite having a number of aquatic species being regulated, illegal trade still persists through the transport of dried or processed products and juvenile species trafficking. In this regard, accurate species identification of illegally traded marine fish stocks by DNA barcoding is deemed to be a more efficient method in regulating and monitoring trade than by morphological means which is very difficult due to the absence of key morphological characters in juveniles and processed products. Here, live juvenile eels (elvers) and dried products of sharks and rays confiscated for illegal trade were identified. Twenty out of 23 (87%) randomly selected "elvers" were identified as Anguilla bicolor pacifica and 3 (13%) samples as Anguilla marmorata. On the other hand, 4 out of 11 (36%) of the randomly selected dried samples of sharks and rays were Manta birostris. The rest of the samples were identified as Alopias pelagicus, Taeniura meyeni, Carcharhinus falciformis, Himantura fai and Mobula japonica. These results confirm that wild juvenile eels and species of manta rays are still being caught in the country regardless of its protected status under Philippine and international laws. It is evident that the illegal trade of protected aquatic species is happening in the guise of dried or processed products thus the need to put emphasis on strengthening conservation measures. This study aims to underscore the importance of accurate species identification in such cases of illegal trade and the effectivity of DNA barcoding as a tool to do this. PMID:24841434

  2. Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart.

    PubMed

    Somyajit, Kumar; Saxena, Sneha; Babu, Sharath; Mishra, Anup; Nagaraju, Ganesh

    2015-11-16

    Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination. However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. Here, we report on the role of RAD51 paralogs in short-term replicative stress devoid of DSBs. We show that RAD51 paralogs localize to nascent DNA and common fragile sites upon replication fork stalling. Strikingly, RAD51 paralogs deficient cells exhibit elevated levels of 53BP1 nuclear bodies and increased DSB formation, the latter being attributed to extensive degradation of nascent DNA at stalled forks. RAD51C and XRCC3 promote the restart of stalled replication in an ATP hydrolysis dependent manner by disengaging RAD51 and other RAD51 paralogs from the halted forks. Notably, we find that Fanconi anemia (FA)-like disorder and breast and ovarian cancer patient derived mutations of RAD51C fails to protect replication fork, exhibit under-replicated genomic regions and elevated micro-nucleation. Taken together, RAD51 paralogs prevent degradation of stalled forks and promote the restart of halted replication to avoid replication fork collapse, thereby maintaining genomic integrity and suppressing tumorigenesis.

  3. Nitrosothiol formation and protection against Fenton chemistry by nitric oxide-induced dinitrosyliron complex formation from anoxia-initiated cellular chelatable iron increase.

    PubMed

    Li, Qian; Li, Chuanyu; Mahtani, Harry K; Du, Jian; Patel, Aashka R; Lancaster, Jack R

    2014-07-18

    Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with (•)NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged (•)NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief (•)NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1-2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief (•)NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of (•)NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of (•)NO.

  4. DNA Vaccine Encoding the Chimeric Form of Schistosoma mansoni Sm-TSP2 and Sm29 Confers Partial Protection against Challenge Infection

    PubMed Central

    Gonçalves de Assis, Natan Raimundo; Batistoni de Morais, Suellen; Figueiredo, Bárbara Castro Pimentel; Ricci, Natasha Delaqua; de Almeida, Leonardo Augusto; da Silva Pinheiro, Carina; Martins, Vicente de Paulo; Oliveira, Sergio Costa

    2015-01-01

    Schistosomiasis is an important parasitic disease worldwide that affects more than 207 million people in 76 countries and causes approximately 250,000 deaths per year. The best long-term strategy to control schistosomiasis is through immunization combined with drug treatment. Due to the ability of DNA vaccines to generate humoral and cellular immune responses, such vaccines are considered a promising approach against schistosomiasis. Sm29 and tetraspanin-2 (Sm-TSP2) are two proteins that are located in the S. mansoni tegument of adult worms and schistosomula and induce high levels of protection through recombinant protein immunization. In this study, we transfected BHK-21 cells with plasmids encoding Sm29, Sm-TSP2 or a chimera containing both genes. Using RT-PCR analysis and western blot, we confirmed that the DNA vaccine constructs were transcribed and translated, respectively, in BHK-21 cells. After immunization of mice, we evaluated the reduction in worm burden. We observed worm burden reductions of 17-22%, 22%, 31-32% and 24-32% in animals immunized with the pUMVC3/Sm29, pUMVC3/SmTSP-2, pUMVC3/Chimera and pUMVC3/Sm29 + pUMVC3/SmTSP-2 plasmids, respectively. We evaluated the humoral response elicited by DNA vaccines, and animals immunized with pUMVC3/Sm29 and pUMVC3/Sm29 + pUMVC3/SmTSP-2 showed higher titers of anti-Sm29 antibodies. The cytokine profile produced by the spleen cells of immunized mice was then evaluated. We observed higher production of Th1 cytokines, such as TNF-α and IFN-γ, in vaccinated mice and no significant production of IL-4 and IL-5. The DNA vaccines tested in this study showed the ability to generate a protective immune response against schistosomiasis, probably through the production of Th1 cytokines. However, future strategies aiming to optimize the protective response induced by a chimeric DNA construct need to be developed. PMID:25942636

  5. DNA Vaccine Encoding the Chimeric Form of Schistosoma mansoni Sm-TSP2 and Sm29 Confers Partial Protection against Challenge Infection.

    PubMed

    Gonçalves de Assis, Natan Raimundo; Batistoni de Morais, Suellen; Figueiredo, Bárbara Castro Pimentel; Ricci, Natasha Delaqua; de Almeida, Leonardo Augusto; da Silva Pinheiro, Carina; Martins, Vicente de Paulo; Oliveira, Sergio Costa

    2015-01-01

    Schistosomiasis is an important parasitic disease worldwide that affects more than 207 million people in 76 countries and causes approximately 250,000 deaths per year. The best long-term strategy to control schistosomiasis is through immunization combined with drug treatment. Due to the ability of DNA vaccines to generate humoral and cellular immune responses, such vaccines are considered a promising approach against schistosomiasis. Sm29 and tetraspanin-2 (Sm-TSP2) are two proteins that are located in the S. mansoni tegument of adult worms and schistosomula and induce high levels of protection through recombinant protein immunization. In this study, we transfected BHK-21 cells with plasmids encoding Sm29, Sm-TSP2 or a chimera containing both genes. Using RT-PCR analysis and western blot, we confirmed that the DNA vaccine constructs were transcribed and translated, respectively, in BHK-21 cells. After immunization of mice, we evaluated the reduction in worm burden. We observed worm burden reductions of 17-22%, 22%, 31-32% and 24-32% in animals immunized with the pUMVC3/Sm29, pUMVC3/SmTSP-2, pUMVC3/Chimera and pUMVC3/Sm29 + pUMVC3/SmTSP-2 plasmids, respectively. We evaluated the humoral response elicited by DNA vaccines, and animals immunized with pUMVC3/Sm29 and pUMVC3/Sm29 + pUMVC3/SmTSP-2 showed higher titers of anti-Sm29 antibodies. The cytokine profile produced by the spleen cells of immunized mice was then evaluated. We observed higher production of Th1 cytokines, such as TNF-α and IFN-γ, in vaccinated mice and no significant production of IL-4 and IL-5. The DNA vaccines tested in this study showed the ability to generate a protective immune response against schistosomiasis, probably through the production of Th1 cytokines. However, future strategies aiming to optimize the protective response induced by a chimeric DNA construct need to be developed.

  6. DNA Vaccine that Targets Hemagglutinin to MHC Class II Molecules Rapidly Induces Antibody-Mediated Protection against Influenza

    PubMed Central

    Mjaaland, Siri; Roux, Kenneth H.; Fredriksen, Agnete Brunsvik

    2013-01-01

    New influenza A viruses with pandemic potential periodically emerge due to viral genomic reassortment. In the face of pandemic threats, production of conventional egg-based vaccines is time consuming and of limited capacity. We have developed in this study a novel DNA vaccine in which viral hemagglutinin (HA) is bivalently targeted to MHC class II (MHC II) molecules on APCs. Following DNA vaccination, transfected cells secreted vaccine proteins that bound MHC II on APCs and initiated adaptive immune responses. A single DNA immunization induced within 8 d protective levels of strain-specific Abs and also cross-reactive T cells. During the Mexican flu pandemic, a targeted DNA vaccine (HA from A/California/07/2009) was generated within 3 wk after the HA sequences were published online. These results suggest that MHC II–targeted DNA vaccines could play a role in situations of pandemic threats. The vaccine principle should be extendable to other infectious diseases. PMID:23956431

  7. Antioxidant, cytotoxic, antitumor, and protective DNA damage metabolites from the red sea brown alga Sargassum sp

    PubMed Central

    Ayyad, Seif-Eldin N.; Ezmirly, Saleh T.; Basaif, Salim A.; Alarif, Walied M.; Badria, Adel F.; Badria, Farid A.

    2011-01-01

    Background: Macroalgae can be viewed as a potential antioxidant and anti-inflammatory sources owing to their capability of producing compounds for its protection from environmental factors such as heat, pollution, stress, oxygen concentration, and UV radiations. Objective: To isolate major compounds which are mainly responsible for the pharmacological activity of brown alga under investigation, Sargassum sp. Materials and Methods: Algal material was air dried, extracted with a mixture of organic solvents, and fractionated with different adsorbents. The structures of obtained pure compounds were elucidated with different spectroscopic techniques, and two pure materials were tested for protection of DNA from damage, antioxidant, antitumor, and cytotoxicity. Results: Four pure compounds were obtained, of which fucosterol (1) and fucoxanthin (4) were tested; it was found that fucoxanthin has strong antioxidant and cytotoxicity against breast cancer (MCF-7) with IC50 = 11.5 μg/ml. Conclusion: The naturally highly conjugated safe compound fucoxanthin could be used as antioxidant and as an antitumor compound. PMID:22022163

  8. Interaction of Iron II Complexes with B-DNA. Insights from Molecular Modeling, Spectroscopy, and Cellular Biology

    PubMed Central

    Gattuso, Hugo; Duchanois, Thibaut; Besancenot, Vanessa; Barbieux, Claire; Assfeld, Xavier; Becuwe, Philippe; Gros, Philippe C.; Grandemange, Stephanie; Monari, Antonio

    2015-01-01

    We report the characterization of the interaction between B-DNA and three terpyridin iron II complexes. Relatively long time-scale molecular dynamics (MD) is used in order to characterize the stable interaction modes. By means of molecular modeling and UV-vis spectroscopy, we prove that they may lead to stable interactions with the DNA duplex. Furthermore, the presence of larger π-conjugated moieties also leads to the appearance of intercalation binding mode. Non-covalent stabilizing interactions between the iron complexes and the DNA are also characterized and evidenced by the analysis of the gradient of the electronic density. Finally, the structural deformations induced on the DNA in the different binding modes are also evidenced. The synthesis and chemical characterization of the three complexes is reported, as well as their absorption spectra in presence of DNA duplexes to prove the interaction with DNA. Finally, their effects on human cell cultures have also been evidenced to further enlighten their biological effects. PMID:26734600

  9. Interaction of Iron II Complexes with B-DNA. Insights from Molecular Modeling, Spectroscopy, and Cellular Biology.

    PubMed

    Gattuso, Hugo; Duchanois, Thibaut; Besancenot, Vanessa; Barbieux, Claire; Assfeld, Xavier; Becuwe, Philippe; Gros, Philippe C; Grandemange, Stephanie; Monari, Antonio

    2015-01-01

    We report the characterization of the interaction between B-DNA and three terpyridin iron II complexes. Relatively long time-scale molecular dynamics (MD) is used in order to characterize the stable interaction modes. By means of molecular modeling and UV-vis spectroscopy, we prove that they may lead to stable interactions with the DNA duplex. Furthermore, the presence of larger π-conjugated moieties also leads to the appearance of intercalation binding mode. Non-covalent stabilizing interactions between the iron complexes and the DNA are also characterized and evidenced by the analysis of the gradient of the electronic density. Finally, the structural deformations induced on the DNA in the different binding modes are also evidenced. The synthesis and chemical characterization of the three complexes is reported, as well as their absorption spectra in presence of DNA duplexes to prove the interaction with DNA. Finally, their effects on human cell cultures have also been evidenced to further enlighten their biological effects. PMID:26734600

  10. Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR.

    PubMed

    Henrich, Timothy J; Gallien, Sebastien; Li, Jonathan Z; Pereyra, Florencia; Kuritzkes, Daniel R

    2012-12-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia were also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  11. Influence of extracellular pH on the cytotoxicity, cellular accumulation, and DNA interaction of novel pH-sensitive 2-aminoalcoholatoplatinum(II) complexes

    PubMed Central

    Valiahdi, Seied Mojtaba; Egger, Alexander E.; Miklos, Walter; Jungwirth, Ute; Meelich, Kristof; Nock, Petra; Berger, Walter; Hartinger, Christian G.; Galanski, Markus; Jakupec, Michael A.; Keppler, Bernhard K.

    2014-01-01

    Extracellular acidity is a frequent pathophysiological condition of solid tumors offering possibilities for improving the tumor selectivity of molecular therapy. This might be accomplished by prodrugs with low systemic toxicity, attaining their full antitumor potency only under acidic conditions, such as bis(2-aminoalcoholato-κ2N,O)platinum(II) complexes that are activated by protonation of alcoholato oxygen, resulting in cleavage of platinum–oxygen bonds. In this work, we examined whether the pH dependency of such compounds is reflected in differential biological activity in vitro. In particular, the pH dependence of cytotoxicity, cellular accumulation, DNA platination, GMP binding, effects on DNA secondary structure, cell cycle alterations, and induction of apoptosis was investigated. Enhanced cytotoxicity of five of these complexes in non-small-cell lung cancer (A549) and colon carcinoma (HT-29) cells at pH 6.0 in comparison with pH 7.4 was confirmed: 50 % growth inhibition concentrations ranged from 42 to 214 μM in A549 cells and from 35 to 87 μM in HT-29 cells at pH 7.4 and decreased at pH 6.0 to 11–50 and 7.3–25 μM, respectively. The effects induced by all five pH-sensitive compounds involve increased 5′-GMP binding, cellular accumulation, and DNA platination as well as stronger effects on DNA secondary structure at pH 6.0 than at pH 7.4. As exemplified by treatment of A549 cells with a 2-amino-4-methyl-1-pentanolato complex, induction of apoptosis is enhanced at pH 6.5. These results confirm the increased reactivity and in vitro activity of these compounds under slightly acidic conditions, encouraging further evaluation of ring-closed aminoalcoholatoplatinum(II) derivatives in solid tumors in vivo. PMID:23354303

  12. Inhibition of DNA methyltransferase or histone deacetylase protects retinal pigment epithelial cells from DNA damage induced by oxidative stress by the stimulation of antioxidant enzymes.

    PubMed

    Tokarz, Paulina; Kaarniranta, Kai; Blasiak, Janusz

    2016-04-01

    Epigenetic modifications influence DNA damage response (DDR). In this study we explored the role of DNA methylation and histone acetylation in DDR in cells challenged with acute or chronic oxidative stress. We used retinal pigment epithelial cells (ARPE-19), which natively are exposed to oxidative stress due to permanent exposure to light and high blood flow. We employed a DNA methyltransferase inhibitor - RG108 (RG), or a histone deacetylase inhibitor - valproic acid (VA). ARPE-19 cells were exposed to tert-butyl hydroperoxide, an acute oxidative stress inducer, or glucose oxidase, which slowly liberates low-doses of hydrogen peroxide in the presence of glucose, creating chronic conditions. VA and RG reduced level of intracellular reactive oxygen species and DNA damage in ARPE-19 cells in normal condition and in oxidative stress. This protective effect of VA and RG was associated with the up-regulated expression of antioxidant enzyme genes: CAT, GPx1, GPx4, SOD1 and SOD2. RG decreased the number of cells in G2/M checkpoint in response to chronic oxidative stress. Neither RG nor VA changed the DNA repair or apoptosis induced by oxidative stress. Therefore, certain epigenetic manipulations may protect ARPE-19 cells from detrimental effects of oxidative stress by modulation of antioxidative enzyme gene expression, which may be further explored in pharmacological studies on oxidative stress-related eye diseases.

  13. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

    PubMed

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

    2014-04-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy.

  14. Cellular accumulation of 18F-labelled boronophenylalanine depending on DNA synthesis and melanin incorporation: a double-tracer microautoradiographic study of B16 melanomas in vivo.

    PubMed

    Kubota, R; Yamada, S; Ishiwata, K; Tada, M; Ido, T; Kubota, K

    1993-04-01

    The cellular distribution of 4-borono-2-[18F]fluoro-L-phenylalanine ([18F]FBPA, an analog of p-boronophenylaline), a potential agent for boron neutron capture therapy (BNCT), and [6-3H]thymidine ([3H]Thd, a DNA precursor) in murine two B16 melanoma sublines and FM3A mammary carcinoma was studied in vivo using double-tracer microautoradiography. Tumour volume, tumour age, cell density in the tissues and the proportion of S phase cells in the cell cycle were the same in the three tumour models. Volume doubling time, which represents tumour growth rate, was fastest in B16F10, followed by B16F1 (P < 0.05), the slowest being in FM3A (P < 0.001). The rate of DNA synthesis in S phase cells corresponded to the volume doubling time. The greatest amount of [18F]FBPA was observed in S phase melanocytes and the lowest amount was found in non-S phase non-melanocytes. The [18F]FBPA accumulation was primarily related to the activity of DNA synthesis and, secondarily, to the degree of pigmentation in melanocytes. The therapeutic efficacy of BNCT with p-boronophenylalanine may be greater in melanoma that exhibits greater DNA synthesis activity and higher melanin content.

  15. Cellular accumulation of 18F-labelled boronophenylalanine depending on DNA synthesis and melanin incorporation: a double-tracer microautoradiographic study of B16 melanomas in vivo.

    PubMed Central

    Kubota, R.; Yamada, S.; Ishiwata, K.; Tada, M.; Ido, T.; Kubota, K.

    1993-01-01

    The cellular distribution of 4-borono-2-[18F]fluoro-L-phenylalanine ([18F]FBPA, an analog of p-boronophenylaline), a potential agent for boron neutron capture therapy (BNCT), and [6-3H]thymidine ([3H]Thd, a DNA precursor) in murine two B16 melanoma sublines and FM3A mammary carcinoma was studied in vivo using double-tracer microautoradiography. Tumour volume, tumour age, cell density in the tissues and the proportion of S phase cells in the cell cycle were the same in the three tumour models. Volume doubling time, which represents tumour growth rate, was fastest in B16F10, followed by B16F1 (P < 0.05), the slowest being in FM3A (P < 0.001). The rate of DNA synthesis in S phase cells corresponded to the volume doubling time. The greatest amount of [18F]FBPA was observed in S phase melanocytes and the lowest amount was found in non-S phase non-melanocytes. The [18F]FBPA accumulation was primarily related to the activity of DNA synthesis and, secondarily, to the degree of pigmentation in melanocytes. The therapeutic efficacy of BNCT with p-boronophenylalanine may be greater in melanoma that exhibits greater DNA synthesis activity and higher melanin content. Images Figure 1 PMID:8471428

  16. Induction of DNA Double-Strand Breaks and Cellular Migration Through Bystander Effects in Cells Irradiated With the Slit-Type Microplanar Beam of the Spring-8 Synchrotron

    SciTech Connect

    Kashino, Genro Kondoh, Takeshi; Nariyama, Nobuteru; Umetani, Keiji; Ohigashi, Takuji; Shinohara, Kunio; Kurihara, Ai; Fukumoto, Manabu; Tanaka, Hiroki; Maruhashi, Akira; Suzuki, Minoru; Kinashi, Yuko; Liu Yong; Masunaga, Shin-ichiro; Watanabe, Masami; Ono, Koji

    2009-05-01

    Purpose: To determine whether glioma cells irradiated with a microplanar X-ray beam exert bystander effects. Methods and Materials: Microplanar beam irradiation of glioma cells in vitro was done using the SPring-8 synchrotron radiation facility. The amount of DNA double-strand breaks (dsbs) was measured by the fluorescence intensity of phosphorylated H2AX or the number of 53BP1 foci. The dose distribution in a cell population exposed to a single microplanar beam was determined by the amount of phosphorylated H2AX-positive cells. Bystander effects were determined by counting the number of 53BP1 foci in nonirradiated cells treated with conditioned medium from cultures of irradiated cells. Results: More DNA dsbs were detected in cells adjacent to an area irradiated by the single beam than in cells in distant, nonirradiated areas as a result of bystander effects caused by scattered X-rays and DNA dsbs. In support of this, more 53BP1 foci were observed in nonirradiated, conditioned medium-treated cells than in control cells (i.e., cells not treated with irradiation or conditioned medium). These results suggest that DNA dsbs were induced in nonirradiated cells by soluble factors in the culture medium. In addition, we observed cellular migration into areas irradiated with peak doses, suggesting that irradiated cells send signals that cause nonirradiated cells to migrate toward damaged cells. Conclusions: Bystander effects are produced by factors secreted as a result of slit-type microplanar X-ray beam irradiation.

  17. Lunasin peptide purified from Solanum nigrum L. protects DNA from oxidative damage by suppressing the generation of hydroxyl radical via blocking fenton reaction.

    PubMed

    Jeong, Jin Boo; De Lumen, Ben O; Jeong, Hyung Jin

    2010-07-01

    Oxidative DNA damage is the most critical factor implicated in carcinogenesis and other disorders. However, the protective effects of lunasin against oxidative DNA damage have not yet reported. In this study, we report here the protective effect of lunasin purified from Solanum nigrum L. against oxidative DNA. Lunasin protected DNA from the oxidative damage induced by Fe(2+) ion and hydroxyl radical. To better understand the mechanism for the protective effect of lunasin against DNA damage, the abilities to chelate Fe(2+), scavenge the generated hydroxyl radical and block the generation of hydroxyl radical were evaluated. Although it did not scavenge generated hydroxyl radical, lunasin blocked the generation of hydroxyl radical by chelating Fe(2+) ion. We conclude that lunasin protects DNA from oxidation by blocking fenton reaction between Fe(2+) and H(2)O(2) by chelating Fe(2+) and that consumption of lunasin may play an important role in the chemoprevention for the oxidative carcinogenesis. PMID:20083341

  18. The Cellular Response to Oxidatively Induced DNA Damage and Polymorphism of Some DNA Repair Genes Associated with Clinicopathological Features of Bladder Cancer.

    PubMed

    Savina, Nataliya V; Nikitchenko, Nataliya V; Kuzhir, Tatyana D; Rolevich, Alexander I; Krasny, Sergei A; Goncharova, Roza I

    2016-01-01

    Genome instability and impaired DNA repair are hallmarks of carcinogenesis. The study was aimed at evaluating the DNA damage response in H2O2-treated lymphocytes using the alkaline comet assay in bladder cancer (BC) patients as compared to clinically healthy controls, elderly persons, and individuals with chronic inflammations. Polymorphism in DNA repair genes involved in nucleotide excision repair (NER) and base excision repair (BER) was studied using the PCR-RFLP method in the Belarusian population to elucidate the possible association of their variations with both bladder cancer risk and clinicopathological features of tumors. The increased level of H2O2-induced DNA damage and a higher proportion of individuals sensitive to oxidative stress were found among BC patients as compared to other groups under study. Heterozygosity in the XPD gene (codon 751) increased cancer risk: OR (95% CI) = 1.36 (1.03-1.81), p = 0.031. The frequency of the XPD 312Asn allele was significantly higher in T ≥ 2 high grade than in T ≥ 2 low grade tumors (p = 0.036); the ERCC6 1097Val/Val genotype was strongly associated with muscle-invasive tumors. Combinations of homozygous wild type alleles occurred with the increased frequency in patients with non-muscle-invasive tumors suggesting that the maintenance of normal DNA repair activity may prevent cancer progression.

  19. The Cellular Response to Oxidatively Induced DNA Damage and Polymorphism of Some DNA Repair Genes Associated with Clinicopathological Features of Bladder Cancer

    PubMed Central

    Savina, Nataliya V.; Nikitchenko, Nataliya V.; Kuzhir, Tatyana D.; Rolevich, Alexander I.; Krasny, Sergei A.; Goncharova, Roza I.

    2016-01-01

    Genome instability and impaired DNA repair are hallmarks of carcinogenesis. The study was aimed at evaluating the DNA damage response in H2O2-treated lymphocytes using the alkaline comet assay in bladder cancer (BC) patients as compared to clinically healthy controls, elderly persons, and individuals with chronic inflammations. Polymorphism in DNA repair genes involved in nucleotide excision repair (NER) and base excision repair (BER) was studied using the PCR-RFLP method in the Belarusian population to elucidate the possible association of their variations with both bladder cancer risk and clinicopathological features of tumors. The increased level of H2O2-induced DNA damage and a higher proportion of individuals sensitive to oxidative stress were found among BC patients as compared to other groups under study. Heterozygosity in the XPD gene (codon 751) increased cancer risk: OR (95% CI) = 1.36 (1.03–1.81), p = 0.031. The frequency of the XPD 312Asn allele was significantly higher in T ≥ 2 high grade than in T ≥ 2 low grade tumors (p = 0.036); the ERCC6 1097Val/Val genotype was strongly associated with muscle-invasive tumors. Combinations of homozygous wild type alleles occurred with the increased frequency in patients with non-muscle-invasive tumors suggesting that the maintenance of normal DNA repair activity may prevent cancer progression. PMID:26649138

  20. Potent inhibition of peroxynitrite-induced DNA strand breakage by ethanol: possible implications for ethanol-mediated cardiovascular protection.

    PubMed

    Cao, Zhuoxiao; Li, Yunbo

    2004-07-01

    Epidemiological studies have conclusively demonstrated that moderate consumption of ethanol is causally associated with a significant reduction in cardiovascular events. However, the exact mechanisms underlying the ethanol-mediated cardiovascular protection remain to be elucidated. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of cardiovascular disorders via its cytotoxic effects, this study was undertaken to investigate if ethanol could inhibit peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite-elicited cytotoxicity. Toward this goal, phiX-174 RF I plasmid DNA was used as an in vitro model to determine the protective effects of ethanol on peroxynitrite-induced DNA strand breaks. Incubation of phiX-174 plasmid DNA with the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1) led to the formation of both single- and double-stranded DNA breaks in a concentration- and time-dependent fashion. The presence of ethanol at concentrations ranging from 0.01 to 1% (w/v) resulted in a significant inhibition of SIN-1-induced DNA strand breaks. Ethanol also showed inhibitory effects on SIN-1-induced DNA strand breakage in the presence of bicarbonate. The inhibition of SIN-1-induced DNA strand breaks by ethanol exhibited a concentration-dependent manner. Notably, a marked inhibition of SIN-1-elicited DNA strand breaks was observed with 0.01% ethanol. Ethanol at 0.01-1% was unable to affect SIN-1-mediated oxygen consumption, indicating that ethanol did not affect the auto-oxidation of SIN-1 to form peroxynitrite. Furthermore, incubation of the plasmid DNA with authentic peroxynitrite resulted in a significant formation of DNA strand breaks, which could be dramatically inhibited by the presence of 0.02-0.1% ethanol. Taken together, this study demonstrates for the first time that ethanol at physiologically relevant concentrations can potently inhibit peroxynitrite-induced DNA strand breakage. In view of

  1. Protective effects of guarana (Paullinia cupana Mart. var. Sorbilis) against DEN-induced DNA damage on mouse liver.

    PubMed

    Fukumasu, H; Avanzo, J L; Heidor, R; Silva, T C; Atroch, A; Moreno, F S; Dagli, M L Z

    2006-06-01

    Guarana (Paullinia cupana Mart. var. Sorbilis) is a plant originally from Brazil, which is rich in tannins. Some tannins are known to present protective effects against DNA damage. This study was performed to investigate the anti-genotoxic/cytotoxic properties of guarana in hepatocytes of mice injected with N-nitrosodiethylamine (DEN). The protective effect of guarana was evaluated both by comet assay and DNA smear fragmentation technique in two month-old female BALB/c mice. These were treated previously with 2.0 mg/g bw of guarana for 16 days and then injected with DEN (160 microg/g body weight) to induce DNA damage. The DEN-only treated group presented higher comet image length than the guarana plus DEN and untreated groups (116.06+/-5.0 microm, 104.09+/-3.3 microm and 93.28+/-14.4 microm, respectively; p<0.01). Guarana treatment presented a 52.54% reduction in comet image length when animals were exposed to DEN (p<0.05). DNA samples from the guarana plus DEN group clearly showed less EtBr fluorescence intensity when compared to the DEN-only group, reinforcing the comet assay data. These results show, for the first time, that guarana has a protective effect against DEN-induced DNA damage in mouse liver.

  2. Timed interactions between viral and cellular replication factors during the initiation of SV40 in vitro DNA replication

    PubMed Central

    Taneja, Poonam; Nasheuer, Heinz-Peter; Hartmann, Hella; Grosse, Frank; Fanning, Ellen; Weisshart, Klaus

    2007-01-01

    The initiation of SV40 (simian virus 40) DNA replication requires the co-operative interactions between the viral Tag (large T-antigen), RPA (replication protein A) and Pol (DNA polymerase α-primase) on the template DNA. Binding interfaces mapped on these enzymes and expressed as peptides competed with the mutual interactions of the native proteins. Prevention of the genuine interactions was accomplished only prior to the primer synthesis step and blocked the assembly of a productive initiation complex. Once the complex was engaged in the synthesis of an RNA primer and its extension, the interfering effects of the peptides ceased, suggesting a stable association of the replication factors during the initiation phase. Specific antibodies were still able to disrupt preformed interactions and inhibited primer synthesis and extension activities, underlining the crucial role of specific protein–protein contacts during the entire initiation process. PMID:17666013

  3. Cellular uptake, cytotoxicity, apoptosis, DNA-binding, photocleavage and molecular docking studies of ruthenium(II) polypyridyl complexes.

    PubMed

    Srishailam, A; Kumar, Yata Praveen; Venkat Reddy, P; Nambigari, Navaneetha; Vuruputuri, Uma; Singh, Surya S; Satyanarayana, S

    2014-03-01

    Three new mononuclear [Ru (phen)2 ptip](2+) (1), [Ru (bpy)2 ptip](2+) (2) and [Ru (dmb)2 ptip](2+) (3) [ptip=(2-(5-phenylthiophen-2-yl)-1H-imidazo[4, 5-f][1,10 phenanthroline, phen=1, 10 phenanthroline, bpy=2, 2' bipyridine, dmb=4, 4'-dimethyl 2, 2' bipyridine] complexes were synthesized and characterised by elemental analysis, IR, NMR and Mass spectra. The DNA-binding behaviours were investigated by electronic absorption titration, luminescence spectra, viscosity measurements and photo-activated cleavage. The DNA-binding constants Kb of complexes 1, 2 and 3 were determined to be 7.0 (± 0.06)× 10(5), 3.87 (± 0.04) × 10(5), 2.79 (±0.07) × 10(5) respectively. The results showed that these complexes interact with CT-DNA by intercalative mode. Cell viability experiments indicated that the Ru(II) complex showed significant dose-dependent cytotoxicity to HeLa tumour cell lines. Further flow cytometry experiments showed that the cytotoxic Ru(II) complex induced apoptosis of HeLa tumour cell lines. Our data demonstrated that the Ru(II) polypyridyl complex binds to DNA and thereby induces apoptosis in tumor cells, suggesting that anti-tumor activity of the Ru(II) complex could be related to its interaction with DNA. The molecular dynamic simulations and docking methods were used to predict the DNA binding affinity of ruthenium complexes and with good visualisation images supporting with experimental results.

  4. CCL21 (SLC) improves tumor protection by a DNA vaccine in a Her2/neu mouse tumor model.

    PubMed

    Nguyen-Hoai, T; Baldenhofer, G; Sayed Ahmed, M S; Pham-Duc, M; Vu, M D; Lipp, M; Dörken, B; Pezzutto, A; Westermann, J

    2012-01-01

    Secondary lymphoid-tissue chemokine (SLC/CCL21) is a CC chemokine that is constitutively expressed in various lymphoid tissues and binds to chemokine receptor CCR7 on mature dendritic cells (DCs) and distinct T-and B-cell sub-populations. In vivo, CCL21 regulates the encounters between DC and T cells and thus is a key regulator of adaptive immune responses. We asked whether CCL21 is able to augment immunogenicity of a DNA-based vaccine against Her2/neu in a Balb/c mouse model with syngeneic Her2/neu+ tumor cells (D2F2/E2). Mice were vaccinated intramuscularly with plasmid DNA (pDNA) on day 1 and boosted on day 15; tumor challenge was performed subcutaneously on day 25. Coexpression of CCL21 and Her-2/neu resulted in induction of a TH1-polarized immune response and substantial improvement of the protective effect of the DNA vaccine. Coexpression of tumor antigen pDNA(Her2/neu) with both pDNA(GM-CSF) and pDNA(CCL21) as adjuvants led to further improvement of protection by the vaccine (70% tumor-free mice on day 35 vs 40% with either adjuvant alone vs 5-10% with tumor antigen alone). Our results show that CCL21 is a potent adjuvant for DNA vaccination, particularly in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF). Clinical use of a pDNA(Her2/neu/CCL21/GM-CSF) vaccine might be particularly promising in minimal residual Her2/neu+ breast cancer.

  5. Protection of radiation induced DNA and membrane damages by total triterpenes isolated from Ganoderma lucidum (Fr.) P. Karst.

    PubMed

    Smina, T P; Maurya, D K; Devasagayam, T P A; Janardhanan, K K

    2015-05-25

    The total triterpenes isolated from the fruiting bodies of Ganoderma lucidum was examined for its potential to prevent γ-radiation induced membrane damage in rat liver mitochondria and microsomes. The effects of total triterpenes on γ-radiation-induced DNA strand breaks in pBR 322 plasmid DNA in vitro and human peripheral blood lymphocytes ex vivo were evaluated. The protective effect of total triterpenes against γ-radiation-induced micronuclei formations in mice bone marrow cells in vivo were also evaluated. The results indicated the significant effectiveness of Ganoderma triterpenes in protecting the DNA and membrane damages consequent to the hazardous effects of radiation. The findings suggest the potential use of Ganoderma triterpenes in radio therapy. PMID:25824410

  6. Protection of radiation induced DNA and membrane damages by total triterpenes isolated from Ganoderma lucidum (Fr.) P. Karst.

    PubMed

    Smina, T P; Maurya, D K; Devasagayam, T P A; Janardhanan, K K

    2015-05-25

    The total triterpenes isolated from the fruiting bodies of Ganoderma lucidum was examined for its potential to prevent γ-radiation induced membrane damage in rat liver mitochondria and microsomes. The effects of total triterpenes on γ-radiation-induced DNA strand breaks in pBR 322 plasmid DNA in vitro and human peripheral blood lymphocytes ex vivo were evaluated. The protective effect of total triterpenes against γ-radiation-induced micronuclei formations in mice bone marrow cells in vivo were also evaluated. The results indicated the significant effectiveness of Ganoderma triterpenes in protecting the DNA and membrane damages consequent to the hazardous effects of radiation. The findings suggest the potential use of Ganoderma triterpenes in radio therapy.

  7. Human T-cell leukemia virus-I tax oncoprotein functionally targets a subnuclear complex involved in cellular DNA damage-response.

    PubMed

    Haoudi, Abdelali; Daniels, Rodney C; Wong, Eric; Kupfer, Gary; Semmes, O John

    2003-09-26

    The virally encoded oncoprotein Tax has been implicated in HTLV-1-mediated cellular transformation. The exact mechanism by which this protein contributes to the oncogenic process is not known. However, it has been hypothesized that Tax induces genomic instability via repression of cellular DNA repair. We examined the effect of de novo Tax expression upon the cell cycle, because appropriate activation of cell cycle checkpoints is essential to a robust damage-repair response. Upon induction of tax expression, Jurkat T-cells displayed a pronounced accumulation in G2/M that was reversible by caffeine. We examined the G2-specific checkpoint signaling response in these cells and found activation of the ATM/chk2-mediated pathway, whereas the ATR/chk1-mediated response was unaffected. Immunoprecipitation with anti-chk2 antibody results in co-precipitation of Tax demonstrating a direct interaction of Tax with a chk2-containing complex. We also show that Tax targets a discrete nuclear site and co-localizes with chk2 and not chk1. This nuclear site, previously identified as Tax Speckled Structures (TSS), also contains the early damage response factor 53BP1. The recruitment of 53BP1 to TSS is dependent upon ATM signaling and requires expression of Tax. Specifically, Tax expression induces redistribution of diffuse nuclear 53BP1 to the TSS foci. Taken together these data suggest that the TSS describe a unique nuclear site involved in DNA damage recognition, repair response, and cell cycle checkpoint activation. We suggest that association of Tax with this multifunctional subnuclear site results in disruption of a subset of the site-specific activities and contributes to cellular genomic instability. PMID:12842897

  8. Analysis of DNA Repair and Protection in the Tardigrade Ramazzottius varieornatus and Hypsibius dujardini after Exposure to UVC Radiation

    PubMed Central

    Horikawa, Daiki D.; Cumbers, John; Sakakibara, Iori; Rogoff, Dana; Leuko, Stefan; Harnoto, Raechel; Arakawa, Kazuharu; Katayama, Toshiaki; Kunieda, Takekazu; Toyoda, Atsushi; Fujiyama, Asao; Rothschild, Lynn J.

    2013-01-01

    Tardigrades inhabiting terrestrial environments exhibit extraordinary resistance to ionizing radiation and UV radiation although little is known about the mechanisms underlying the resistance. We found that the terrestrial tardigrade Ramazzottius varieornatus is able to tolerate massive doses of UVC irradiation by both being protected from forming UVC-induced thymine dimers in DNA in a desiccated, anhydrobiotic state as well as repairing the dimers that do form in the hydrated animals. In R. varieornatus accumulation of thymine dimers in DNA induced by irradiation with 2.5 kJ/m2 of UVC radiation disappeared 18 h after the exposure when the animals were exposed to fluorescent light but not in the dark. Much higher UV radiation tolerance was observed in desiccated anhydrobiotic R. varieornatus compared to hydrated specimens of this species. On the other hand, the freshwater tardigrade species Hypsibius dujardini that was used as control, showed much weaker tolerance to UVC radiation than R. varieornatus, and it did not contain a putative phrA gene sequence. The anhydrobiotes of R. varieornatus accumulated much less UVC-induced thymine dimers in DNA than hydrated one. It suggests that anhydrobiosis efficiently avoids DNA damage accumulation in R. varieornatus and confers better UV radiation tolerance on this species. Thus we propose that UV radiation tolerance in tardigrades is due to the both high capacities of DNA damage repair and DNA protection, a two-pronged survival strategy. PMID:23762256

  9. Arg-Gingipain A DNA Vaccine Induces Protective Immunity against Infection by Porphyromonas gingivalis in a Murine Model

    PubMed Central

    Yonezawa, Hideo; Ishihara, Kazuyuki; Okuda, Katsuji

    2001-01-01

    Arginine-specific cysteine proteinases (RgpA and RgpB) produced by the periodontal pathogen Porphyromonas gingivalis are suspected virulence factors and are involved in interrupting host defense mechanisms as well as in penetrating and destroying periodontal connective tissues. To induce a protective immune response against P. gingivalis, we constructed an rgpA DNA vaccine. BALB/c mice were immunized intradermally by Gene Gun with plasmid DNA carrying rgpA. Antibody responses against P. gingivalis were determined by an enzyme-linked immunosorbent assay. The rgpA DNA vaccine induced high levels of serum antibodies against P. gingivalis. Sera from the rgpA DNA vaccine-immunized mice diminished the proteolytic activity of RgpA and RgpB and inhibited the binding of P. gingivalis to a type I collagen sponge. Moreover, the sera effectively reduced the hemagglutination of P. gingivalis, indicating that the hemagglutinin activity of the organism is associated with RgpA. We found with a murine abscess model that mice immunized with the rgpA DNA vaccine were resistant to an invasive P. gingivalis W50 challenge. These results suggest that the rgpA DNA vaccine induced specific antibodies against the enzyme and that this vaccine could confer protective immunity against P. gingivalis infection. PMID:11292699

  10. Analysis of DNA repair and protection in the Tardigrade Ramazzottius varieornatus and Hypsibius dujardini after exposure to UVC radiation.

    PubMed

    Horikawa, Daiki D; Cumbers, John; Sakakibara, Iori; Rogoff, Dana; Leuko, Stefan; Harnoto, Raechel; Arakawa, Kazuharu; Katayama, Toshiaki; Kunieda, Takekazu; Toyoda, Atsushi; Fujiyama, Asao; Rothschild, Lynn J

    2013-01-01

    Tardigrades inhabiting terrestrial environments exhibit extraordinary resistance to ionizing radiation and UV radiation although little is known about the mechanisms underlying the resistance. We found that the terrestrial tardigrade Ramazzottius varieornatus is able to tolerate massive doses of UVC irradiation by both being protected from forming UVC-induced thymine dimers in DNA in a desiccated, anhydrobiotic state as well as repairing the dimers that do form in the hydrated animals. In R. varieornatus accumulation of thymine dimers in DNA induced by irradiation with 2.5 kJ/m(2) of UVC radiation disappeared 18 h after the exposure when the animals were exposed to fluorescent light but not in the dark. Much higher UV radiation tolerance was observed in desiccated anhydrobiotic R. varieornatus compared to hydrated specimens of this species. On the other hand, the freshwater tardigrade species Hypsibius dujardini that was used as control, showed much weaker tolerance to UVC radiation than R. varieornatus, and it did not contain a putative phrA gene sequence. The anhydrobiotes of R. varieornatus accumulated much less UVC-induced thymine dimers in DNA than hydrated one. It suggests that anhydrobiosis efficiently avoids DNA damage accumulation in R. varieornatus and confers better UV radiation tolerance on this species. Thus we propose that UV radiation tolerance in tardigrades is due to the both high capacities of DNA damage repair and DNA protection, a two-pronged survival strategy.

  11. Analysis of DNA repair and protection in the Tardigrade Ramazzottius varieornatus and Hypsibius dujardini after exposure to UVC radiation.

    PubMed

    Horikawa, Daiki D; Cumbers, John; Sakakibara, Iori; Rogoff, Dana; Leuko, Stefan; Harnoto, Raechel; Arakawa, Kazuharu; Katayama, Toshiaki; Kunieda, Takekazu; Toyoda, Atsushi; Fujiyama, Asao; Rothschild, Lynn J

    2013-01-01

    Tardigrades inhabiting terrestrial environments exhibit extraordinary resistance to ionizing radiation and UV radiation although little is known about the mechanisms underlying the resistance. We found that the terrestrial tardigrade Ramazzottius varieornatus is able to tolerate massive doses of UVC irradiation by both being protected from forming UVC-induced thymine dimers in DNA in a desiccated, anhydrobiotic state as well as repairing the dimers that do form in the hydrated animals. In R. varieornatus accumulation of thymine dimers in DNA induced by irradiation with 2.5 kJ/m(2) of UVC radiation disappeared 18 h after the exposure when the animals were exposed to fluorescent light but not in the dark. Much higher UV radiation tolerance was observed in desiccated anhydrobiotic R. varieornatus compared to hydrated specimens of this species. On the other hand, the freshwater tardigrade species Hypsibius dujardini that was used as control, showed much weaker tolerance to UVC radiation than R. varieornatus, and it did not contain a putative phrA gene sequence. The anhydrobiotes of R. varieornatus accumulated much less UVC-induced thymine dimers in DNA than hydrated one. It suggests that anhydrobiosis efficiently avoids DNA damage accumulation in R. varieornatus and confers better UV radiation tolerance on this species. Thus we propose that UV radiation tolerance in tardigrades is due to the both high capacities of DNA damage repair and DNA protection, a two-pronged survival strategy. PMID:23762256

  12. The Mbd4 DNA glycosylase protects mice from inflammation-driven colon cancer and tissue injury

    PubMed Central

    Yu, Amy Marie; Calvo, Jennifer A.; Muthupalani, Suresh; Samson, Leona D.

    2016-01-01

    Much of the global cancer burden is associated with longstanding inflammation accompanied by release of DNA-damaging reactive oxygen and nitrogen species. Here, we report that the Mbd4 DNA glycosylase is protective in the azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model of inflammation-driven colon cancer. Mbd4 excises T and U from T:G and U:G mismatches caused by deamination of 5-methylcytosine and cytosine. Since the rate of deamination is higher in inflamed tissues, we investigated the role of Mbd4 in inflammation-driven tumorigenesis. In the AOM/DSS assay, Mbd4−/− mice displayed more severe clinical symptoms, decreased survival, and a greater tumor burden than wild-type (WT) controls. The increased tumor burden in Mbd4−/− mice did not arise from impairment of AOM-induced apoptosis in the intestinal crypt. Histopathological analysis indicated that the colonic epithelium of Mbd4−/− mice is more vulnerable than WT to DSS-induced tissue damage. We investigated the role of the Mbd4−/− immune system in AOM/DSS-mediated carcinogenesis by repeating the assay on WT and Mbd4−/− mice transplanted with WT bone marrow. Mbd4−/− mice with WT bone marrow behaved similarly to Mbd4−/− mice. Together, our results indicate that the colonic epithelium of Mbd4−/− mice is more vulnerable to DSS-induced injury, which exacerbates inflammation-driven tissue injury and cancer. PMID:27086921

  13. Exploration of cellular DNA lesion, DNA-binding and biocidal ordeal of novel curcumin based Knoevenagel Schiff base complexes incorporating tryptophan: Synthesis and structural validation

    NASA Astrophysics Data System (ADS)

    Chandrasekar, Thiravidamani; Raman, Natarajan

    2016-07-01

    A few novel Schiff base transition metal complexes of general formula [MLCl] (where, L = Schiff base, obtained by the condensation reaction of Knoevenagel condensate of curcumin, L-tryptophan and M = Cu(II), Ni(II), Co(II), and Zn(II)), were prepared by stencil synthesis. They were typified using UV-vis, IR, EPR spectral techniques, micro analytical techniques, magnetic susceptibility and molar conductivity. Geometry of the metal complexes was examined and recognized as square planar. DNA binding and viscosity studies revealed that the metal(II) complexes powerfully bound via an intercalation mechanism with the calf thymus DNA. Gel-electrophoresis technique was used to investigate the DNA cleavage competence of the complexes and they establish to approve the cleavage of pBR322 DNA in presence of oxidant H2O2. This outcome inferred that the synthesized complexes showed better nuclease activity. Moreover, the complexes were monitored for antimicrobial activities. The results exposed that the synthesized compounds were forceful against all the microbes under exploration.

  14. Amifostine protection against induced DNA damage in gamma-irradiated Escherichia coli cells depend on recN DNA repair gene product activity.

    PubMed

    Almeida, Eliseo; Fuentes, Jorge Luis; Cuetara, Elizabeth; Prieto, Elio; Llagostera, Montserrat

    2010-04-01

    Amifostine is the most effective radioprotector known and the only one accepted for clinical use in cancer radiotherapy. In this work, the antigenotoxic effect of amifostine against gamma-rays was studied in Escherichia coli cells deficient in DNA damage repair activities. Assays of irradiated cells treated with amifostine showed that the drug reduced the genotoxicity induced by radiation in E. coli wild-type genotypes and in uvr, recF, recB, recB-recC-recF mutant strains, but not in recN defective cells. Thus, the mechanism of DNA protection by amifostine against gamma-radiation-induced genotoxicity appears to involve participation of the RecN protein that facilitates repair of DNA double-strand breaks. The results are discussed in relation to amifostine's chemopreventive potential.

  15. Arginine conjugates of metallo-supramolecular cylinders prescribe helicity and enhance DNA junction binding and cellular activity.

    PubMed

    Cardo, Lucia; Sadovnikova, Victoria; Phongtongpasuk, Siriporn; Hodges, Nikolas J; Hannon, Michael J

    2011-06-21

    The conjugation of arginine residues at the ends of a metallo-supramolecular triple-helical cylinder enables absolute control over the helicity of the cylinder core, and boosts the DNA junction recognition by the complexes and their activity against a cancer cell line.

  16. Protective immunity and lack of histopathological damage two years after DNA vaccination against infectious hematopoietic necrosis virus in trout

    USGS Publications Warehouse

    Kurath, Gael; Garver, Kyle A.; Corbeil, Serge; Elliott, Diane G.; Anderson, Eric D.; LaPatra, Scott E.

    2006-01-01

    The DNA vaccine pIHNw-G encodes the glycoprotein of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). Vaccine performance in rainbow trout was measured 3, 6, 13, 24, and 25 months after vaccination. At three months all fish vaccinated with 0.1 μg pIHNw-G had detectable neutralizing antibody (NAb) and they were completely protected from lethal IHNV challenge with a relative percent survival (RPS) of 100% compared to control fish. Viral challenges at 6, 13, 24, and 25 months post-vaccination showed protection with RPS values of 47–69%, while NAb seroprevalence declined to undetectable levels. Passive transfer experiments with sera from fish after two years post-vaccination were inconsistent but significant protection was observed in some cases. The long-term duration of protection observed here defined a third temporal phase in the immune response to IHNV DNA vaccination, characterized by reduced but significant levels of protection, and decline or absence of detectable NAb titers. Examination of multiple tissues showed an absence of detectable long-term histopathological damage due to DNA vaccination.

  17. Protective effects of a Modified Vaccinia Ankara-based vaccine candidate against Crimean-Congo Haemorrhagic Fever virus require both cellular and humoral responses.

    PubMed

    Dowall, Stuart D; Graham, Victoria A; Rayner, Emma; Hunter, Laura; Watson, Robert; Taylor, Irene; Rule, Antony; Carroll, Miles W; Hewson, Roger

    2016-01-01

    Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. There is no approved vaccine currently available against CCHF. The most promising candidate, which has previously been shown to confer protection in the small animal model, is a modified Vaccinia Ankara virus vector expressing the CCHF viral glycoprotein (MVA-GP). It has been shown that MVA-GP induces both humoral and cellular immunogenicity. In the present study, sera and T-lymphocytes were passively and adoptively transferred into recipient mice prior to challenge with CCHF virus. Results demonstrated that mediators from both arms of the immune system were required to demonstrate protective effects against lethal challenge.

  18. Protective effects of a Modified Vaccinia Ankara-based vaccine candidate against Crimean-Congo Haemorrhagic Fever virus require both cellular and humoral responses

    PubMed Central

    Dowall, Stuart D.; Graham, Victoria A.; Rayner, Emma; Hunter, Laura; Watson, Robert; Taylor, Irene; Rule, Antony; Carroll, Miles W.; Hewson, Roger

    2016-01-01

    Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. There is no approved vaccine currently available against CCHF. The most promising candidate, which has previously been shown to confer protection in the small animal model, is a modified Vaccinia Ankara virus vector expressing the CCHF viral glycoprotein (MVA-GP). It has been shown that MVA-GP induces both humoral and cellular immunogenicity. In the present study, sera and T-lymphocytes were passively and adoptively transferred into recipient mice prior to challenge with CCHF virus. Results demonstrated that mediators from both arms of the immune system were required to demonstrate protective effects against lethal challenge. PMID:27272940

  19. Measurement of DNA damage after exposure to electromagnetic radiation in the cellular phone communication frequency band (835.62 and 847.74 MHz).

    PubMed

    Malyapa, R S; Ahern, E W; Straube, W L; Moros, E G; Pickard, W F; Roti Roti, J L

    1997-12-01

    Mouse C3H 10T1/2 fibroblasts and human glioblastoma U87MG cells were exposed to cellular phone communication frequency radiations to investigate whether such exposure produces DNA damage in in vitro cultures. Two types of frequency modulations were studied: frequency-modulated continuous-wave (FMCW), with a carrier frequency of 835.62 MHz, and code-division multiple-access (CDMA) centered on 847.74 MHz. Exponentially growing (U87MG and C3H 10T1/2 cells) and plateau-phase (C3H 10T1/2 cells) cultures were exposed to either FMCW or CDMA radiation for varying periods up to 24 h in specially designed radial transmission lines (RTLs) that provided relatively uniform exposure with a specific absorption rate (SAR) of 0.6 W/kg. Temperatures in the RTLs were monitored continuously and maintained at 37 +/- 0.3 degrees C. Sham exposure of cultures in an RTL (negative control) and 137Cs gamma-irradiated samples (positive control) were included with every experiment. The alkaline comet assay as described by Olive et al. (Exp. Cell Res. 198, 259-269, 1992) was used to measure DNA damage. No significant differences were observed between the test group exposed to FMCW or CDMA radiation and the sham-treated negative controls. Our results indicate that exposure of cultured mammalian cells to cellular phone communication frequencies under these conditions at an SAR of 0.6 W/kg does not cause DNA damage as measured by the alkaline comet assay. PMID:9399708

  20. Antioxidant activity of Coriandrum sativum and protection against DNA damage and cancer cell migration

    PubMed Central

    2013-01-01

    Background Coriandrum sativum is a popular culinary and medicinal herb of the Apiaceae family. Health promoting properties of this herb have been reported in pharmacognostical, phytochemical and pharmacological studies. However, studies on C. sativum have always focused on the aerial parts of the herb and scientific investigation on the root is limited. The aim of this research was to investigate the antioxidant and anticancer activities of C. sativum root, leaf and stem, including its effect on cancer cell migration, and its protection against DNA damage, with special focus on the roots. Methods Powdered roots, leaves and stems of C. sativum were extracted through sequential extraction using hexane, dichloromethane, ethyl acetate, methanol and water. Total phenolic content, FRAP and DPPH radical scavenging activities were measured. Anti-proliferative activitiy on the breast cancer cell line, MCF-7, was assayed using the MTT assay. Activities of the antioxidant enzymes, catalase, superoxide dismutase, glutathione peroxidase, and of the caspases-3, -8 and -9 were assayed on treatment with the extract. Cell cycle progression was analysed using flow cytometry. The scratch motility assay was used to assess inhibition of MCF-7 cell migration. DNA damage in 3 T3-L1 fibroblasts was evaluated by the comet assay. The components in the extract were identified by HPLC and GC-MS. Results The ethyl acetate extract of C. sativum roots showed the highest antiproliferative activity on MCF-7 cells (IC50 = 200.0 ± 2.6 μg/mL) and had the highest phenolic content, FRAP and DPPH scavenging activities among the extracts. C. sativum root inhibited DNA damage and prevented MCF-7 cell migration induced by H2O2, suggesting its potential in cancer prevention and inhibition of metastasis. The extract exhibited anticancer activity in MCF-7 cells by affecting antioxidant enzymes possibly leading to H2O2 accumulation, cell cycle arrest at the G2/M phase and apoptotic cell death by

  1. The Telomere Binding Protein Cdc13 and the Single-Stranded DNA Binding Protein RPA Protect Telomeric DNA from Resection by Exonucleases.

    PubMed

    Greetham, Matthew; Skordalakes, Emmanuel; Lydall, David; Connolly, Bernard A

    2015-09-25

    The telomere is present at the ends of all eukaryotic chromosomes and usually consists of repetitive TG-rich DNA that terminates in a single-stranded 3' TG extension and a 5' CA-rich recessed strand. A biochemical assay that allows the in vitro observation of exonuclease-catalyzed degradation (resection) of telomeres has been developed. The approach uses an oligodeoxynucleotide that folds to a stem-loop with a TG-rich double-stranded region and a 3' single-stranded extension, typical of telomeres. Cdc13, the major component of the telomere-specific CST complex, strongly protects the recessed strand from the 5'→3' exonuclease activity of the model exonuclease from bacteriophage λ. The isolated DNA binding domain of Cdc13 is less effective at shielding telomeres. Protection is specific, not being observed in control DNA lacking the specific TG-rich telomere sequence. RPA, the eukaryotic single-stranded DNA binding protein, also inhibits telomere resection. However, this protein is non-specific, equally hindering the degradation of non-telomere controls.

  2. S100A1 DNA-based Inotropic Therapy Protects Against Proarrhythmogenic Ryanodine Receptor 2 Dysfunction

    PubMed Central

    Ritterhoff, Julia; Völkers, Mirko; Seitz, Andreas; Spaich, Kristin; Gao, Erhe; Peppel, Karsten; Pleger, Sven T; Zimmermann, Wolfram H; Friedrich, Oliver; Fink, Rainer H A; Koch, Walter J; Katus, Hugo A; Most, Patrick

    2015-01-01

    Restoring expression levels of the EF-hand calcium (Ca2+) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca2+ handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca2+ resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca2+ leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca2+- and β-adrenergic receptor-triggered proarrhythmogenic SR Ca2+ leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca2+ leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca2+ leak in HF, combining antiarrhythmic potency with chronic inotropic actions. PMID:26005840

  3. A dominant-negative form of the major human abasic endonuclease enhances cellular sensitivity to laboratory and clinical DNA-damaging agents.

    PubMed

    McNeill, Daniel R; Wilson, David M

    2007-01-01

    Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the primary enzyme in mammals for the repair of abasic sites in DNA, as well as a variety of 3' damages that arise upon oxidation or as products of enzymatic processing. If left unrepaired, APE1 substrates can promote mutagenic and cytotoxic outcomes. We describe herein a dominant-negative form of APE1 that lacks detectable nuclease activity and binds substrate DNA with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two amino acid substitutions at active site residues Glu(96) (changed to Gln) and Asp(210) (changed to Asn). In vitro biochemical assays reveal that ED impedes wild-type APE1 AP site incision function, presumably by binding AP-DNA and blocking normal lesion processing. Moreover, tetracycline-regulated (tet-on) expression of ED in Chinese hamster ovary cells enhances the cytotoxic effects of the laboratory DNA-damaging agents, methyl methanesulfonate (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). This MMS-induced, ED-dependent cell killing coincides with a hyperaccumulation of AP sites, implying that excessive DNA damage is the cause of cell death. Because an objective of the study was to identify a protein reagent that could be used in targeted gene therapy protocols, the effects of ED on cellular sensitivity to a number of chemotherapeutic compounds was tested. We show herein that ED expression sensitizes Chinese hamster ovary cells to the killing effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as carmustine) and the chain terminating nucleoside analogue dideoxycytidine (also known as zalcitabine), but not to the radiomimetic bleomycin, the nucleoside analogue beta-D-arabinofuranosylcytosine (also known as cytarabine), the topoisomerase inhibitors camptothecin and etoposide, or the cross-linking agents mitomycin C and cisplatin. Transient expression of ED in the human cancer cell line NCI-H1299 enhanced cellular

  4. DNA-Free Recombinant SV40 Capsids Protect Mice from Acute Renal Failure by Inducing Stress Response, Survival Pathway and Apoptotic Arrest

    PubMed Central

    Abd-El-Latif, Mahmoud; Pizov, Galina; Eden, Arieh; Haviv, Yosef S.; Oppenheim, Ariella

    2008-01-01

    Viruses induce signaling and host defense during infection. Employing these natural trigger mechanisms to combat organ or tissue failure is hampered by harmful effects of most viruses. Here we demonstrate that SV40 empty capsids (Virus Like Particles-VLPs), with no DNA, induce host Hsp/c70 and Akt-1 survival pathways, key players in cellular survival mechanisms. We postulated that this signaling might protect against organ damage in vivo. Acute kidney injury (AKI) was chosen as target. AKI is critical, prevalent disorder in humans, caused by nephrotoxic agents, sepsis or ischemia, via apoptosis/necrosis of renal tubular cells, with high morbidity and mortality. Systemic administration of VLPs activated Akt-1 and upregulated Hsp/c70 in vivo. Experiments in mercury-induced AKI mouse model demonstrated that apoptosis, oxidative stress and toxic renal failure were significantly attenuated by pretreatment with capsids prior to the mercury insult. Survival rate increased from 12% to >60%, with wide dose response. This study demonstrates that SV40 VLPs, devoid of DNA, may potentially be used as prophylactic agent for AKI. We anticipate that these finding may be projected to a wide range of organ failure, using empty capsids of SV40 as well as other viruses. PMID:18714386

  5. Cell-permeable intrinsic cellular inhibitors of apoptosis protect and rescue intestinal epithelial cells from radiation-induced cell death.

    PubMed

    Matsuzaki-Horibuchi, Shiori; Yasuda, Takeshi; Sakaguchi, Nagako; Yamaguchi, Yoshihiro; Akashi, Makoto

    2015-01-01

    One of the important mechanisms for gastrointestinal (GI) injury following high-dose radiation exposure is apoptosis of epithelial cells. X-linked inhibitor of apoptosis (XIAP) and cellular IAP2 (cIAP2) are intrinsic cellular inhibitors of apoptosis. In order to study the effects of exogenously added IAPs on apoptosis in intestinal epithelial cells, we constructed bacterial expression plasmids containing genes of XIAP (full-length, BIR2 domain and BIR3-RING domain with and without mutations of auto-ubiquitylation sites) and cIAP2 proteins fused to a protein-transduction domain (PTD) derived from HIV-1 Tat protein (TAT) and purified these cell-permeable recombinant proteins. When the TAT-conjugated IAPs were added to rat intestinal epithelial cells IEC6, these proteins were effectively delivered into the cells and inhibited apoptosis, even when added after irradiation. Our results suggest that PTD-mediated delivery of IAPs may have clinical potential, not only for radioprotection but also for rescuing the GI system from radiation injuries.

  6. Possible prebiotic significance of polyamines in the condensation, protection, encapsulation, and biological properties of DNA

    NASA Technical Reports Server (NTRS)

    Baeza, Isabel; Ibanez, Miguel; Wong, Carlos; Chavez, Pedro; Gariglio, Patricio; Oro, J.

    1992-01-01

    While DNA which has undergone ionic condensation with Co(3+)(NH3)6 is resistant to the action of the endonuclase DNAse I, in much the same way as DNA condensed with spermidine, it was significantly less active in transcription with the E. coli RNA polymerase than DNA-spermidine condensed forms. Although both compacted forms of DNA were more efficiently encapsulated into neutral liposomes, negatively charged liposomes were seldom formed in the presence of the present, positive ion-condensed DNA; spermidine is accordingly proposed as a plausible prebiotic DNA-condensing agent. Attention is given to the relevance of the polyimide-nucleic acids complexes in the evolution of life.

  7. A tomato chloroplast-targeted DnaJ protein protects Rubisco activity under heat stress.

    PubMed

    Wang, Guodong; Kong, Fanying; Zhang, Song; Meng, Xia; Wang, Yong; Meng, Qingwei

    2015-06-01

    Photosynthesis is one of the biological processes most sensitive to heat stress in plants. Carbon assimilation, which depends on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is one of the major sites sensitive to heat stress in photosynthesis. In this study, the roles of a tomato (Solanum lycopersicum) chloroplast-targeted DnaJ protein (SlCDJ2) in resisting heat using sense and antisense transgenic tomatoes were examined. SlCDJ2 was found to be uniformly distributed in the thylakoids and stroma of the chloroplasts. Under heat stress, sense plants exhibited higher chlorophyll contents and fresh weights, and lower accumulation of reactive oxygen species (ROS) and membrane damage. Moreover, Rubisco activity, Rubisco large subunit (RbcL) content, and CO2 assimilation capacity were all higher in sense plants and lower in antisense plants compared with wild-type plants. Thus, SlCDJ2 contributes to maintenance of CO2 assimilation capacity mainly by protecting Rubisco activity under heat stress. SlCDJ2 probably achieves this by keeping the levels of proteolytic enzymes low, which prevents accelerated degradation of Rubisco under heat stress. Furthermore, a chloroplast heat-shock protein 70 was identified as a binding partner of SlCDJ2 in yeast two-hybrid assays. Taken together, these findings establish a role for SlCDJ2 in maintaining Rubisco activity in plants under heat stress. PMID:25801077

  8. Protective effect of black raspberry seed containing anthocyanins against oxidative damage to DNA, protein, and lipid.

    PubMed

    Choi, Mi-Hee; Shim, Soon-Mi; Kim, Gun-Hee

    2016-02-01

    This study aimed to determine bioactive components and radical scavenging capacity of black raspberry seed extracts as byproducts obtaining during the juice (FSE) and wine (WSE) making process. Cyanidin-3-O-rutinoside was identified as a major anthocyanin and the total anthocyanin contents of fresh and wine seed were 78.24 and 41.61 mg/100 g of dry weight, respectively. The total phenolic and flavonoid contents of FSE and WSE were 2.31 g gallic acid equivalent (GAE) and 360.95 mg catechin equivalent (CE), and 2.44 g GAE and 379.54 mg CE per 100 g dry weight, respectively. The oxygen radical absorbance capacity (ORAC) values were 1041.9 μM TE/g for FSE and 1060.4 μM TE/g for WSE. Pretreatment of the FSE and WSE inhibited the generation of intracellular reactive oxygen species (ROS), DNA and protein damage induced by hydroxyl radicals, and Fe(3+)/ascorbic acid-induced lipid peroxidation in a dose dependent manner. WSE more effectively protected from oxidative damage than FSE. Results from the current study suggest that black raspberry seeds as byproducts from juice and wine processing could be potential sources for natural antioxidants. PMID:27162401

  9. Antioxidant and DNA damage protecting potentials of polysaccharide extracted from Phellinus baumii using a delignification method.

    PubMed

    Jin, Qun-Li; Zhang, Zuo-Fa; Lv, Guo-Ying; Cai, Wei-Ming; Cheng, Jun-Wen; Wang, Jian-Gong; Fan, Lei-Fa

    2016-11-01

    A delignification method was employed to extract the polysaccharide from the fruiting body of Phellinus baumii. The three parameters, processing temperature, ratio of water to raw material and amount of acetic acid every time were optimized using the Box-Behnken design. As a result, the optimal extraction conditions were: processing temperature 70.3°C, ratio of water to raw material of 34.7mL/g and amount of acetic acid of 0.32mL every time. Under these conditions, the highest yield of polysaccharide (10.28%) was obtained. The main fraction (PPB-2) purified from PPB was composed of fucose, arabinose, galactose, glucose, xylose and mannose, while glucose was the predominant monosaccharide. PPB-2 exhibited noticeable antioxidant activity and strong protection against oxidative DNA damage. These findings implied that acid-chlorite delignification was a superior method to extract the polysaccharide from P. baumii and PPB-2 may be useful for cancer chemoprevention. PMID:27516306

  10. A tomato chloroplast-targeted DnaJ protein protects Rubisco activity under heat stress.

    PubMed

    Wang, Guodong; Kong, Fanying; Zhang, Song; Meng, Xia; Wang, Yong; Meng, Qingwei

    2015-06-01

    Photosynthesis is one of the biological processes most sensitive to heat stress in plants. Carbon assimilation, which depends on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is one of the major sites sensitive to heat stress in photosynthesis. In this study, the roles of a tomato (Solanum lycopersicum) chloroplast-targeted DnaJ protein (SlCDJ2) in resisting heat using sense and antisense transgenic tomatoes were examined. SlCDJ2 was found to be uniformly distributed in the thylakoids and stroma of the chloroplasts. Under heat stress, sense plants exhibited higher chlorophyll contents and fresh weights, and lower accumulation of reactive oxygen species (ROS) and membrane damage. Moreover, Rubisco activity, Rubisco large subunit (RbcL) content, and CO2 assimilation capacity were all higher in sense plants and lower in antisense plants compared with wild-type plants. Thus, SlCDJ2 contributes to maintenance of CO2 assimilation capacity mainly by protecting Rubisco activity under heat stress. SlCDJ2 probably achieves this by keeping the levels of proteolytic enzymes low, which prevents accelerated degradation of Rubisco under heat stress. Furthermore, a chloroplast heat-shock protein 70 was identified as a binding partner of SlCDJ2 in yeast two-hybrid assays. Taken together, these findings establish a role for SlCDJ