Genistein protects hematopoietic stem cells against G-CSF-induced DNA damage.

PubMed

Souza, Liliana R; Silva, Erica; Calloway, Elissa; Kucuk, Omer; Rossi, Michael; McLemore, Morgan L

2014-05-01

Granulocyte colony-stimulating factor (G-CSF) has been used to treat neutropenia in various clinical settings. Although clearly beneficial, there are concerns that the chronic use of G-CSF in certain conditions increases the risk of myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). The most striking example is in severe congenital neutropenia (SCN). Patients with SCN develop MDS/AML at a high rate that is directly correlated to the cumulative lifetime dosage of G-CSF. Myelodysplastic syndrome and AML that arise in these settings are commonly associated with chromosomal deletions. We have demonstrated in this study that chronic G-CSF treatment in mice results in expansion of the hematopoietic stem cell (HSC) population. In addition, primitive hematopoietic progenitors from G-CSF-treated mice show evidence of DNA damage as demonstrated by an increase in double-strand breaks and recurrent chromosomal deletions. Concurrent treatment with genistein, a natural soy isoflavone, limits DNA damage in this population. The protective effect of genistein seems to be related to its preferential inhibition of G-CSF-induced proliferation of HSCs. Importantly, genistein does not impair G-CSF-induced proliferation of committed hematopoietic progenitors, nor diminishes neutrophil production. The protective effect of genistein was accomplished with plasma levels that are attainable through dietary supplementation.

The Protective Effect of Whole Honey and Phenolic Extract on Oxidative DNA Damage in Mice Lymphocytes Using Comet Assay.

PubMed

Cheng, Ni; Wang, Yuan; Cao, Wei

2017-12-01

In this study, the antioxidant activity and the protective effect against hydrogen peroxide-induced DNA damage were assessed for five honeys of different botanical origin. Seven phenolic acids were detected in the honey samples. Ferulic acid was the most abundant phenolic acid detected in longan honey, jujube honey and buckwheat honey. Ellagic acid, p-hydroxybenzoic acid and protocatechuic acid were the main phenolic acids detected in vitex honey. Of all honey samples tested, the highest total phenolic content and antioxidant activity were found in buckwheat honey, whereas the lowest total phenolic content and antioxidant activity were found in locust honey. Treatment with hydrogen peroxide induced a 62% increase in tail DNA in mice lymphocytes, and all studied honeys significantly inhibited this effect (P < 0.05). The buckwheat honey with higher antioxidant capability also exhibited super protective effect than others. Phenolic extracts of honey displayed greater protective effects than whole honey in comet assay. The hydrogen peroxide-generated increase in 8-hydroxy-2-deoxyguanosine (8-OHdG) was effectively inhibited by the honeys studied (P < 0.05). Moreover, a dose-effect relationship between honey concentration and its protective effect was clearly observed in this study. It can be deduced that phenolic acids of honey can penetrate into lymphocytes and protect DNA from oxidative damage by scavenging hydrogen peroxide and/or chelating ferrous ions.

Muscarinic Receptor Activation Protects Cells from Apoptotic Effects of DNA Damage, Oxidative Stress, and Mitochondrial Inhibition*

PubMed Central

De Sarno, Patrizia; Shestopal, Svetlana A.; King, Taj D.; Zmijewska, Anna; Song, Ling; Jope, Richard S.

2006-01-01

The impact of muscarinic receptor stimulation was examined on apoptotic signaling induced by DNA damage, oxidative stress, and mitochondrial impairment. Exposure of human neuroblastoma SH-SY5Y cells to the DNA-damaging agent camptothecin increased p53 levels, activated caspase-3, and caused cell death. Pretreatment with oxotremorine-M, a selective agonist of muscarinic receptors that are expressed endogenously in these cells, did not affect the accumulation of p53 but greatly attenuated caspase-3 activation and protected from cell death to nearly the same extent as treatment with a general caspase inhibitor. Treatment with 50–200 μm H2O2 caused the activation of caspase-3 beginning after 2–3 h, followed by eventual cell death. Oxotremorine-M pretreatment protected cells from H2O2-inducedcaspase-3 activation and death, and this was equivalent to protection afforded by a caspase inhibitor. Muscarinic receptor stimulation also protected cells from caspase-3 activation induced by exposure to rotenone, a mitochondrial complex 1 inhibitor, but no protection was evident from staurosporine-induced caspase-3 activation. The mechanism of protection afforded by muscarinic receptor activation from camptothecin-induced apoptotic signaling involved blockade of mitochondrial cytochrome c release associated with a bolstering of mitochondrial bcl-2 levels and blockade of the translocation of Bax to mitochondria. Likely the most proximal of these events to muscarinic receptor activation, mitochondrial Bax accumulation, also was attenuated by oxotremorine-M treatment after treatment with H2O2 or rotenone. These results demonstrate that stimulation of muscarinic receptors provides substantial protection from DNA damage, oxidative stress, and mitochondrial impairment, insults that may be encountered by neurons in development, aging, or neurodegenerative diseases. These findings suggest that neurotransmitter-induced signaling bolsters survival mechanisms, and inadequate

DNA-protective activities of hyperforin and aristoforin.

PubMed

Ševčovičová, A; Šemeláková, M; Plšíková, J; Loderer, D; Imreová, P; Gálová, E; Kožurková, M; Miadoková, E; Fedoročko, P

2015-04-01

The aim of this study was to explain the molecular mechanisms of action of hyperforin, a phluoroglucinol derivative found in Hypericum perforatum L. and its more stable derivative aristoforin. DNA-topology assay revealed partial DNA-protective activities of hyperforin and aristoforin against Fe(2+)-induced DNA breaks. In order to assess molecular mechanisms underlying DNA-protective activity, the potential antioxidant activity of hyperforin and aristoforin was investigated using DPPH and OH scavenging assays, reducing power assay and Fe(2+)-chelating assay. We also studied interaction of hyperforin and aristoforin with DNA using established protocols for fluorescence titration. The ability of the studied compounds to relax topoisomerase I with electrophoretic techniques was investigated. The reduction in the fluorescence of hyperforin indicated an interaction between hyperforin and DNA with a binding constant of 0.2×10(8)M(-1). We suggest that a mechanism of hyperforin/aristoforin DNA-protective abilities is based on free radicals (mainly OH) scavenging activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Assessment of antioxidative, chelating, and DNA-protective effects of selected essential oil components (eugenol, carvacrol, thymol, borneol, eucalyptol) of plants and intact Rosmarinus officinalis oil.

PubMed

Horvathova, Eva; Navarova, Jana; Galova, Eliska; Sevcovicova, Andrea; Chodakova, Lenka; Snahnicanova, Zuzana; Melusova, Martina; Kozics, Katarina; Slamenova, Darina

2014-07-16

Selected components of plant essential oils and intact Rosmarinus officinalis oil (RO) were investigated for their antioxidant, iron-chelating, and DNA-protective effects. Antioxidant activities were assessed using four different techniques. DNA-protective effects on human hepatoma HepG2 cells and plasmid DNA were evaluated with the help of the comet assay and the DNA topology test, respectively. It was observed that whereas eugenol, carvacrol, and thymol showed high antioxidative effectiveness in all assays used, RO manifested only antiradical effect and borneol and eucalyptol did not express antioxidant activity at all. DNA-protective ability against hydrogen peroxide (H2O2)-induced DNA lesions was manifested by two antioxidants (carvacrol and thymol) and two compounds that do not show antioxidant effects (RO and borneol). Borneol was able to preserve not only DNA of HepG2 cells but also plasmid DNA against Fe(2+)-induced damage. This paper evaluates the results in the light of experiences of other scientists.

Immune protection duration and efficacy stability of DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2 against coccidiosis.

PubMed

Song, Xiaokai; Zhao, Xiaofang; Xu, Lixin; Yan, Ruofeng; Li, Xiangrui

2017-04-01

In our previous study, an effective DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2 was constructed. In the present study, the immunization dose of the DNA vaccine pVAX1.0-TA4-IL-2 was further optimized. With the optimized dose, the dynamics of antibodies induced by the DNA vaccine was determined using indirect ELISA. To evaluate the immune protection duration of the DNA vaccine, two-week-old chickens were intramuscularly immunized twice and the induced efficacy was evaluated by challenging with E. tenella at 5, 9, 13, 17 and 21weeks post the last immunization (PLI) separately. To evaluate the efficacy stability of the DNA vaccine, two-week-old chickens were immunized with 3 batches of the DNA vaccine, and the induced efficacy was evaluated by challenging with E. tenella. The results showed that the optimal dose was 25μg. The induced antibody level persisted until 10weeks PPI. For the challenge time of 5 and 9weeks PLI, the immunization resulted in ACIs of 182.28 and 162.23 beyond 160, showing effective protection. However, for the challenge time of 13, 17 and 21weeks PLI, the immunization resulted in ACIs below 160 which means poor protection. Therefore, the immune protection duration of the DNA vaccination was at least 9weeks PLI. DNA immunization with three batches DNA vaccine resulted in ACIs of 187.52, 191.57 and 185.22, which demonstrated that efficacies of the three batches DNA vaccine were effective and stable. Overall, our results indicate that DNA vaccine pVAX1.0-TA4-IL-2 has the potential to be developed as effective vaccine against coccidiosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Protective effects of buckwheat honey on DNA damage induced by hydroxyl radicals.

PubMed

Zhou, Juan; Li, Peng; Cheng, Ni; Gao, Hui; Wang, Bini; Wei, Yahui; Cao, Wei

2012-08-01

To understand the antioxidant properties of buckwheat honeys, we investigated their antioxidant effects on hydroxyl radical-induced DNA breaks in the non-site-specific and site-specific systems, the physicochemical properties, antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical scavenging activity, chelating, and reducing power assays), total phenolic content and individual phenolic acids were also determined. Total phenolic content of buckwheat honeys ranged from 774 to 1694 mg PA/kg, and p-hydroxybenzoic and p-coumaric acids proved to be the main components in buckwheat honeys. All the buckwheat honey samples possess stronger capability to protect DNA in the non-site-specific systems than in the site-specific systems from being damaged by hydroxyl radicals. In the non-site-specific and site-specific system, buckwheat honeys samples prevented ()OH-induced DNA breaks by 21-78% and 5-31% over control value, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

Protective effect of gangliosides on DNA in human spermatozoa exposed to cryopreservation.

PubMed

Gavella, Mirjana; Lipovac, Vaskresenija; Garaj-Vrhovac, Verica; Gajski, Goran

2012-01-01

Gangliosides, the sialic acid-containing glycosphyngolipids, are amphiphilic compounds which in micellar form affect the properties and functions of a cellular membrane. The aim of this study was to test whether exogenous gangliosides supplied to cryopreservation media before freezing could protect sperm cells from cryopreservation-induced DNA damage assessed by Comet assay. Additionally, to investigate whether gangliosides were also able to reduce membrane integrity damage, malonaldialdehyde as a measure of lipid peroxidation and sperm-specific lactate dehydrogenase-C4 activity as an enzyme marker of sperm membrane leakage were determined. The monosialogangliosides (GM1) and trisialogangliosides (GT1b) were examined at a concentration of 100 μM, which was above their respective critical micellar concentrations. Exogenous gangliosides were not found to protect sperm membrane from lipid peroxidation. However, a freezing-/thawing-induced increase in Comet parameters was equally significantly prevented by the presence of both GM1 and GT1b (P < .05), indicating that the ceramide moiety, rather than the polar groups, is involved in the protective ability of gangliosides. The observed phenomena suggest that ganglioside micelles could modulate hydrophobic properties of the sperm membrane responsible for better tolerance to DNA fragmentation, thus protecting DNA integrity from cryopreservation-induced damage.

Protective effects of folic acid on DNA damage and DNA methylation levels induced by N-methyl- N'-nitro- N-nitrosoguanidine in Kazakh esophageal epithelial cells.

PubMed

Chen, Y; Feng, H; Chen, D; Abuduwaili, K; Li, X; Zhang, H

2018-01-01

The protective effects of folic acid on DNA damage and DNA methylation induced by N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) in Kazakh esophageal epithelial cells were investigated using a 3 × 3 factorial design trial. The cells were cultured in vitro and exposed to media containing different concentrations of folic acid and MNNG, after which growth indices were detected. DNA damage levels were measured using comet assays, and genome-wide DNA methylation levels (MLs) were measured using high-performance liquid chromatography. The DNA methylation of methylenetetrahydrofolate reductase (MTHFR) and folate receptor- α (FR α) genes was detected by bisulfite sequencing polymerase chain reaction (PCR). The results showed significant increases in tail DNA concentration, tail length, and Olive tail moment ( p < 0.01); a significant reduction of genome-wide DNA MLs ( p < 0.01); and an increase in the methylation frequencies of MTHFR and FR α genes. In particular, significant differences were observed in the promoter regions of both genes ( p < 0.01). Our study indicated that a reduction in folic acid concentration promotes DNA damage and DNA methylation in Kazakh esophageal epithelial cells upon MNNG exposure. Thus, sufficient folic acid levels could play a protective role against the damage induced by this compound.

Gene Gun Bombardment with DNA-Coated Golden Particles Enhanced the Protective Effect of a DNA Vaccine Based on Thioredoxin Glutathione Reductase of Schistosoma japonicum

PubMed Central

Cao, Yan; Zhao, Bin; Han, Yanhui; Zhang, Juan; Li, Xuezhen; Qiu, Chunhui; Wu, Xiujuan; Hong, Yang; Ai, Dezhou; Lin, Jiaojiao; Fu, Zhiqiang

2013-01-01

Schistosomiasis, caused by infection with Schistosoma species, remains an important parasitic zoonosis. Thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR) plays an important role in the development of the parasite and for its survival. Here we present a recombinant plasmid DNA vaccine, pVAX1/SjTGR, to estimate its protection against S. japonicum in BALB/c mice. The DNA vaccine administrated by particle bombardment induced higher protection than by intramuscular injection. All animals vaccinated with pVAX1/SjTGR developed significant specific anti-SjTGR antibodies than control groups. Moreover, animals immunized by gene gun exhibited a splenocyte proliferative response, with an increase in IFN-γ and IL-4. The recombinant plasmid administrated by gene gun achieved a medium protective efficacy of 27.83–38.83% (P < 0.01) of worm reduction and 40.38–44.51% (P < 0.01) of liver egg count reduction. It suggests that different modes of administering a DNA vaccine can influence the protective efficacy induced by the vaccine. Interestingly, from the enzymatic activity results, we found that worms obtained from pVAX1/SjTGR-vaccinated animals expressed lower enzymatic activity than the control group and the antibodies weakened the enzymatic activity of SjTGR in vitro, too. It implies that the high-level antibodies may contribute to the protective effects. PMID:23509820

[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.

In vitro protective effects of an aqueous extract of Clitoria ternatea L. flower against hydrogen peroxide-induced cytotoxicity and UV-induced mtDNA damage in human keratinocytes.

PubMed

Zakaria, N N A; Okello, E J; Howes, M-J; Birch-Machin, M A; Bowman, A

2018-06-01

The traditional practice of eating the flowers of Clitoria ternatea L. or drinking their infusion as herbal tea in some of the Asian countries is believed to promote a younger skin complexion and defend against skin aging. This study was conducted to investigate the protective effect of C. ternatea flower water extract (CTW) against hydrogen peroxide-induced cytotoxicity and ultraviolet (UV)-induced mitochondrial DNA (mtDNA) damage in human keratinocytes. The protective effect against hydrogen peroxide-induced cytotoxicity was determined by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and mtDNA damage induced by UV was determined by polymerase chain reaction. Preincubation of HaCaT with 100, 250, and 500 μg/ml CTW reduced cytotoxicity effects of H 2 O 2 compared with control (H 2 O 2 alone). CTW also significantly reduced mtDNA damage in UV-exposed HaCaT (p < .05). CTW was chemically-characterized using high resolution liquid chromatography-mass spectrometry. The main compounds detected were assigned as anthocyanins derived from delphinidin, including polyacylated ternatins, and flavonol glycosides derived from quercetin and kaempferol. These results demonstrated the protective effects of C. ternatea flower extracts that contain polyacylated anthocyanins and flavonol glycosides as major constituents, against H 2 O 2 and UV-induced oxidative stress on skin cells, and may provide some explanation for the putative traditional and cosmetic uses of C. ternatea flower against skin aging. Copyright © 2018 John Wiley & Sons, Ltd.

Privacy rules for DNA databanks. Protecting coded 'future diaries'.

PubMed

Annas, G J

1993-11-17

In privacy terms, genetic information is like medical information. But the information contained in the DNA molecule itself is more sensitive because it contains an individual's probabilistic "future diary," is written in a code that has only partially been broken, and contains information about an individual's parents, siblings, and children. Current rules for protecting the privacy of medical information cannot protect either genetic information or identifiable DNA samples stored in DNA databanks. A review of the legal and public policy rationales for protecting genetic privacy suggests that specific enforceable privacy rules for DNA databanks are needed. Four preliminary rules are proposed to govern the creation of DNA databanks, the collection of DNA samples for storage, limits on the use of information derived from the samples, and continuing obligations to those whose DNA samples are in the databanks.

Hippophae leaf extract concentration regulates antioxidant and prooxidant effects on DNA.

PubMed

Saini, Manu; Tiwari, Sandhya; Prasad, Jagdish; Singh, Surender; Kumar, M S Yogendra; Bala, Madhu

2010-03-01

Extracts from Hippophae leaves constitute some commonly consumed beverages such as tea and wine. We had developed an extract of Hippophae leaves (SBL-1), which was rich in quercetin, had antimutagenic effects, radioprotective effects, and countered radiation-induced gene conversion in Saccharomyces cerevisiae. This study was designed to investigate the action of SBL-1 on guanine cytosine (GC)-rich nascent and mouse genomic DNA in vitro. The human and mouse liver DNA have about 43% GC content. Our results showed that at small concentration SBL-1 protected nascent as well as genomic DNA, while at large concentration SBL-1 damaged both types of DNA. The concentration of SBL-1 that protected DNA also demonstrated higher free radical scavenging activity. The reducing power of SBL-1 was greater than its free radical scavenging activity. The greater reducing power may have reduced the trace metals present in the SBL-1, leading to generation of hydroxyl radicals via Fenton reaction. The increased proportion of unscavenged hydroxyl radicals with increase in SBL-1 concentration may have been responsible for DNA damage or prooxidant effect of SBL-1 in vitro. This study suggests that the dietary supplements prepared from Hippophae should have low metal content.

Electroporation of a multivalent DNA vaccine cocktail elicits a protective immune response against anthrax and plague.

PubMed

Albrecht, Mark T; Livingston, Brian D; Pesce, John T; Bell, Matt G; Hannaman, Drew; Keane-Myers, Andrea M

2012-07-06

Electroporation of DNA vaccines represents a platform technology well positioned for the development of multivalent biodefense vaccines. To evaluate this hypothesis, three vaccine constructs were produced using codon-optimized genes encoding Bacillus anthracis Protective Antigen (PA), and the Yersinia pestis genes LcrV and F1, cloned into pVAX1. A/J mice were immunized on a prime-boost schedule with these constructs using the electroporation-based TriGrid Delivery System. Immunization with the individual pDNA vaccines elicited higher levels of antigen-specific IgG than when used in combination. DNA vaccine effectiveness was proven, the pVAX-PA titers were toxin neutralizing and fully protective against a lethal B. anthracis spore challenge when administered alone or co-formulated with the plague pDNA vaccines. LcrV and F1 pVAX vaccines against plague were synergistic, resulting in 100% survival, but less protective individually and when co-formulated with pVAX-PA. These DNA vaccine responses were Th1/Th2 balanced with high levels of IFN-γ and IL-4 in splenocyte recall assays, contrary to complimentary protein Alum vaccinations displaying a Th2 bias with increased IL-4 and low levels of IFN-γ. These results demonstrate the feasibility of electroporation to deliver and maintain the overall efficacy of an anthrax-plague DNA vaccine cocktail whose individual components have qualitative immunological differences when combined. Published by Elsevier Ltd.

ZnO Nanoparticles Protect RNA from Degradation Better than DNA.

PubMed

McCall, Jayden; Smith, Joshua J; Marquardt, Kelsey N; Knight, Katelin R; Bane, Hunter; Barber, Alice; DeLong, Robert K

2017-11-08

Gene therapy and RNA delivery require a nanoparticle (NP) to stabilize these nucleic acids when administered in vivo. The presence of degradative hydrolytic enzymes within these environments limits the nucleic acids' pharmacologic activity. This study compared the effects of nanoscale ZnO and MgO in the protection afforded to DNA and RNA from degradation by DNase, serum or tumor homogenate. For double-stranded plasmid DNA degradation by DNase, our results suggest that the presence of MgO NP can protect DNA from DNase digestion at an elevated temperature (65 °C), a biochemical activity not present in ZnO NP-containing samples at any temperature. In this case, intact DNA was remarkably present for MgO NP after ethidium bromide staining and agarose gel electrophoresis where these same stained DNA bands were notably absent for ZnO NP. Anticancer RNA, polyinosinic-polycytidylic acid (poly I:C) is now considered an anti-metastatic RNA targeting agent and as such there is great interest in its delivery by NP. For it to function, the NP must protect it from degradation in serum and the tumor environment. Surprisingly, ZnO NP protected the RNA from degradation in either serum-containing media or melanoma tumor homogenate after gel electrophoretic analysis, whereas the band was much more diminished in the presence of MgO. For both MgO and ZnO NP, buffer-dependent rescue from degradation occurred. These data suggest a fundamental difference in the ability of MgO and ZnO NP to stabilize nucleic acids with implications for DNA and RNA delivery and therapy.

Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved

PubMed Central

Long, Hannah K.; King, Hamish W.; Patient, Roger K.; Odom, Duncan T.; Klose, Robert J.

2016-01-01

DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species. PMID:27084945

Fisetin Protects DNA Against Oxidative Damage and Its Possible Mechanism.

PubMed

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

2016-06-01

The paper tries to assess the protective effect of fisetin against •OH-induced DNA damage, then to investigate the possible mechanism. 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 Cu(2+)-reducing power assays. 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 Cu(2+)-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. 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.

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

Evaluation of Biological and Physical Protection against Nuclease Degradation of Clay-Bound Plasmid DNA

PubMed Central

Demanèche, Sandrine; Jocteur-Monrozier, Lucile; Quiquampoix, Hervé; Simonet, Pascal

2001-01-01

In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium Acinetobacter sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules. PMID:11133458

Protection of CpG islands from DNA methylation is DNA-encoded and evolutionarily conserved.

PubMed

Long, Hannah K; King, Hamish W; Patient, Roger K; Odom, Duncan T; Klose, Robert J

2016-08-19

DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed in vivo and to discover whether mechanisms governing CGI formation are evolutionarily conserved. Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Block Copolymer Micellization as a Protection Strategy for DNA Origami.

PubMed

Agarwal, Nayan P; Matthies, Michael; Gür, Fatih N; Osada, Kensuke; Schmidt, Thorsten L

2017-05-08

DNA nanotechnology enables the synthesis of nanometer-sized objects that can be site-specifically functionalized with a large variety of materials. For these reasons, DNA-based devices such as DNA origami are being considered for applications in molecular biology and nanomedicine. However, many DNA structures need a higher ionic strength than that of common cell culture buffers or bodily fluids to maintain their integrity and can be degraded quickly by nucleases. To overcome these deficiencies, we coated several different DNA origami structures with a cationic poly(ethylene glycol)-polylysine block copolymer, which electrostatically covered the DNA nanostructures to form DNA origami polyplex micelles (DOPMs). This straightforward, cost-effective, and robust route to protect DNA-based structures could therefore enable applications in biology and nanomedicine where unprotected DNA origami would be degraded. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

‘Protected DNA Probes’ capable of strong hybridization without removal of base protecting groups

PubMed Central

Ohkubo, Akihiro; Kasuya, Rintaro; Sakamoto, Kazushi; Miyata, Kenichi; Taguchi, Haruhiko; Nagasawa, Hiroshi; Tsukahara, Toshifumi; Watanobe, Takuma; Maki, Yoshiyuki; Seio, Kohji; Sekine, Mitsuo

2008-01-01

We propose a new strategy called the ‘Protected DNA Probes (PDP) method’ in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac4C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac6az8c7A). It was found that PDP containing ac4C and ac6az8c7A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness. PMID:18272535

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

PubMed

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. Copyright © 2012 Elsevier Inc. All rights reserved.

[Protective Effect of S-isopentenyl-L-cysteine against DNA Damage in Irradiated Mice].

PubMed

Zheng, Qi-sheng; Yu, Guang-yun; He, Xin; Jiang, Ming; Chu, Xiao-fei; Zhao, Shu-yi; Fan, Sai-jun; Liu, Pei-xun

2015-10-01

To evaluate the protective effect of S-isopentenyl-L-cysteine,a new cysteine derivative,on DNA damage induced by radiation by using acute radiation injury animal models. Forty ICR mice were randomly divided into five groups:the control group,1.0Gy gamma irradiation group,1.0Gy gamma irradiation combined with S-isopentenyl-L-cysteine group,7.2Gy gamma irradiation group,and 7.2Gy gamma irradiation combined with S-isopentenyl-L-cysteine group,with 8 mice in each group.The comet assay and bone marrow polychromatic micronucleus experiments were performed to evaluate the double-strand DNA breaks in ICR mice exposed to 1.0 and 7.2Gy gamma-ray, respectively. The tail DNA percentage,tail length,tail moment,and olive tail moment of peripheral blood lymphocytes in 7.2Gy gamma irradiation group were significantly higher than that of the control group (P<0.01).And it was also observed that above experimental indexes of 7.2Gy gamma irradiation combined with S-isopentenyl-L-cysteine group was significantly less than that of 7.2Gy gamma irradiation group (P<0.05). In addition,the micronucleus rate of 1.0Gy gamma irradiation group and 7.2Gy gamma irradiation group were both significantly higher than in the control group (P<0.01). In addition,in mice given S-isopentenyl-L-cysteine before irradiation,the micronucleus rate of ICR mice exposed to 1.0 and 7.2Gy gamma-ray decreased from (39.5000 ± 3.3141)‰ to (28.1667±4.1345)‰ (P=0.033) and from (76.5000 ± 4.6242)‰ to (22.8333 ± 3.6553)‰(P=0.000),respectively. The bone marrow polychromatic micronucleus experiment indicated that the value of polychromatic erythrocyte (PCE)/normochromatic erythrocyte(NCE) of ICR mice exposed to 1.0 and 7.2Gy gamma-ray was less than the control group(P<0.05). Meanwhile,after irradiating by certain dose,the value of PCE/NCE in mice given S-isopentenyl-L-cysteine before irradiation was significantly higher than the corresponding groups (P<0.05). S-isopentenyl-L-cysteine has a good protective

Protective effects of a freeze-dried extract of vegetables and fruits on the hydroxyl radical-mediated oxidative damage of DNA and decrease of erythrocytes deformability.

PubMed

Wang, Hsiao-Ning; Liu, Tsan-Zon; Chen, Ya-Lei; Shiuan, David

2007-01-01

The protective effects of a freeze-dried extracts of vegetables and fruits (BauYuan; BY) on the hydroxyl radical-mediated DNA strand breakages and the structural integrity of human red blood cells (RBCs) were investigated. First, the supercoiled plasmid (pEGFP-C1) DNA was subjected to oxidation damage by an ascorbate-fortified Fenton reaction and the protective effects were analyzed by agarose gel electrophoresis. In the absence of BY extracts, exposure of the high-throughput .OH-generating system (Fe2+ concentration >1.0 microM) caused a complete fragmentation of DNA. Supplementation of BY extract (1 mg/mL) to the plasmid DNA prior to the exposure could prevent it significantly. In contrast, as the plasmid exposed to a low-grade .OH-generating system (Fe2+<0.1 microM), the BY extract (1 mg/mL) provided an almost complete protection. Next, the cell deformabilities were measured to assess the protection effects of various BY extracts on human erythrocytes exposed to the oxidative insults. We found that both the aqueous extract and the organic solvent-derived extracts could strongly protect human RBCs from the reactive oxygen species (ROS)-mediated decrease in the deformability indices. The results implicated that the BY extracts could effectively protect the cell membrane integrity via scavenging ROS which enabling RBCs to maintain a balance of water content and surface area to prevent the drop of cell deformability.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rezvanfar, Mohammad Amin; Rezvanfar, Mohammad Ali; Shahverdi, Ahmad Reza

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 peroxidasemore » (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

Glycoprotein-Specific Antibodies Produced by DNA Vaccination Protect Guinea Pigs from Lethal Argentine and Venezuelan Hemorrhagic Fever

PubMed Central

Golden, Joseph W.; Maes, Piet; Kwilas, Steven A.; Ballantyne, John

2016-01-01

mitigate the severity of disease caused by arenaviruses, particularly species found in South America. Because of variations in potency of the human-derived product, limited availability, and safety concerns, this treatment option has essentially been abandoned. Accordingly, despite this approach being an effective postinfection treatment option, research on novel approaches to produce potent polyclonal antibody-based therapies have been deficient. Here we show that DNA-based vaccine technology can be used to make potently neutralizing antibodies in rabbits that exclusively target the glycoproteins of several human-pathogenic arenaviruses found in South America, including JUNV, MACV, GTOV, and SABV. These antibodies protected guinea pigs from lethal disease when given post-virus challenge. We also generated a purified antibody cocktail with antibodies targeting three arenaviruses and demonstrated protective efficacy against all three targets. Our findings demonstrate that use of the DNA vaccine technology could be used to produce candidate antiarenavirus neutralizing antibody-based products. PMID:26792737

Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines.

PubMed

Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

2016-01-01

Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540, 1080, and 2161 ng/ml B equivalents) concentrations

American ginseng tea protects cellular DNA within 2 h from consumption: results of a pilot study in healthy human volunteers.

PubMed

Szeto, Yim Tong; Sin, Yuk Shan Pauline; Pak, Sok Cheon; Kalle, Wouter

2015-01-01

The acute genoprotective effect of Panax quinquefolius (American ginseng) has been investigated. The experiment was carried out to explore the DNA protective effect after a single dose of American ginseng tea bag infusion. Fourteen subjects (6 males and 8 females) were recruited in this study. Seven of them (3 males and 4 females) were asked to drink a cup of freshly prepared American ginseng infusions. Water was taken by the remaining subjects as the control group. Blood samples of both groups were taken before and 2 h post-ingestion. The blood samples were challenged with ultraviolet B irradiation followed by using comet assay. Completed slides were stained with Giemsa stain and DNA damage was assessed. Results showed a significant decrease in comet score after American ginseng supplementation and no change in the control group. The current study demonstrated a cup of American ginseng infusion could protect cellular DNA from oxidative stress at least within 2 h.

Clostridium perfringens beta toxin DNA prime-protein boost elicits enhanced protective immune response in mice.

PubMed

Solanki, Amit Kumar; Bhatia, Bharati; Kaushik, Himani; Deshmukh, Sachin K; Dixit, Aparna; Garg, Lalit C

2017-07-01

Clostridium perfringens beta toxin (CPB) is the primary pathogenic factor responsible for necrotic enteritis in sheep, cattle and humans. Owing to rapid progression of the disease, vaccination is the only possible recourse to avoid high mortality in animal farms and huge economic losses. The present study reports evaluation of a cpb gene-based DNA vaccine encoding the beta toxin of C. perfringens with homologous as well as heterologous booster strategy. Immunization strategy employing heterologous booster with heat-inactivated rCPB mounted stronger immune response when compared to that generated by homologous booster. Antibody isotyping and cytokine ELISA demonstrated the immune response to be Th1-biased mixed immune response. While moderate protection of immunized BALB/c and C57BL/6 mice against rCPB challenge was observed with homologous booster strategy, heterologous booster strategy led to complete protection. Thus, beta toxin-based DNA vaccine using the heterologous prime-boosting strategy was able to generate better immune response and conferred greater degree of protection against high of dose rCPB challenge than homologous booster regimen, making it an effective vaccination approach against C. perfringens beta toxin.

DNA condensation by chiral alpha-methylated polyamine analogues and protection of cellular DNA from oxidative damage.

PubMed

Nayvelt, Irina; Hyvönen, Mervi T; Alhonen, Leena; Pandya, Ipsit; Thomas, Thresia; Khomutov, Alex R; Vepsäläinen, Jouko; Patel, Rajesh; Keinänen, Tuomo A; Thomas, T J

2010-01-11

Polyamines are essential molecules supporting the structure, conformation, and function of nucleic acids and proteins. We studied stereoisomers of alpha,alpha'-dimethylated spermine [(R,R)-Me(2)Spm, (S,S)-Me(2)Spm, (R,S)-Me(2)Spm] for their ability to provoke DNA condensation and protect DNA from damage. (R,R)- and (R,S)-Me(2)Spm displayed more efficient condensing ability than spermine, with significantly lower EC(50) (concentration for 50% compaction) values (p < or = 0.01). However, spermine exerted slightly more duplex stabilization than Me(2)Spm. Condensation resulted in nanoparticles with hydrodynamic radii between 39.6 and 48.4 nm, and electron microscopy showed the presence of toroids and spheroids. Natural polyamines and stereoisomers of Me(2)Spm protected DNA against DNase digestion and oxidative stress in vitro and against etoposide and oxidative stress in DU145 cells but afforded little protection against UV-C irradiation. Our findings indicate that Me(2)Spm stereoisomers are efficient DNA packaging agents with potential applications in gene delivery. Our study also reveals stereospecificity in DNA interaction and protection against cellular stress.

Suppression of antitumour protective cytotoxic T lymphocyte responses to a human papillomavirus 16 E7 DNA vaccine by coinjection of interleukin-12 complementary DNA: involvement of nitric oxide in immune suppression

PubMed Central

Sin, Jeong-Im

2009-01-01

Interleukin-12 (IL-12) has been shown to enhance cellular immunity in vitro and in vivo. The beneficial roles of IL-12 as a DNA vaccine adjuvant have been commonly observed. Here the impact of IL-12 complementary DNA (cDNA) as an adjuvant for a human papillomavirus (HPV) type 16 E7 DNA vaccine is investigated in a mouse tumour model. Coinjection of E7 DNA vaccine with IL-12 cDNA completely suppressed antigen-specific cytotoxic T-lymphocyte (CTL) responses, leading to a complete loss of antitumour protection from a tumour cell challenge. In addition, antigen-specific antibody and T helper cell proliferative responses were also suppressed by IL-12 cDNA coinjection. This inhibition was observed over different IL-12 cDNA doses. Furthermore, separate leg injections of IL-12 and E7 cDNAs suppressed antigen-specific CTL and tumour protective responses, but not antibody and T helper cell proliferative responses, suggesting different pathways for suppression of these two separate responses. Further knockout animal studies demonstrated that interferon-γ and nitric oxide are not directly associated with suppression of antigen-specific antibody responses by IL-12 cDNA coinjection. However, nitric oxide was found to be involved in suppression of antigen-specific CTL and tumour protective responses by IL-12 cDNA coinjection. These data suggest that coinjection of IL-12 cDNA results in suppression of E7-specific CTL responses through nitric oxide, leading to a loss of antitumour resistance in this DNA vaccine model. This study further shows that the adjuvant effect of IL-12 is dependent on the antigen types tested. PMID:19740332

Suppression of antitumour protective cytotoxic T lymphocyte responses to a human papillomavirus 16 E7 DNA vaccine by coinjection of interleukin-12 complementary DNA: involvement of nitric oxide in immune suppression.

PubMed

Sin, Jeong-Im

2009-09-01

Interleukin-12 (IL-12) has been shown to enhance cellular immunity in vitro and in vivo. The beneficial roles of IL-12 as a DNA vaccine adjuvant have been commonly observed. Here the impact of IL-12 complementary DNA (cDNA) as an adjuvant for a human papillomavirus (HPV) type 16 E7 DNA vaccine is investigated in a mouse tumour model. Coinjection of E7 DNA vaccine with IL-12 cDNA completely suppressed antigen-specific cytotoxic T-lymphocyte (CTL) responses, leading to a complete loss of antitumour protection from a tumour cell challenge. In addition, antigen-specific antibody and T helper cell proliferative responses were also suppressed by IL-12 cDNA coinjection. This inhibition was observed over different IL-12 cDNA doses. Furthermore, separate leg injections of IL-12 and E7 cDNAs suppressed antigen-specific CTL and tumour protective responses, but not antibody and T helper cell proliferative responses, suggesting different pathways for suppression of these two separate responses. Further knockout animal studies demonstrated that interferon-gamma and nitric oxide are not directly associated with suppression of antigen-specific antibody responses by IL-12 cDNA coinjection. However, nitric oxide was found to be involved in suppression of antigen-specific CTL and tumour protective responses by IL-12 cDNA coinjection. These data suggest that coinjection of IL-12 cDNA results in suppression of E7-specific CTL responses through nitric oxide, leading to a loss of antitumour resistance in this DNA vaccine model. This study further shows that the adjuvant effect of IL-12 is dependent on the antigen types tested.

DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations.

PubMed Central

Fynan, E F; Webster, R G; Fuller, D H; Haynes, J R; Santoro, J C; Robinson, H L

1993-01-01

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 micrograms 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 micrograms 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. Images Fig. 1 PMID:8265577

VEZF1 Elements Mediate Protection from DNA Methylation

PubMed Central

Strogantsev, Ruslan; Gaszner, Miklos; Hair, Alan; Felsenfeld, Gary; West, Adam G.

2010-01-01

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state. PMID:20062523

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.

[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.

DIRECT BINDING OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE TO TELOMERIC DNA PROTECTS TELOMERES AGAINST CHEMOTHERAPY-INDUCED RAPID DEGRADATION

PubMed Central

Demarse, Neil A.; Ponnusamy, Suriyan; Spicer, Eleanor K.; Apohan, Elif; Baatz, John E.; Ogretmen, Besim; Davies, Christopher

2009-01-01

GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel shift assays, we show that recombinant GAPDH binds directly with high affinity (Kd = 45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats and that nucleotides T1, G5 and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA: GAPDH), and GAPDH appears to form a high-molecular weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition, and this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells. PMID:19800890

Glycoprotein-Specific Antibodies Produced by DNA Vaccination Protect Guinea Pigs from Lethal Argentine and Venezuelan Hemorrhagic Fever.

PubMed

Golden, Joseph W; Maes, Piet; Kwilas, Steven A; Ballantyne, John; Hooper, Jay W

2016-01-20

severity of disease caused by arenaviruses, particularly species found in South America. Because of variations in potency of the human-derived product, limited availability, and safety concerns, this treatment option has essentially been abandoned. Accordingly, despite this approach being an effective postinfection treatment option, research on novel approaches to produce potent polyclonal antibody-based therapies have been deficient. Here we show that DNA-based vaccine technology can be used to make potently neutralizing antibodies in rabbits that exclusively target the glycoproteins of several human-pathogenic arenaviruses found in South America, including JUNV, MACV, GTOV, and SABV. These antibodies protected guinea pigs from lethal disease when given post-virus challenge. We also generated a purified antibody cocktail with antibodies targeting three arenaviruses and demonstrated protective efficacy against all three targets. Our findings demonstrate that use of the DNA vaccine technology could be used to produce candidate antiarenavirus neutralizing antibody-based products. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines

PubMed Central

Yılmaz, Sezen; Ustundag, Aylin; Cemiloglu Ulker, Ozge; Duydu, Yalcın

2016-01-01

Objective Many studies have been published on the antioxidative effects of boric acid (BA) and sodium borates in in vitro studies. However, the boron (B) concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentra- tion range relevant to humans. The aim of this study was to investigate the protective ef- fects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast) cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. Materials and Methods In this experimental study, comet assay and neutral red uptake (NRU) assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2). Results The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 µM). These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA) the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Conclusion Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54, 108, 540

Investigations of antioxidant-mediated protection and mitigation of radiation-induced DNA damage and lipid peroxidation in murine skin.

PubMed

Jelveh, Salomeh; Kaspler, Pavel; Bhogal, Nirmal; Mahmood, Javed; Lindsay, Patricia E; Okunieff, Paul; Doctrow, Susan R; Bristow, Robert G; Hill, Richard P

2013-08-01

Radioprotection and mitigation effects of the antioxidants, Eukarion (EUK)-207, curcumin, and the curcumin analogs D12 and D68, on radiation-induced DNA damage or lipid peroxidation in murine skin were investigated. These antioxidants were studied because they have been previously reported to protect or mitigate against radiation-induced skin reactions. DNA damage was assessed using two different assays. A cytokinesis-blocked micronucleus (MN) assay was performed on primary skin fibroblasts harvested from the skin of C3H/HeJ male mice 1 day, 1 week and 4 weeks after 5 Gy or 10 Gy irradiation. Local skin or whole body irradiation (100 kVp X-rays or caesium (Cs)-137 γ-rays respectively) was performed. DNA damage was further quantified in keratinocytes by immunofluorescence staining of γ-histone 2AX (γ-H2AX) foci in formalin-fixed skin harvested 1 hour or 1 day post-whole body irradiation. Radiation-induced lipid peroxidation in the skin was investigated at the same time points as the MN assay by measuring malondialdehyde (MDA) with a Thiobarbituric acid reactive substances (TBARS) assay. None of the studied antioxidants showed significant mitigation of skin DNA damage induced by local irradiation. However, when EUK-207 or curcumin were delivered before irradiation they provided some protection against DNA damage. In contrast, all the studied antioxidants demonstrated significant mitigating and protecting effects on radiation-induced lipid peroxidation at one or more of the three time points after local skin irradiation. Our results show no evidence for mitigation of DNA damage by the antioxidants studied in contrast to mitigation of lipid peroxidation. Since these agents have been reported to mitigate skin reactions following irradiation, the data suggest that changes in lipid peroxidation levels in skin may reflect developing skin reactions better than residual post-irradiation DNA damage in skin cells. Further direct comparison studies are required to confirm

Enhanced Immune Response and Protective Effects of Nano-chitosan-based DNA Vaccine Encoding T Cell Epitopes of Esat-6 and FL against Mycobacterium Tuberculosis Infection

PubMed Central

Feng, Ganzhu; Jiang, Qingtao; Xia, Mei; Lu, Yanlai; Qiu, Wen; Zhao, Dan; Lu, Liwei; Peng, Guangyong; Wang, Yingwei

2013-01-01

Development of a novel and effective vaccine against Mycobacterium tuberculosis (M.tb) is a challenging for preventing TB infection. In this study, a novel nanoparticle-based recombinant DNA vaccine was developed, which contains Esat-6 three T cell epitopes (Esat-6/3e) and fms-like tyrosine kinase 3 ligand (FL) genes (termed Esat-6/3e-FL), and was enveloped with chitosan (CS) nanoparticles (nano-chitosan). The immunologic and protective efficacy of the nano-chitosan-based DNA vaccine (termed nano-Esat-6/3e-FL) was assessed in C57BL/6 mice after intramuscular prime vaccination with the plasmids DNA and nasal boost with the Esat-6/3e peptides. The results showed that the immunized mice remarkably elicited enhanced T cell responses and protection against M.tb H37Rv challenge. These findings indicate that the nano-chitosan can significantly elevate the immunologic and protective effects of the DNA vaccine, and the nano-Esat-6/3e-FL is a useful vaccine for preventing M.tb infection in mice. PMID:23637790

The fruit extract of Berberis crataegina DC: exerts potent antioxidant activity and protects DNA integrity.

PubMed

Charehsaz, Mohammad; Sipahi, Hande; Celep, Engin; Üstündağ, Aylin; Cemiloğlu Ülker, Özge; Duydu, Yalçın; Aydın, Ahmet; Yesilada, Erdem

2015-04-17

Dried fruits of Berberis crataegina (Berberidaceae) have been frequently consumed as food garniture in Turkish cuisine, while its fruit paste has been used to increase stamina and in particular to prevent from cardiovascular dysfunctions in Northeastern Black Sea region of Turkey. This study investigated this folkloric information in order to explain the claimed healing effects as well as to evaluate possible risks. Total phenolic, flavonoid and proanthocyanidin contents and antioxidant capacity of the methanolic fruit extract were evaluated through several in vitro assays. The cytotoxic and genotoxic effects of B. crataegina fruit extract were also assessed in both cervical cancer cell line (HeLa) and human peripheral blood lymphocytes. The extract showed protective effects against ferric-induced oxidative stress and had a relatively good antioxidant activity. It also ameliorated the H2O2 mediated DNA damage in lymphocytes, suggesting the protective effect against oxidative DNA damage. The methanolic extract of B. crataegina fruits may be a potential antioxidant nutrient and also may exert a protective role against lipid peroxidation as well as oxidative DNA damage.

Evaluation of antimutagenic and protective effects of Parkinsonia aculeata L. leaves against H2O2 induced damage in pBR322 DNA.

PubMed

Sharma, Sonia; Sharma, Sushant; Vig, Adarsh Pal

2016-01-01

The in vitro antimutagenic and DNA protecting potential of organic (methanol, hexane, n-butanol) and aqueous extract/fractions of Parkinsonia aculeata L. (Fabaceae) was investigated by employing Ames assay and DNA nicking assay. DNA damage by hydroxyl radicals was effectively inhibited by all the extract/fractions. A marked antimutagenic effect was observed against 4-Nitro-o-phenylenediamine and sodium azide (direct acting mutagens) and 2-Aminofluorene (indirect acting mutagen) in TA98 and TA100 strains of Salmonella typhimurium. In Ames assay, two different modes of experiments i.e. pre-incubation and co-incubation were performed and it was observed that all the extract/fractions showed better results in the pre-incubation as compared to co- incubation mode. Out of all the extract/fractions tested, n-butanol fraction was found to be the most effective in preventing DNA damage and inhibiting mutagenesis. UHPLC analysis of extract/fractions revealed presence of polyphenols such as gallic acid, catechin, chlorogenic acid, caffeic acid, umbelliferone, coumaric acid, rutin, and ellagic acid etc. DNA protecting and antimutagenic activity of this plant could be attributed to presence of these polyphenols. The results of this study indicate the presence of potent antioxidant factors in Parkinsonia aculeata L, which are being explored further for their mechanism of action.

Protective mechanisms of p53-p21-pRb proteins against DNA damage-induced cell death.

PubMed

Garner, Elizabeth; Raj, Kenneth

2008-02-01

There have been innumerate demonstrations of p53's activity as a tumour suppressor protein with the ability to stimulate cell signalling that can lead to cell cycle arrest and cell death in the event of DNA damage. Despite the solid body of evidence to support these properties of p53, reports have emerged that suggest a role for p53 in protecting cells from cell death. Our recent report highlighted a mechanism by which p53 activity can promote cell survival in the event of DNA damage. Here we present the various mechanisms that are activated by p53 signalling that can confer protection to cells with damaged DNA and emphasise the practical and clinical implications of a more balanced and context-dependent understanding of p53's pro-apoptotic and pro-survival activities.

Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination.

PubMed

Boyer, J D; Ugen, K E; Wang, B; Agadjanyan, M; Gilbert, L; Bagarazzi, M L; Chattergoon, M; Frost, P; Javadian, A; Williams, W V; Refaeli, Y; Ciccarelli, R B; McCallus, D; Coney, L; Weiner, D B

1997-05-01

Novel approaches for the generation of more effective vaccines for HIV-1 are of significant importance. In this report we analyze the immunogenicity and efficacy of an HIV-1 DNA vaccine encoding env, rev and gag/pol in a chimpanzee model system. The immunized animals developed specific cellular and humoral immune responses. Animals were challenged with a heterologous chimpanzee titered stock of HIV-1 SF2 virus and followed for 48 weeks after challenge. Polymerase chain reaction coupled with reverse transcription (RT-PCR) results indicated infection in the control animal, whereas those animals vaccinated with the DNA constructs were protected from the establishment of infection. These studies serve as an important benchmark for the use of DNA vaccine technology for the production of protective immune responses.

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5′-DNA end

PubMed Central

Sato, Koichi; Shimomuki, Mayo; Katsuki, Yoko; Takahashi, Daisuke; Kobayashi, Wataru; Ishiai, Masamichi; Miyoshi, Hiroyuki; Takata, Minoru; Kurumizaka, Hitoshi

2016-01-01

The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork. PMID:27694619

Effective Protection Induced by a Monovalent DNA Vaccine against Dengue Virus (DV) Serotype 1 and a Bivalent DNA Vaccine against DV1 and DV2 in Mice.

PubMed

Zheng, Xiaoyan; Chen, Hui; Wang, Ran; Fan, Dongying; Feng, Kaihao; Gao, Na; An, Jing

2017-01-01

Dengue virus (DV) is the causal pathogen of dengue fever, which is one of the most rapidly spread mosquito-borne disease worldwide and has become a severe public health problem. Currently, there is no specific treatment for dengue; thus, a vaccine would be an effective countermeasure to reduce the morbidity and mortality. Although, the chimeric Yellow fever dengue tetravalent vaccine has been approved in some countries, it is still necessary to develop safer, more effective, and less costly vaccines. In this study, a DNA vaccine candidate pVAX1-D1ME expressing the prME protein of DV1 was inoculated in BALB/c mice via intramuscular injection or electroporation, and the immunogenicity and protection were evaluated. Compared with traditional intramuscular injection, administration with 50 μg pVAX1-D1ME via electroporation with three immunizations induced persistent humoral and cellular immune responses and effectively protected mice against lethal DV1 challenge. In addition, immunization with a bivalent vaccine consisting of pVAX1-D1ME and pVAX1-D2ME via electroporation generated a balanced IgG response and neutralizing antibodies against DV1 and DV2 and could protect mice from lethal challenge with DV1 and DV2. This study sheds new light on developing a dengue tetravalent DNA vaccine.

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.

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.

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

Antioxidant and DNA damage protective properties of anthocyanin-rich extracts from Hibiscus and Ocimum: a comparative study.

PubMed

Sarkar, Biswatrish; Kumar, Dhananjay; Sasmal, Dinakar; Mukhopadhyay, Kunal

2014-01-01

Anthocyanin extracts (AEs) from Ocimum tenuiflorum (leaf), Hibiscus rosa-sinensis (petal) and Hibiscus sabdariffa (calyx) were investigated and compared for in vitro antioxidant activity and DNA damage protective property. Total phenolic content (TPC) and total anthocyanin content (TAC) of the AEs were determined and the major anthocyanins were characterised. In vitro antioxidant activities were assessed by ferric-reducing antioxidant power (FRAP) assay, 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical-scavenging activity, 2-deoxy-D-ribose degradation assay and lipid peroxidation assay. The protective property of the AEs was also examined against oxidative DNA damage by H2O2 and UV using pUC19 plasmid. All the AEs particularly those from O. tenuiflorum demonstrated efficient antioxidant activity and protected DNA from damage. Strong correlation between antioxidant capacity and TPC and TAC was observed. Significant correlation between antioxidant capacity and TPC and TAC ascertained that phenolics and anthocyanins were the major contributors of antioxidant activity.

Chemotherapeutic Drug-Conjugated Microbeads Demonstrate Preferential Binding to Methylated Plasmid DNA.

PubMed

Lin, Kevin N; Grandhi, Taraka Sai Pavan; Goklany, Sheba; Rege, Kaushal

2018-04-10

Plasmid DNA (pDNA) is an attractive therapeutic biomolecule in several diseases including cancer, AIDS, cystic fibrosis, Parkinson's disease, and Alzheimer's disease. Increasing demand for plasmid DNA as a therapeutic biomolecule for transgene expression or vaccine applications necessitate novel approaches to bioprocessing. The synthesis, characterization and evaluation of aminoglycoside-derived hydrogel microbeads (Amikabeads) for pDNA binding is described previously. Here, the generation and evaluation of novel chemotherapeutic drug-conjugated microbeads for application in pDNA binding and recovery is described. Chemotherapeutic drug-conjugated Amikabeads demonstrate higher binding of methylated pDNA compared to unmethylated pDNA in presence of high salt concentrations. Desorption of plasmids from drug-conjugated microbeads is facilitated by the use of organic modifiers. The observed differences in binding methylated versus unmethylated DNA can make drug-conjugated microbeads useful in diagnostic as well as therapeutic applications. These results demonstrate that anti-cancer drugs represent a diverse set of ligands that may be exploited for molecular engineering of novel DNA binding materials for applications in delivery, diagnostics, and biomanufacturing. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protection of cadmium chloride induced DNA damage by Lamiaceae plants

PubMed Central

Thirugnanasampandan, Ramaraj; Jayakumar, Rajarajeswaran

2011-01-01

Objective To analyze the total phenolic content, DNA protecting and radical scavenging activity of ethanolic leaf extracts of three Lamiaceae plants, i.e. Anisomelos malabarica (A. malabarica), Leucas aspera (L. aspera) and Ocimum basilicum (O. basilicum). Methods The total polyphenols and flavonoids were analyzed in the ethanolic leaf extracts of the lamiaceae plants. To determine the DNA protecting activity, various concentrations of the plant extracts were prepared and treated on cultured HepG2 human lung cancer cells. The pretreated cells were exposed to H2O2 to induce DNA damage through oxidative stress. Comet assay was done and the tail length of individual comets was measured. Nitric oxide and superoxide anion scavenging activities of lamiaceae plants were analyzed. Results Among the three plant extracts, the highest amount of total phenolic content was found in O. basilicum (189.33 mg/g), whereas A. malabarica showed high levels of flavonoids (10.66 mg/g). O. basilicum also showed high levels of DNA protecting (85%) and radical scavenging activity. Conclusions The results of this study shows that bioactive phenols present in lamiaceae plants may prevent carcinogenesis through scavenging free radicals and inhibiting DNA damage. PMID:23569799

The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response

PubMed Central

Cicconi, Alessandro; Micheli, Emanuela; Vernì, Fiammetta; Jackson, Alison; Gradilla, Ana Citlali; Cipressa, Francesca; Raimondo, Domenico; Bosso, Giuseppe; Wakefield, James G.; Ciapponi, Laura; Cenci, Giovanni; Gatti, Maurizio

2017-01-01

Abstract Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin. PMID:27940556

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.

Protective roles of single-wall carbon nanotubes in ultrasonication-induced DNA base damage.

PubMed

Petersen, Elijah J; Tu, Xiaomin; Dizdaroglu, Miral; Zheng, Ming; Nelson, Bryant C

2013-01-28

The overall level of ultrasonication-induced DNA damage is reduced in the presence of single-wall carbon nanotubes (SWCNTs), particularly for DNA lesions formed by one-electron reduction of intermediate radicals. The protective role of SWCNTs observed in this work suggests a contrary view to the general idea that carbon nanotubes have damaging effects on biomolecules. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protection of Rhesus Monkeys by a DNA Prime/Poxvirus Boost Malaria Vaccine Depends on Optimal DNA Priming and Inclusion of Blood Stage Antigens

PubMed Central

Weiss, Walter R.; Kumar, Anita; Jiang, George; Williams, Jackie; Bostick, Anthony; Conteh, Solomon; Fryauff, David; Aguiar, Joao; Singh, Manmohan; O'Hagan, Derek T.; Ulmer, Jeffery B.; Richie, Thomas L.

2007-01-01

Background We have previously described a four antigen malaria vaccine consisting of DNA plasmids boosted by recombinant poxviruses which protects a high percentage of rhesus monkeys against Plasmodium knowlesi (Pk) malaria. This is a multi-stage vaccine that includes two pre-erythrocytic antigens, PkCSP and PkSSP2(TRAP), and two erythrocytic antigens, PkAMA-1 and PkMSP-1(42kD). The present study reports three further experiments where we investigate the effects of DNA dose, timing, and formulation. We also compare vaccines utilizing only the pre-erythrocytic antigens with the four antigen vaccine. Methodology In three experiments, rhesus monkeys were immunized with malaria vaccines using DNA plasmid injections followed by boosting with poxvirus vaccine. A variety of parameters were tested, including formulation of DNA on poly-lactic co-glycolide (PLG) particles, varying the number of DNA injections and the amount of DNA, varying the interval between the last DNA injection to the poxvirus boost from 7 to 21 weeks, and using vaccines with from one to four malaria antigens. Monkeys were challenged with Pk sporozoites given iv 2 to 4 weeks after the poxvirus injection, and parasitemia was measured by daily Giemsa stained blood films. Immune responses in venous blood samples taken after each vaccine injection were measured by ELIspot production of interferon-γ, and by ELISA. Conclusions 1) the number of DNA injections, the formulation of the DNA plasmids, and the interval between the last DNA injection and the poxvirus injection are critical to vaccine efficacy. However, the total dose used for DNA priming is not as important; 2) the blood stage antigens PkAMA-1 and PkMSP-1 were able to protect against high parasitemias as part of a genetic vaccine where antigen folding is not well defined; 3) immunization with PkSSP2 DNA inhibited immune responses to PkCSP DNA even when vaccinations were given into separate legs; and 4) in a counter-intuitive result, higher

Lithium chloride protects retinal neurocytes from nutrient deprivation by promoting DNA non-homologous end-joining

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhuang Jing; Li Fan; Liu Xuan

2009-03-13

Lithium chloride is a therapeutic agent for treatment of bipolar affective disorders. Increasing numbers of studies have indicated that lithium has neuroprotective effects. However, the molecular mechanisms underlying the actions of lithium have not been fully elucidated. This study aimed to investigate whether lithium chloride produces neuroprotective function by improving DNA repair pathway in retinal neurocyte. In vitro, the primary cultured retinal neurocytes (85.7% are MAP-2 positive cells) were treated with lithium chloride, then cultured with serum-free media to simulate the nutrient deprived state resulting from ischemic insult. The neurite outgrowth of the cultured cells increased significantly in a dose-dependentmore » manner when exposed to different levels of lithium chloride. Genomic DNA electrophoresis demonstrated greater DNA integrity of retinal neurocytes when treated with lithium chloride as compared to the control. Moreover, mRNA and protein levels of Ligase IV (involved in DNA non-homologous end-joining (NHEJ) pathway) in retinal neurocytes increased with lithium chloride. The end joining activity assay was performed to determine the role of lithium on NHEJ in the presence of extract from retinal neurocytes. The rejoining levels in retinal neurocytes treated with lithium were significantly increased as compared to the control. Furthermore, XRCC4, the Ligase IV partner, and the transcriptional factor, CREB and CTCF, were up-regulated in retinal cells after treating with 1.0 mM lithium chloride. Therefore, our data suggest that lithium chloride protects the retinal neural cells from nutrient deprivation in vitro, which may be similar to the mechanism of cell death in glaucoma. The improvement in DNA repair pathway involving in Ligase IV might have an important role in lithium neuroprotection. This study provides new insights into the neural protective mechanisms of lithium chloride.« less

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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Protective role of quercetin against copper(II)-induced oxidative stress: A spectroscopic, theoretical and DNA damage study.

PubMed

Jomova, Klaudia; Lawson, Michael; Drostinova, Lenka; Lauro, Peter; Poprac, Patrik; Brezova, Vlasta; Michalik, Martin; Lukes, Vladimir; Valko, Marian

2017-12-01

The radical scavenging and metal chelating properties of flavonoids indicate that they may play a protective role in diseases with perturbed metal homeostasis such as Alzheimer's disease. In this work we investigated the effect of the coordination of quercetin to copper(II) in view of the formation of ROS in Cu-catalyzed Fenton reaction. ABTS and DPPH assays confirmed that the copper(II)-quercetin complex exhibits a stronger radical scavenging activity than does quercetin alone. EPR spin trapping experiments have shown that chelation of quercetin to copper significantly suppressed the formation of hydroxyl radicals in the Cu(II)-Fenton reaction. DNA damage experiments revealed a protective effect for quercetin, but only at higher stoichiometric ratios of quercetin relative to copper. DNA protective effect of quercetin against ROS attack was described by two mechanisms. The first mechanism lies in suppressed formation of ROS due to the decreased catalytic action of copper in the Fenton reaction, as a consequence of its chelation and direct scavenging of ROS by free quercetin. Since the Cu-quercetin complex intercalates into DNA, the second mechanism was attributed to a suppressed intercalating ability of the Cu-quercetin complex due to the mildly intercalating free quercetin into DNA, thus creating a protective wall against stronger intercalators. Copyright © 2017 Elsevier Ltd. All rights reserved.

A DNA vaccine delivered by dermal electroporation fully protects cynomolgus macaques against Lassa fever.

PubMed

Cashman, Kathleen A; Wilkinson, Eric R; Shaia, Carl I; Facemire, Paul R; Bell, Todd M; Bearss, Jeremy J; Shamblin, Joshua D; Wollen, Suzanne E; Broderick, Kate E; Sardesai, Niranjan Y; Schmaljohn, Connie S

2017-12-02

Lassa virus (LASV) is an ambisense RNA virus in the Arenaviridae family and is the etiological agent of Lassa fever, a severe hemorrhagic disease endemic to West and Central Africa. 1,2 There are no US Food and Drug Administration (FDA)-licensed vaccines available to prevent Lassa fever. 1,2 in our previous studies, we developed a gene-optimized DNA vaccine that encodes the glycoprotein precursor gene of LASV (Josiah strain) and demonstrated that 3 vaccinations accompanied by dermal electroporation protected guinea pigs from LASV-associated illness and death. Here, we describe an initial efficacy experiment in cynomolgus macaque nonhuman primates (NHPs) in which we followed an identical 3-dose vaccine schedule that was successful in guinea pigs, and a follow-on experiment in which we used an accelerated vaccination strategy consisting of 2 administrations, spaced 4 weeks apart. In both studies, all of the LASV DNA-vaccinated NHPs survived challenge and none of them had measureable, sustained viremia or displayed weight loss or other disease signs post-exposure. Three of 10 mock-vaccinates survived exposure to LASV, but all of them became acutely ill post-exposure and remained chronically ill to the study end point (45 d post-exposure). Two of the 3 survivors experienced sensorineural hearing loss (described elsewhere). These results clearly demonstrate that the LASV DNA vaccine combined with dermal electroporation is a highly effective candidate for eventual use in humans.

DNA damage protection against free radicals of two antioxidant neolignan glucosides from sugarcane molasses.

PubMed

Asikin, Yonathan; Takahashi, Makoto; Mizu, Masami; Takara, Kensaku; Oku, Hirosuke; Wada, Koji

2016-03-15

Sugarcane molasses is a potential by-product of the sugarcane manufacturing industry that is rich in antioxidant materials. The present study aimed to obtain antioxidative compounds from sugarcane molasses and to evaluate their ability to protect DNA from oxidative damage. Two neolignan glucosides were isolated from sugarcane molasses using bioassay and UV spectra monitoring-guided fractionation. The compounds were elucidated as (7R,8S)-dehydrodiconiferyl alcohol-4-O-β-d-glucoside (1) and (7S,8R)-simulanol-9'-O-β-d-glucoside (2). Neolignan glucoside 2 protected against DNA damage caused by free radicals more effectively than did neolignan glucoside 1 (13.62 and 9.08 µmol L(-1) for peroxyl and hydroxyl radicals, respectively, compared to 48.07 and 14.42 µmol L(-1) ). Additionally, neolignan glucoside 2 exhibited superior DNA protection against free radicals compared with various known antioxidative compounds, including p-coumaric acid, ferulic acid, vanillic acid and epigallocatechin gallate. The isolated neolignan glucosides from sugarcane molasses are able to protect DNA from oxidative damage caused by free radicals. This is the first identification of these two compounds in sugarcane molasses. The sugarcane molasses can therefore be used as potential nutraceutical preventative agents, and the findings may foster the utilization of this by-product as a bioresource-based product. © 2015 Society of Chemical Industry. Copyright © 2015 Society of Chemical Industry.

A study of DNA protective effect of orange juice supplementation.

PubMed

Szeto, Yim Tong; To, Tai Lun; Pak, Sok Cheon; Kalle, Wouter

2013-05-01

The potential acute genoprotective effect of orange juice supplementation was investigated. Six healthy subjects (aged 33 to 60 years; 3 women and 3 men) were asked to drink 400 mL of commercial orange juice, which contained 100 mg vitamin C and 40.8 g sugar. Venous blood (2 mL) was taken before and 2 h after ingestion (test trial). A week later, the subjects were asked to repeat the trial by drinking 400 mL water with 100 mg vitamin C and 40.8 g glucose (control trial). Lymphocytes isolated from blood samples underwent comet assay on the day of collection. Pre- and postingestion DNA damage scores were measured in both the test and control trials. Results showed that there was a significant decrease in DNA damage induced by hydrogen peroxide after 2 h of supplementation with orange juice, and no change in baseline DNA damage. There was no significant decrease in the DNA damage in lymphocytes in the control trial.

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

2016-05-27

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.

UVA-potentiated damage to calf thymus DNA by Fenton reaction system and protection by para-aminobenzoic acid.

PubMed

Shih, M K; Hu, M L

1996-03-01

Calf thymus DNA was irradiated with low-intensity UVA (main output at 365 nm, 2 mW cm-2 or 36 kJ m-2 for 30 min), and the role of metal ions, hydrogen peroxide and reactive oxygen species (ROS) was examined. DNA damage was measured as thiobarbituric acid-reactive substances (possibly from degradation of deoxyribose) and as changes in ethidium bromide-DNA fluorescence due to unwinding from strand breaks. Under the present experimental conditions, UVA alone or in the presence of H2O2 had no effect on DNA but slightly enhanced the damage by iron/EDTA. Ultraviolet A strongly enhanced DNA damage (ca four- to five-fold) by the Fenton reaction system (50 microM Fe2+/100 microM EDTA + 0.5 mM H2O2). The results suggest that the Fenton reaction system was "photosensitized" to damage DNA by low-intensity UVA radiation. The enhanced damage by UVA was attributed in part to the reduction of Fe3+ to Fe2+. Ultraviolet A had no effect when iron (ferric or ferrous) ions were replaced by Cu2+, Zn2+, Mn2+ or Cd2+. The ROS involved in the UVA-enhanced damage to DNA by the Fenton reagents were OH and, to a lesser extent, superoxide anions. The UVA-potentiated DNA damage by the Fenton reaction system was then used to examine the protective effect of para-aminobenzoate (PABA), a UVB-absorbing sunscreen that protects against photocarcinogenesis in hairless mice. The results show that PABA and mannitol dose-dependently inhibited the damage with concentrations required for 50% inhibition at 0.1 mM and 3 mM, respectively. The protection by PABA was attributed to its radical-scavenging ability because PABA does not absorb light in the UVA region. These findings may be relevant to the biological damage by UVA and suggest that PABA is useful in protection against photocarcinogenesis by wide-range UV radiation.

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.

DNA Protection Protein, a Novel Mechanism of Radiation Tolerance: Lessons from Tardigrades

PubMed Central

Hashimoto, Takuma; Kunieda, Takekazu

2017-01-01

Genomic DNA stores all genetic information and is indispensable for maintenance of normal cellular activity and propagation. Radiation causes severe DNA lesions, including double-strand breaks, and leads to genome instability and even lethality. Regardless of the toxicity of radiation, some organisms exhibit extraordinary tolerance against radiation. These organisms are supposed to possess special mechanisms to mitigate radiation-induced DNA damages. Extensive study using radiotolerant bacteria suggested that effective protection of proteins and enhanced DNA repair system play important roles in tolerability against high-dose radiation. Recent studies using an extremotolerant animal, the tardigrade, provides new evidence that a tardigrade-unique DNA-associating protein, termed Dsup, suppresses the occurrence of DNA breaks by radiation in human-cultured cells. In this review, we provide a brief summary of the current knowledge on extremely radiotolerant animals, and present novel insights from the tardigrade research, which expand our understanding on molecular mechanism of exceptional radio-tolerability. PMID:28617314

The effect of co-administration of DNA carrying chicken interferon-gamma gene on protection of chickens against infectious bursal disease by DNA-mediated vaccination.

PubMed

Hsieh, Ming Kun; Wu, Ching Ching; Lin, Tsang Long

2006-11-17

The purpose of the present study was to determine whether DNA vaccination by co-administration of DNA coding for chicken interferon-gamma (IFN-gamma) gene and DNA encoding for the VP243 gene of IBDV could enhance immune response and protection efficacy of chickens against challenge by IBDV. Plasmids carrying VP243 gene of IBDV strain variant E (VE) (P/VP243/E) and chicken IFN-gamma gene (P/cIFN-gamma) were constructed, respectively. One-day-old chickens were intramuscularly injected with P/VP243/E, or P/cIFN-gamma, or both once, twice, or three times into the thigh muscle of one leg or the thigh muscles of two separate legs at weekly intervals. Chickens were orally challenged with IBDV strain VE at 3 weeks of age and observed for 10 days. Chickens receiving two plasmids in the same site two times had significantly higher (P<0.05) bursal lesion scores and significantly lower (P<0.05) bursa weight/body weight ratios than those that only received P/VP243/E two or three times. Chickens inoculated with two plasmids separately in the thigh muscles of different legs or P/VP243/E two times had 33-50% protection and those receiving two plasmids in the same sites did not have any protection against IBD. The enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers to IBDV of chickens in the groups with three doses of P/VP243/E were significantly higher (P<0.05) than those in groups receiving two doses of P/VP243/E or P/VP243/E and P/cIFN-gamma. Chickens protected by DNA vaccination did not have detectable IBDV antigen in the bursae as determined by immunofluorescent antibody assay (IFA). The results indicated that co-administration of plasmid encoding chicken IFN-gamma gene with plasmid encoding a large segment gene of the IBDV did not enhance immune response and protection against challenge by IBDV.

Curcumin protects against cytotoxic and inflammatory effects of quartz particles but causes oxidative DNA damage in a rat lung epithelial cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Hui; Berlo, Damien van; Shi Tingming

2008-02-15

Chronic inhalation of high concentrations of respirable quartz particles has been implicated in various lung diseases including lung fibrosis and cancer. Generation of reactive oxygen species (ROS) and oxidative stress is considered a major mechanism of quartz toxicity. Curcumin, a yellow pigment from Curcuma longa, has been considered as nutraceutical because of its strong anti-inflammatory, antitumour and antioxidant properties. The aim of our present study was to investigate whether curcumin can protect lung epithelial cells from the cytotoxic, genotoxic and inflammatory effects associated with quartz (DQ12) exposure. Electron paramagnetic resonance (EPR) measurements using the spin-trap DMPO demonstrated that curcumin reducesmore » hydrogen peroxide-dependent hydroxyl-radical formation by quartz. Curcumin was also found to reduce quartz-induced cytotoxicity and cyclooxygenase 2 (COX-2) mRNA expression in RLE-6TN rat lung epithelial cells (RLE). Curcumin also inhibited the release of macrophage inflammatory protein-2 (MIP-2) from RLE cells as observed upon treatment with interleukin-1 beta (IL-1{beta}) and tumour necrosis factor-alpha (TNF{alpha}). However, curcumin failed to protect the RLE cells from oxidative DNA damage induced by quartz, as shown by formamidopyrimidine glycosylase (FPG)-modified comet assay and by immunocytochemistry for 8-hydroxydeoxyguanosine. In contrast, curcumin was found to be a strong inducer of oxidative DNA damage itself at non-cytotoxic and anti-inflammatory concentrations. In line with this, curcumin also enhanced the mRNA expression of the oxidative stress response gene heme oxygenase-1 (ho-1). Curcumin also caused oxidative DNA damage in NR8383 rat alveolar macrophages and A549 human lung epithelial cells. Taken together, these observations indicate that one should be cautious in considering the potential use of curcumin in the prevention or treatment of lung diseases associated with quartz exposure.« less

In vitro effects of simultaneous exposure to platinum and cadmium on the activity of antioxidant enzymes and DNA damage and potential protective effects of selenium and zinc.

PubMed

Tariba, Blanka; Živković, Tanja; Gajski, Goran; Gerić, Marko; Gluščić, Valentina; Garaj-Vrhovac, Vera; Peraica, Maja; Pizent, Alica

2017-04-01

Circulating platinum (Pt) is detectable in the blood of Pt-treated cancer patients for over a decade after the treatment. Prolonged exposure to Pt, in combination with adverse compounds from nutrition and lifestyle, such as cadmium (Cd), could increase the risk from second cancers. The aim of this study was to investigate the effects of simultaneous exposure to Cd- and Pt-compounds on oxidative and DNA damage and the possible protective effects of zinc (Zn) and selenium (Se). The aqueous solutions of PtCl 4 , CdCl 2  ×   H 2 O, ZnCl 2 and Na 2 SeO 3 were added, alone or in combination, to whole blood and isolated erythrocytes to produce the final concentrations of 2000 μg/L of Pt, 8 μg/L of Cd, 100 μg/L of Se, and 1000 μg/L of Zn. The activity of copper, zinc-superoxide dismutase, glutathione peroxidase and glutathione in whole blood was determined after 1 h exposure in in vitro conditions. The induction of DNA strand-breaks in human peripheral blood leukocytes was determined with the alkaline comet assay after 24 h exposure. Exposure to Pt and/or Cd decreased the activities of antioxidant enzymes and elevated DNA damage compared to control. A statistically significant change in the activity of both enzymes and in the induction of DNA strand-breaks was observed in the cells treated with Pt + Cd combination, while the addition of Se and/or Zn resulted in partial recovery of these effects. The results indicate that combined exposure to Pt and Cd could disrupt antioxidant protection of the organism and increase DNA damage, whereas Se and Zn could partially ameliorate these harmful effects.

Protective effects of lemongrass (Cymbopogon citratus STAPF) essential oil on DNA damage and carcinogenesis in female Balb/C mice.

PubMed

Bidinotto, Lucas T; Costa, Celso A R A; Salvadori, Daisy M F; Costa, Mirtes; Rodrigues, Maria A M; Barbisan, Luís F

2011-08-01

This study investigated the protective effect of oral treatment with lemongrass (Cymbopogon citratus STAPF) essential oil (LGEO) on leukocyte DNA damage induced by N-methyl-N-nitrosurea (MNU). Also, the anticarcinogenic activity of LGEO was investigated in a multi-organ carcinogenesis bioassay induced by 7,12-dimethylbenz(a)antracene, 1,2-dimethylhydrazine and N-butyl-N-(4-hydroxibuthyl)nitrosamine in Balb/C female Balb/c mice (DDB-initiated mice). In the short-term study, the animals were allocated into three groups: vehicle group (negative control), MNU group (positive control) and LGEO 500 mg kg⁻¹ (five times per week for 5 weeks) plus MNU group (test group). Blood samples were collected to analyze leukocyte DNA damage by comet assay 4 h after each MNU application at the end of weeks 3 and 5. The LGEO 500 mg kg⁻¹ treated group showed significantly lower (P < 0.01) leukocyte DNA damage than its respective positive group exposed to MNU alone at week 3. In the medium-term study, DDB-initiated mice were allocated into three groups: vehicle group (positive control) and LGEO 125 or 500 mg kg⁻¹ (five times per week for 6 weeks; test groups). At week 20, all animals were euthanized and mammary glands, colon and urinary bladder were processed for histopathological analyses for detection of preneoplastic and neoplastic lesions. A slight non-significant effect of treatment with LGEO 500 mg kg⁻¹ in reducing development of alveolar and ductal mammary hyperplasia was found (P = 0.075). Our findings indicate that lemongrass essential oil provided protective action against MNU-induced DNA damage and a potential anticarcinogenic activity against mammary carcinogenesis in DDB-initiated female Balb/C mice. Copyright © 2010 John Wiley & Sons, Ltd.

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

Vector optimization and needle-free intradermal application of a broadly protective polyvalent influenza A DNA vaccine for pigs and humans

PubMed Central

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

The principal phenolic and alcoholic components of wine protect human lymphocytes against hydrogen peroxide- and ionizing radiation-induced DNA damage in vitro.

PubMed

Greenrod, William; Fenech, Michael

2003-03-01

We have tested the hypothesis that the alcoholic and phenolic components of wine are protective against the DNA-damaging and cytotoxic effects of hydrogen peroxide and gamma-radiation in vitro. The components of wine tested were ethanol, glycerol, a mixture of the phenolic compounds catechin and caffeic acid and tartaric acid, all at concentrations that were 2.5 or 10.0% of the concentration in a typical Australian white wine (Riesling). These components were tested individually or combined as a mixture and compared to a white wine stripped of polyphenols, as well as a Hanks balanced salt solution control, which was the diluent for the wine components. The effect of the components was tested in lymphocytes, using the cytokinesis-block micronucleus assay, after 30 min incubation in plasma or whole blood for the hydrogen peroxide or gamma-radiation challenge, respectively. The results obtained showed that ethanol, glycerol, the catechin-caffeic acid mixture, the mixture of all components and the stripped white wine significantly reduced the DNA-damaging effects of hydrogen peroxide and gamma-radiation (P = 0.043-0.001, ANOVA). The strongest protective effect against DNA damage by gamma-irradiation was observed for the catechin-caffeic acid mixture and the mixture of all components (30 and 32% reduction, respectively). These two treatments as well as ethanol produced the strongest protective effects against DNA damage by hydrogen peroxide (24, 25 and 18%, respectively). The protection provided by the mixture did not account for the expected additive protective effects of the individual components. Ethanol was the only component that significantly increased baseline DNA damage rate, however, this effect was negated in the mixture. In conclusion, our results suggest that the main phenolic and alcoholic components of wine can reduce the DNA-damaging effects of two important oxidants, i.e. hydrogen peroxide and ionizing radiation, in this physiologically relevant in vitro

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

YAP activation protects urothelial cell carcinoma from treatment-induced DNA damage.

PubMed

Ciamporcero, E; Shen, H; Ramakrishnan, S; Yu Ku, S; Chintala, S; Shen, L; Adelaiye, R; Miles, K M; Ullio, C; Pizzimenti, S; Daga, M; Azabdaftari, G; Attwood, K; Johnson, C; Zhang, J; Barrera, G; Pili, R

2016-03-24

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 knockdown 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.

Suppressive Effects on the Immune Response and Protective Immunity to a JEV DNA Vaccine by Co-administration of a GM-CSF-Expressing Plasmid in Mice

PubMed Central

Chen, Hui; Gao, Na; Fan, Dongying; Wu, Jiangman; Zhu, Junping; Li, Jieqiong; Wang, Juan; Chen, Yanlei; An, Jing

2012-01-01

As a potential cytokine adjuvant of DNA vaccines, granulocyte-macrophage colony–stimulating factor (GM-CSF) has received considerable attention due to its essential role in the recruitment of antigen-presenting cells, differentiation and maturation of dendritic cells. However, in our recent study of a Japanese encephalitis virus (JEV) DNA vaccine, co-inoculation of a GM-CSF plasmid dramatically suppressed the specific IgG response and resulted in decreased protection against JEV challenge. It is known that GM-CSF has been used in clinic to treat neutropenia for repopulating myeloid cells, and as an adjuvant in vaccine studies; it has shown various effects on the immune response. Therefore, in this study, we characterized the suppressive effects on the immune response to a JEV DNA vaccine by the co-administration of the GM-CSF-expressing plasmid and clarified the underlying mechanisms of the suppression in mice. Our results demonstrated that co-immunization with GM-CSF caused a substantial dampening of the vaccine-induced antibody responses. The suppressive effect was dose- and timing-dependent and likely related to the immunogenicity of the antigen. The suppression was associated with the induction of immature dendritic cells and the expansion of regulatory T cells but not myeloid-derived suppressor cells. Collectively, our findings not only provide valuable information for the application of GM-CSF in clinic and using as a vaccine adjuvant but also offer further insight into the understanding of the complex roles of GM-CSF. PMID:22493704

Programmed Protection of Foreign DNA from Restriction Allows Pathogenicity Island Exchange during Pneumococcal Transformation

PubMed Central

Johnston, Calum; Martin, Bernard; Granadel, Chantal; Polard, Patrice; Claverys, Jean-Pierre

2013-01-01

In bacteria, transformation and restriction-modification (R-M) systems play potentially antagonistic roles. While the former, proposed as a form of sexuality, relies on internalized foreign DNA to create genetic diversity, the latter degrade foreign DNA to protect from bacteriophage attack. The human pathogen Streptococcus pneumoniae is transformable and possesses either of two R-M systems, DpnI and DpnII, which respectively restrict methylated or unmethylated double-stranded (ds) DNA. S. pneumoniae DpnII strains possess DpnM, which methylates dsDNA to protect it from DpnII restriction, and a second methylase, DpnA, which is induced during competence for genetic transformation and is unusual in that it methylates single-stranded (ss) DNA. DpnA was tentatively ascribed the role of protecting internalized plasmids from DpnII restriction, but this seems unlikely in light of recent results establishing that pneumococcal transformation was not evolved to favor plasmid exchange. Here we validate an alternative hypothesis, showing that DpnA plays a crucial role in the protection of internalized foreign DNA, enabling exchange of pathogenicity islands and more generally of variable regions between pneumococcal isolates. We show that transformation of a 21.7 kb heterologous region is reduced by more than 4 logs in dpnA mutant cells and provide evidence that the specific induction of dpnA during competence is critical for full protection. We suggest that the integration of a restrictase/ssDNA-methylase couplet into the competence regulon maintains protection from bacteriophage attack whilst simultaneously enabling exchange of pathogenicicy islands. This protective role of DpnA is likely to be of particular importance for pneumococcal virulence by allowing free variation of capsule serotype in DpnII strains via integration of DpnI capsule loci, contributing to the documented escape of pneumococci from capsule-based vaccines. Generally, this finding is the first evidence for a

Protective effect of dry olive leaf extract in adrenaline induced DNA damage evaluated using in vitro comet assay with human peripheral leukocytes.

PubMed

Cabarkapa, Andrea; Zivković, Lada; Zukovec, Dijana; Djelić, Ninoslav; Bajić, Vladan; Dekanski, Dragana; Spremo-Potparević, Biljana

2014-04-01

Excessive release of stress hormone adrenaline is accompanied by generation of reactive oxygen species which may cause disruption of DNA integrity leading to cancer and age-related disorders. Phenolic-rich plant product dry olive leaf extract (DOLE) is known to modulate effects of various oxidants in human cells. The aim was to evaluate the effect of commercial DOLE against adrenaline induced DNA damage in human leukocytes by using comet assay. Peripheral blood leukocytes from 6 healthy subjects were treated in vitro with three final concentrations of DOLE (0.125, 0.5, and 1mg/mL) for 30 min at 37°C under two different protocols, pretreatment and post-treatment. Protective effect of DOLE was assessed from its ability to attenuate formation of DNA lesions induced by adrenaline. Compared to cells exposed only to adrenaline, DOLE displayed significant reduction (P<0.001) of DNA damage at all three concentrations and under both experimental protocols. Pearson correlation analysis revealed a significant positive association between DOLE concentration and leukocytes DNA damage (P<0.05). Antigenotoxic effect of the extract was more pronounced at smaller concentrations. Post-treatment with 0.125 mg/mL DOLE was the most effective against adrenaline genotoxicity. Results indicate genoprotective and antioxidant properties in dry olive leaf extract, strongly supporting further explorations of its underlying mechanisms of action. Copyright © 2014 Elsevier Ltd. All rights reserved.

DNA mismatch repair and oligonucleotide end-protection promote base-pair substitution distal from a CRISPR/Cas9-induced DNA break

PubMed Central

Harmsen, Tim; Klaasen, Sjoerd; van de Vrugt, Henri; te Riele, Hein

2018-01-01

Abstract Single-stranded oligodeoxyribonucleotide (ssODN)-mediated repair of CRISPR/Cas9-induced DNA double-strand breaks (DSB) can effectively be used to introduce small genomic alterations in a defined locus. Here, we reveal DNA mismatch repair (MMR) activity is crucial for efficient nucleotide substitution distal from the Cas9-induced DNA break when the substitution is instructed by the 3′ half of the ssODN. Furthermore, protecting the ssODN 3′ end with phosphorothioate linkages enhances MMR-dependent gene editing events. Our findings can be exploited to optimize efficiencies of nucleotide substitutions distal from the DSB and imply that oligonucleotide-mediated gene editing is effectuated by templated break repair. PMID:29447381

Protective effects of melatonin against 12C6+ beam irradiation-induced oxidative stress and DNA injury in the mouse brain

NASA Astrophysics Data System (ADS)

Wu, Z. H.; Zhang, H.; Wang, X. Y.; Yang, R.; Liu, B.; Liu, Y.; Zhao, W. P.; Feng, H. Y.; Xue, L. G.; Hao, J. F.; Niu, B. T.; Wang, Z. H.

2012-01-01

The purpose of this experiment was to estimate the protective effects of melatonin against radiation-induced brain damages in mice induced by heavy ion beams. Kun-Ming mice were randomly divided into five groups: normal control group, irradiation control group, and three different doses of melatonin (5, 10, and 20 mg/kg, i.p.) treated groups. Apart from the normal control group, the other four groups were exposed to whole-body 4.0 Gy carbon ion beam irradiation (approximately 0.5 Gy/min) after i.p. administration of normal saline or melatonin 1 h before irradiation. The oxidative redox status of brain tissue was assessed by measurement of malondiadehyde (MDA) levels, total superoxide dismutase (T-SOD), cytosolic superoxide dismutase (Cu/ZnSOD, SOD1) and mitochondrial superoxide dismutase (MnSOD, SOD2) activities at 8 h after irradiation. DNA damages were determined using the Comet assay and apoptosis and cell cycle distribution were detected by flow cytometric analyses. A dramatic dose-dependent decrease in MDA levels, tail moment, rates of tailing cells, and apoptosis, and a dose-dependent increase in T-SOD and SOD2 activities, in brain tissues in the melatonin-treated groups were detected compared with the irradiation only group. Furthermore, flow cytometric analysis demonstrated that the percentage of brain cells in the G0/G1 phase decreased significantly, while those in the S and G2/M stage increased dramatically, with mice pretreated with melatonin compared to the irradiation control group. These data indicate that melatonin has protective effects against irradiation-induced brain injury, and that its underlying protective mechanisms may relate to modulation of oxidative stress induced by heavy ionirradiation.

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.

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.

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

Comparison of potential protection conferred by three immunization strategies (protein/protein, DNA/DNA, and DNA/protein) against Brucella infection using Omp2b in BALB/c Mice.

PubMed

Golshani, Maryam; Rafati, Sima; Nejati-Moheimani, Mehdi; Ghasemian, Melina; Bouzari, Saeid

2016-12-25

In the present study, immunogenicity and protective efficacy of the Brucella outer membrane protein 2b (Omp2b) was evaluated in BALB/c mice using Protein/Protein, DNA/DNA and DNA/Protein vaccine strategies. Immunization of mice with three vaccine regimens elicited a strong specific IgG response (higher IgG2a titers over IgG1 titers) and provided Th1-oriented immune response. Vaccination of BALB/c mice with the DNA/Pro regimen induced higher levels of IFN-γ/IL-2 and conferred more protection levels against B. melitenisis and B. abortus challenge than did the protein or DNA alone. In conclusion, Omp2b is able to stimulate specific immune responses and to confer cross protection against B. melitensis and B. abortus infection. Therefore, it could be introduced as a new potential candidate for the development of a subunit vaccine against Brucella infection. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA-based adaptive immunity protect host from infection-associated periodontal bone resorption via recognition of Porphyromonas gingivalis virulence component.

PubMed

Han, Xiaozhe; LaRosa, Karen B; Kawai, Toshihisa; Taubman, Martin A

2014-01-03

Porphyromonas gingivalis (Pg) is one of a constellation of oral organisms associated with human chronic periodontitis. While adaptive immunity to periodontal pathogen proteins has been investigated and is an important component of periodontal bone resorption, the effect of periodontal pathogen DNA in eliciting systemic and mucosal antibody and modulating immune responses has not been investigated. Rowett rats were locally injected with whole genomic Pg DNA in alum. Escherichia coli (Ec) genomic DNA, Fusobacterium nucleatum (Fn) genomic DNA, and saline/alum injected rats served as controls. After various time points, serum IgG and salivary IgA antibody to Ec, Fn or Pg were detected by ELISA. Serum and salivary antibody reactions with Pg surface antigens were determined by Western blot analyses and the specific antigen was identified by mass spectrometry. Effects of genomic DNA immunization on Pg bacterial colonization and experimental periodontal bone resorption were also evaluated. Sera from Pg DNA, Ec DNA and Fn DNA-injected rats did not react with Ec or Fn bacteria. Serum IgG antibody levels to Pg and Pg surface extracts were significantly higher in animals immunized with Pg DNA as compared to the control groups. Rats injected with Pg DNA demonstrated a strong serum IgG and salivary IgA antibody reaction solely to Pg fimbrillin (41kDa), the major protein component of Pg fimbriae. In the Pg DNA-immunized group, the numbers of Pg bacteria in oral cavity and the extent of periodontal bone resorption were significantly reduced after Pg infection. This study suggests that infected hosts may select specific genes from whole genomic DNA of the periodontal pathogen for transcription and presentation. The results indicate that the unique gene selected can initiate a host protective immune response to the parent bacterium. Copyright © 2013 Elsevier Ltd. All rights reserved.

The protective function of noncoding DNA in genome defense of eukaryotic male germ cells.

PubMed

Qiu, Guo-Hua; Huang, Cuiqin; Zheng, Xintian; Yang, Xiaoyan

2018-04-01

Peripheral and abundant noncoding DNA has been hypothesized to protect the genome and the central protein-coding sequences against DNA damage in somatic genome. In the cytosol, invading exogenous nucleic acids may first be deactivated by small RNAs encoded by noncoding DNA via mechanisms similar to the prokaryotic CRISPR-Cas system. In the nucleus, the radicals generated by radiation in the cytosol, radiation energy and invading exogenous nucleic acids are absorbed, blocked and/or reduced by peripheral heterochromatin, and damaged DNA in heterochromatin is removed and excluded from the nucleus to the cytoplasm through nuclear pore complexes. To further strengthen the hypothesis, this review summarizes the experimental evidence supporting the protective function of noncoding DNA in the genome of male germ cells. Based on these data, this review provides evidence supporting the protective role of noncoding DNA in the genome defense of sperm genome through similar mechanisms to those of the somatic genome.

Protective activity of Hertia cheirifolia extracts against DNA damage, lipid peroxidation and protein oxidation.

PubMed

Kada, Seoussen; Bouriche, Hamama; Senator, Abderrahmane; Demirtaş, Ibrahim; Özen, Tevfik; Çeken Toptanci, Bircan; Kızıl, Göksel; Kızıl, Murat

2017-12-01

Hertia cheirifolia L. (Asteraceae), a perennial shrub widely distributed in Northern Africa, is traditionally used to treat inflammatory disorders. The protective effect of methanol (Met E) and aqueous (Aq E) extracts of Hertia cheirifolia against DNA, lipid and protein oxidation was investigated. Different concentrations (50-1000 μg/mL) of Hertia cheirifolia aerial part extracts were examined against DNA, lipid and protein oxidation induced by H 2 O 2  + UV, FeSO 4 , and Fe 3+ /H 2 O 2 -ascorbic acid, respectively. The DPPH • , metal ion chelating, reducing power and β-carotene bleaching tests were conducted. Both extracts were rich in polyphenols, flavonoids and tannins, and were able to scavenge DPPH • with IC 50 values of 138 and 197 μg/mL, respectively. At 300 μg/mL, Aq E exerted stronger chelating effect (99%) than Met E (69%). However, Met E reducing power (IC 50  =   61 μg/mL) was more than that of Aq E (IC 50  =   193 μg/mL). Both extracts protected from β-carotene bleaching by 74% and 94%, respectively, and inhibited linoleic acid peroxidation. The inhibitory activity of Aq E extract (64%) was twice more than that of Met E (32%). Interestingly, both extracts protected DNA against the cleavage by about 96-98%. At 1 mg/mL, Met E and Aq E restored protein band intensity by 94-99%. Hertia cheirifolia exhibits potent antioxidant activity and protects biomolecules against oxidative damage; hence, it may serve as potential source of natural antioxidant for pharmaceutical applications and food preservation. This is the first report on the protective activity of this plant against biomolecule oxidation.

Identification of Western Equine Encephalitis Virus Structural Proteins That Confer Protection after DNA Vaccination▿

PubMed Central

Gauci, Penelope J.; Wu, Josh Q. H.; Rayner, George A.; Barabé, Nicole D.; Nagata, Leslie P.; Proll, David F.

2010-01-01

DNA vaccines encoding different portions of the structural proteins of western equine encephalitis virus were tested for the efficacy of their protection in a 100% lethal mouse model of the virus. The 6K-E1 structural protein encoded by the DNA vaccine conferred complete protection against challenge with the homologous strain and limited protection against challenge with a heterologous strain. PMID:19923571

Binding characteristics and protective capacity of cyanidin-3-glucoside and its aglycon to calf thymus DNA.

PubMed

Zhang, Chao; Guo, Xiaofei; Cai, Wenqian; Ma, Yue; Zhao, Xiaoyan

2015-04-01

The binding characteristics and protective capacity of cyanidin (Cy) and cyanidin-3-glucoside (C3G) to calf thymus DNA were explored for the first time. The Cy and C3G gave a bathochromic shift to the ultraviolet-visible spectra of the DNA, indicating the formation of the DNA-Cy and DNA-C3G complexes. The complexes were formed by an intercalative binding mode based on the results of the fluorescence spectra and competitive binding analysis. Meanwhile, the Cy and C3G protected the DNA from the damage induced by the hydroxyl radical. The binding capacity and protective capacity of the C3G were stronger than that of the Cy. Furthermore, the formation of the DNA-anthocyanin complexes was spontaneous when the hydrogen bond and hydrophobic force played a key role. Hence, the Cy and C3G could protect the DNA automatically from the damage induced by the hydroxyl radical. © 2015 Institute of Food Technologists®

A single portion of blueberry (Vaccinium corymbosum L) improves protection against DNA damage but not vascular function in healthy male volunteers.

PubMed

Del Bó, Cristian; Riso, Patrizia; Campolo, Jonica; Møller, Peter; Loft, Steffen; Klimis-Zacas, Dorothy; Brambilla, Ada; Rizzolo, Anna; Porrini, Marisa

2013-03-01

It has been suggested that anthocyanin-rich foods may exert antioxidant effects and improve vascular function as demonstrated mainly in vitro and in the animal model. Blueberries are rich sources of anthocyanins and we hypothesized that their intake could improve cell protection against oxidative stress and affect endothelial function in humans. The aim of the study was to investigate the effect of one portion (300 g) of blueberries on selected markers of oxidative stress and antioxidant protection (endogenous and oxidatively induced DNA damage) and of vascular function (changes in peripheral arterial tone and plasma nitric oxide levels) in male subjects. In a randomized cross-over design, separated by a wash out period ten young volunteers received one portion of blueberries ground by blender or one portion of a control jelly. Before and after consumption (at 1, 2, and 24 hours), blood samples were collected and used to evaluate anthocyanin absorption (through mass spectrometry), endogenous and H(2)O(2)-induced DNA damage in blood mononuclear cells (through the comet assay), and plasma nitric oxide concentrations (through a fluorometric assay). Peripheral arterial function was assessed by means of Endo-PAT 2000. Blueberries significantly reduced (P < .01) H(2)O(2)-induced DNA damage (-18%) 1 hour after blueberry consumption compared to control. No significant differences were observed for endogenous DNA damage, peripheral arterial function and nitric oxide levels after blueberry intake. In conclusion, one portion of blueberries seems sufficient to improve cell antioxidant defense against DNA damage, but further studies are necessary to understand their role on vascular function. Copyright © 2013 Elsevier Inc. All rights reserved.

Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice.

PubMed

Campos-Neto, A; Webb, J R; Greeson, K; Coler, R N; Skeiky, Y A W; Reed, S G

2002-06-01

We have recently shown that a cocktail containing two leishmanial recombinant antigens (LmSTI1 and TSA) and interleukin-12 (IL-12) as an adjuvant induces solid protection in both a murine and a nonhuman primate model of cutaneous leishmaniasis. However, because IL-12 is difficult to prepare, is expensive, and does not have the stability required for a vaccine product, we have investigated the possibility of using DNA as an alternative means of inducing protective immunity. Here, we present evidence that the antigens TSA and LmSTI1 delivered in a plasmid DNA format either as single genes or in a tandem digene construct induce equally solid protection against Leishmania major infection in susceptible BALB/c mice. Immunization of mice with either TSA DNA or LmSTI1 DNA induced specific CD4(+)-T-cell responses of the Th1 phenotype without a requirement for specific adjuvant. CD8 responses, as measured by cytotoxic-T-lymphocyte activity, were generated after immunization with TSA DNA but not LmSTI1 DNA. Interestingly, vaccination of mice with TSA DNA consistently induced protection to a much greater extent than LmSTI1 DNA, thus supporting the notion that CD8 responses might be an important accessory arm of the immune response for acquired resistance against leishmaniasis. Moreover, the protection induced by DNA immunization was specific for infection with Leishmania, i.e., the immunization had no effect on the course of infection of the mice challenged with an unrelated intracellular pathogen such as Mycobacterium tuberculosis. Conversely, immunization of BALB/c mice with a plasmid DNA that is protective against challenge with M. tuberculosis had no effect on the course of infection of these mice with L. major. Together, these results indicate that the protection observed with the leishmanial DNA is mediated by acquired specific immune response rather than by the activation of nonspecific innate immune mechanisms. In addition, a plasmid DNA containing a fusion construct

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity.

PubMed

Eickhoff, Christopher S; Zhang, Xiuli; Vasconcelos, Jose R; Motz, R Geoffrey; Sullivan, Nicole L; O'Shea, Kelly; Pozzi, Nicola; Gohara, David W; Blase, Jennifer R; Di Cera, Enrico; Hoft, Daniel F

2016-09-01

Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4+ and CD8+ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8+ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8+ T cell epitope enhance subdominant pathogen-specific CD8+ T cell responses. More specifically, CD8+ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8+ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T

In vitro antioxidant properties, DNA damage protective activity, and xanthine oxidase inhibitory effect of cajaninstilbene acid, a stilbene compound derived from pigeon pea [Cajanus cajan (L.) Millsp.] leaves.

PubMed

Wu, Nan; Kong, Yu; Fu, Yujie; Zu, Yuangang; Yang, Zhiwei; Yang, Mei; Peng, Xiao; Efferth, Thomas

2011-01-12

The antioxidant properties, DNA damage protective activities, and xanthine oxidase (XOD) inhibitory effect of cajaninstilbene acid (CSA) derived from pigeon pea leaves were studied in the present work. Compared with resveratrol, CSA showed stronger antioxidant properties, DNA damage protective activity, and XOD inhibition activity. The IC(50) values of CSA for superoxide radical scavenging, hydroxyl radical scavenging, nitric oxide scavenging, reducing power, lipid peroxidation, and XOD inhibition were 19.03, 6.36, 39.65, 20.41, 20.58, and 3.62 μM, respectively. CSA possessed good protective activity from oxidative DNA damage. Furthermore, molecular docking indicated that CSA was more potent than resveratrol or allopurinol to interact with the active site of XOD (calculated free binding energy: -229.71 kcal mol(-1)). On the basis of the results, we conclude that CSA represents a valuable natural antioxidant source and may potentially be applicable in health food industry.

Frying oils with high natural or added antioxidants content, which protect against postprandial oxidative stress, also protect against DNA oxidation damage.

PubMed

Rangel-Zuñiga, Oriol A; Haro, Carmen; Tormos, Carmen; Perez-Martinez, Pablo; Delgado-Lista, Javier; Marin, Carmen; Quintana-Navarro, Gracia M; Cerdá, Concha; Sáez, Guillermo T; Lopez-Segura, Fernando; Lopez-Miranda, Jose; Perez-Jimenez, Francisco; Camargo, Antonio

2017-06-01

Using sunflower oil as frying oil increases postprandial oxidative stress, which is considered the main endogenous source of DNA oxidative damage. We aimed to test whether the protective effect of virgin olive oil and oil models with added antioxidants against postprandial oxidative stress may also protect against DNA oxidative damage. Twenty obese people received four breakfasts following a randomized crossover design consisting of different oils [virgin olive oil (VOO), sunflower oil (SFO), and a mixed seed oil (SFO/canola oil) with added dimethylpolysiloxane (SOX) or natural antioxidants from olives (SOP)], which were subjected to 20 heating cycles. We observed the postprandial increase in the mRNA levels of p53, OGG1, POLB, and GADD45b after the intake of the breakfast prepared with SFO and SOX, and an increase in the expression of MDM2, APEX1, and XPC after the intake of the breakfast prepared with SFO, whereas no significant changes at the postprandial state were observed after the intake of the other breakfasts (all p values <0.05). We observed lower 8-OHdG postprandial levels after the intake of the breakfast prepared with VOO and SOP than after the intake of the breakfast prepared with SFO and SOX (all p values <0.05). Our results support the beneficial effect on DNA oxidation damage of virgin olive oil and the oil models with added antioxidants, as compared to the detrimental use of sunflower oil, which induces p53-dependent DNA repair pathway activation.

Protective efficacy of a Treponema pallidum Gpd DNA vaccine vectored by chitosan nanoparticles and fused with interleukin-2.

PubMed

Zhao, Feijun; Wang, Shiping; Zhang, Xiaohong; Gu, Weiming; Yu, Jian; Liu, Shuangquan; Zeng, Tiebing; Zhang, Yuejun; Wu, Yimou

2012-02-01

In the present study, immunomodulatory responses of a DNA vaccine constructed by fusing Treponema pallidum (Tp) glycerophosphodiester phosphodiesterase (Gpd) to interleukin-2 (IL-2) and using chitosan (CS) nanoparticles as vectors were investigated. New Zealand white rabbits were immunized by intramuscular inoculation of control DNAs, Tp Gpd DNA vaccine, or Gpd-IL-2 fusion DNA vaccine, which were vectored by CS nanoparticles. Levels of the anti-Gpd antibodies and levels of IL-2 and interferon-γ in rabbits were increased upon inoculation of Gpd-IL-2 fusion DNA vaccine, when compared with the inoculation with Gpd DNA vaccine, with CS vectoring increasing the effects. The Gpd-IL-2 fusion DNA vaccine efficiently enhanced the antigen-specific lymphocyte proliferative response. When the rabbits were challenged intradermally with 10(5) Tp (Nichols) spirochetes, the Gpd-IL-2 fusion DNA vaccine conferred better protection than the Gpd DNA vaccine (P < 0.05), as characterized by lower detectable amounts of dark field positive lesions (17.5%), lower ulcerative lesion scores (15%), and faster recovery. Individuals treated with the Tp Gpd-IL-2 fusion DNA vaccine vectored by CS nanoparticles had the lowest amounts of dark field positive lesions (10%) and ulcerations (5%) observed and the fastest recovery (42 days). These results indicate that the Gpd-IL-2 fusion DNA vaccine vectored by CS nanoparticles can efficiently induce Th1-dominant immune responses, improve protective efficacy against Tp spirochete infection, and effectively attenuate development of syphilitic lesions.

Pseudomonas aeruginosa phage PaP1 DNA polymerase is an A-family DNA polymerase demonstrating ssDNA and dsDNA 3'-5' exonuclease activity.

PubMed

Liu, Binyan; Gu, Shiling; Liang, Nengsong; Xiong, Mei; Xue, Qizhen; Lu, Shuguang; Hu, Fuquan; Zhang, Huidong

2016-08-01

Most phages contain DNA polymerases, which are essential for DNA replication and propagation in infected host bacteria. However, our knowledge on phage-encoded DNA polymerases remains limited. This study investigated the function of a novel DNA polymerase of PaP1, which is the lytic phage of Pseudomonas aeruginosa. PaP1 encodes its sole DNA polymerase called Gp90 that was predicted as an A-family DNA polymerase with polymerase and 3'-5' exonuclease activities. The sequence of Gp90 is homologous but not identical to that of other A-family DNA polymerases, such as T7 DNA polymerases (Pol) and DNA Pol I. The purified Gp90 demonstrated a polymerase activity. The processivity of Gp90 in DNA replication and its efficiency in single-dNTP incorporation are similar to those of T7 Pol with processive thioredoxin (T7 Pol/trx). Gp90 can degrade ssDNA and dsDNA in 3'-5' direction at a similar rate, which is considerably lower than that of T7 Pol/trx. The optimized conditions for polymerization were a temperature of 37 °C and a buffer consisting of 40 mM Tris-HCl (pH 8.0), 30 mM MgCl2, and 200 mM NaCl. These studies on DNA polymerase encoded by PaP1 help advance our knowledge on phage-encoded DNA polymerases and elucidate PaP1 propagation in infected P. aeruginosa.

DNA vaccine-derived human IgG produced in transchromosomal bovines protect in lethal models of hantavirus pulmonary syndrome.

PubMed

Hooper, Jay W; Brocato, Rebecca L; Kwilas, Steven A; Hammerbeck, Christopher D; Josleyn, Matthew D; Royals, Michael; Ballantyne, John; Wu, Hua; Jiao, Jin-an; Matsushita, Hiroaki; Sullivan, Eddie J

2014-11-26

Polyclonal immunoglobulin-based medical products have been used successfully to treat diseases caused by viruses for more than a century. We demonstrate the use of DNA vaccine technology and transchromosomal bovines (TcBs) to produce fully human polyclonal immunoglobulins (IgG) with potent antiviral neutralizing activity. Specifically, two hantavirus DNA vaccines [Andes virus (ANDV) DNA vaccine and Sin Nombre virus (SNV) DNA vaccine] were used to produce a candidate immunoglobulin product for the prevention and treatment of hantavirus pulmonary syndrome (HPS). A needle-free jet injection device was used to vaccinate TcB, and high-titer neutralizing antibodies (titers >1000) against both viruses were produced within 1 month. Plasma collected at day 10 after the fourth vaccination was used to produce purified α-HPS TcB human IgG. Treatment with 20,000 neutralizing antibody units (NAU)/kg starting 5 days after challenge with ANDV protected seven of eight animals, whereas zero of eight animals treated with the same dose of normal TcB human IgG survived. Likewise, treatment with 20,000 NAU/kg starting 5 days after challenge with SNV protected immunocompromised hamsters from lethal HPS, protecting five of eight animals. Our findings that the α-HPS TcB human IgG is capable of protecting in animal models of lethal HPS when administered after exposure provides proof of concept that this approach can be used to develop candidate next-generation polyclonal immunoglobulin-based medical products without the need for human donors, despeciation protocols, or inactivated/attenuated vaccine antigen. Copyright © 2014, American Association for the Advancement of Science.

Yeast aconitase binds and provides metabolically coupled protection to mitochondrial DNA.

PubMed

Chen, Xin Jie; Wang, Xiaowen; Butow, Ronald A

2007-08-21

Aconitase (Aco1p) is a multifunctional protein: It is an enzyme of the tricarboxylic acid cycle. In animal cells, Aco1p also is a cytosolic protein binding to mRNAs to regulate iron metabolism. In yeast, Aco1p was identified as a component of mtDNA nucleoids. Here we show that yeast Aco1p protects mtDNA from excessive accumulation of point mutations and ssDNA breaks and suppresses reductive recombination of mtDNA. Aconitase binds to both ds- and ssDNA, with a preference for GC-containing sequences. Therefore, mitochondria are opportunistic organelles that seize proteins, such as metabolic enzymes, for construction of the nucleoid, an mtDNA maintenance/segregation apparatus.

Heterogeneity in the A33 protein impacts the cross-protective efficacy of a candidate smallpox DNA vaccine.

PubMed

Golden, Joseph W; Hooper, Jay W

2008-07-20

We previously developed a gene-based vaccine, termed 4pox, which targets four orthopoxvirus proteins (A33, L1, B5, and A27). Because any subunit orthopoxvirus vaccine must protect against multiple species of orthopoxviruses, we are interested in understanding the cross-protective potential of our 4pox vaccine target immunogens. In our current studies, we focused on the A33 immunogen. We found one monoclonal antibody against A33, MAb-1G10, which could not bind the monkeypox virus A33 ortholog, A35. MAb-1G10 binding could be rescued if A35 amino acids 118 and 120 were substituted with those from A33. MAb-1G10 has been shown to protect mice from VACV challenge, thus our findings indicated a protective epitope differs among orthopoxviruses. Accordingly, we tested the cross-protective efficacy of a DNA vaccine consisting of A35R against VACV challenge and compared it to vaccination with A33R DNA. Mice vaccinated with A35R had greater mortality and more weight loss compared to those vaccinated with A33R. These findings demonstrate that despite high homology between A33R orthologs, amino acid differences can impact cross-protection. Furthermore, our results caution that adequate cross-protection by any pan-orthopoxvirus subunit vaccine will require not only careful evaluation of cross-protective immunity, but also of targeting of multiple orthopoxvirus immunogens.

Forensic identification of CITES protected slimming cactus (Hoodia) using DNA barcoding.

PubMed

Gathier, Gerard; van der Niet, Timotheus; Peelen, Tamara; van Vugt, Rogier R; Eurlings, Marcel C M; Gravendeel, Barbara

2013-11-01

Slimming cactus (Hoodia), found only in southwestern Africa, is a well-known herbal product for losing weight. Consequently, Hoodia extracts are sought-after worldwide despite a CITES Appendix II status. The failure to eradicate illegal trade is due to problems with detecting and identifying Hoodia using morphological and chemical characters. Our aim was to evaluate the potential of molecular identification of Hoodia based on DNA barcoding. Screening of nrITS1 and psbA-trnH DNA sequences from 26 accessions of Ceropegieae resulted in successful identification, while conventional chemical profiling using DLI-MS led to inaccurate detection and identification of Hoodia. The presence of Hoodia in herbal products was also successfully established using DNA sequences. A validation procedure of our DNA barcoding protocol demonstrated its robustness to changes in PCR conditions. We conclude that DNA barcoding is an effective tool for Hoodia detection and identification which can contribute to preventing illegal trade. © 2013 American Academy of Forensic Sciences.

Demonstrating Interactions of Transcription Factors with DNA by Electrophoretic Mobility Shift Assay.

PubMed

Yousaf, Nasim; Gould, David

2017-01-01

Confirming the binding of a transcription factor with a particular DNA sequence may be important in characterizing interactions with a synthetic promoter. Electrophoretic mobility shift assay is a powerful approach to demonstrate the specific DNA sequence that is bound by a transcription factor and also to confirm the specific transcription factor involved in the interaction. In this chapter we describe a method we have successfully used to demonstrate interactions of endogenous transcription factors with sequences derived from endogenous and synthetic promoters.

Oral Vaccination with a DNA Vaccine Encoding Capsid Protein of Duck Tembusu Virus Induces Protection Immunity

PubMed Central

Shen, Haoyue; Jia, Renyong; Wang, Mingshu; Chen, Shun; Zhu, Dekang; Liu, Mafeng; Zhao, Xinxin; Yang, Qiao; Wu, Ying; Liu, Yunya; Zhang, Ling; Yin, Zhongqiong; Jing, Bo

2018-01-01

The emergence of duck tembusu virus (DTMUV), a new member of the Flavivirus genus, has caused great economical loss in the poultry industry in China. Since the outbreak and spread of DTMUV is hard to control in a clinical setting, an efficient and low-cost oral delivery DNA vaccine SL7207 (pVAX1-C) based on the capsid protein of DTMUV was developed and evaluated in this study. The antigen capsid protein was expressed from the DNA vaccine SL7207 (pVAX1-C), both in vitro and in vivo. The humoral and cellular immune responses in vivo were observed after oral immunization with the SL7207 (pVAX1-C) DNA vaccine. High titers of the specific antibody against the capsid protein and the neutralizing antibody against the DTMUV virus were both detected after inoculation. The ducks were efficiently protected from lethal DTMUV exposure by the SL7207 (pVAX1-C) vaccine in this experiment. Taken together, we demonstrated that the capsid protein of DTMUV possesses a strong immunogenicity against the DTMUV infection. Moreover, an oral delivery of the DNA vaccine SL7207 (pVAX1-C) utilizing Salmonella SL7207 was an efficient way to protect the ducks against DTMUV infection and provides an economic and fast vaccine delivery strategy for a large scale clinical use. PMID:29642401

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. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Protective Mechanism of Visible Red Light in Normal Human Dermal Fibroblasts: Enhancement of GADD45A-Mediated DNA Repair Activity.

PubMed

Kim, Yeo Jin; Kim, Hyoung-June; Kim, Hye Lim; Kim, Hyo Jeong; Kim, Hyun Soo; Lee, Tae Ryong; Shin, Dong Wook; Seo, Young Rok

2017-02-01

The phototherapeutic effects of visible red light on skin have been extensively investigated, but the underlying biological mechanisms remain poorly understood. We aimed to elucidate the protective mechanism of visible red light in terms of DNA repair of UV-induced oxidative damage in normal human dermal fibroblasts. The protective effect of visible red light on UV-induced DNA damage was identified by several assays in both two-dimensional and three-dimensional cell culture systems. With regard to the protective mechanism of visible red light, our data showed alterations in base excision repair mediated by growth arrest and DNA damage inducible, alpha (GADD45A). We also observed an enhancement of the physical activity of GADD45A and apurinic/apyrimidinic endonuclease 1 (APE1) by visible red light. Moreover, UV-induced DNA damages were diminished by visible red light in an APE1-dependent manner. On the basis of the decrease in GADD45A-APE1 interaction in the activating transcription factor-2 (ATF2)-knockdown system, we suggest a role for ATF2 modulation in GADD45A-mediated DNA repair upon visible red light exposure. Thus, the enhancement of GADD45A-mediated base excision repair modulated by ATF2 might be a potential protective mechanism of visible red light. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Loss of PUMA protects the ovarian reserve during DNA-damaging chemotherapy and preserves fertility.

PubMed

Nguyen, Quynh-Nhu; Zerafa, Nadeen; Liew, Seng H; Morgan, F Hamish; Strasser, Andreas; Scott, Clare L; Findlay, Jock K; Hickey, Martha; Hutt, Karla J

2018-05-23

Female gametes are stored in the ovary in structures called primordial follicles, the supply of which is non-renewable. It is well established that DNA-damaging cancer treatments can deplete the ovarian reserve of primordial follicles, causing premature ovarian failure and infertility. The precise mechanisms underlying this chemotherapy-driven follicle loss are unclear, and this has limited the development of targeted ovarian-protective agents. To address this fundamental knowledge gap, we used gene deletion mouse models to examine the role of the DNA damage-induced pro-apoptotic protein, PUMA, and its transcriptional activator TAp63, in primordial follicle depletion caused by treatment with cyclophosphamide or cisplatin. Cyclophosphamide caused almost complete destruction of the primordial follicle pool in adult wild-type (WT) mice, and a significant destructive effect was also observed for cisplatin. In striking contrast, Puma -/- mice retained 100% of their primordial follicles following either genotoxic treatment. Furthermore, elimination of PUMA alone completely preserved fertility in cyclophosphamide-treated mice, indicating that oocytes rescued from DNA damage-induced death can repair themselves sufficiently to support reproductive function and offspring health. Primordial follicles were also protected in TAp63 -/- mice following cisplatin treatment, but not cyclophosphamide, suggesting mechanistic differences in the induction of apoptosis and depletion of the ovarian reserve in response to these different chemotherapies. These studies identify PUMA as a crucial effector of apoptosis responsible for depletion of primordial follicles following exposure to cyclophosphamide or cisplatin, and this indicates that inhibition of PUMA may be an effective ovarian-protective strategy during cancer treatment in women.

Effects of different extenders on DNA integrity of boar spermatozoa following freezing-thawing.

PubMed

Hu, Jian-hong; Li, Qing-wang; Jiang, Zhong-liang; Li, Wen-ye

2008-12-01

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing-thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P<0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100mM trehalose, but cryopreservation could increase the degree of DNA damage (P<0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.

Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Juanjuan; Zhang, Yu; Xu, Wentao, E-mail: xuwentaoboy@sina.com

Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did notmore » affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ{sub m}). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers

PubMed Central

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-01-01

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks. PMID:26563448

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers.

PubMed

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-11-13

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks.

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection

PubMed Central

Abeyta, Antonio; Castella, Maria; Jacquemont, Celine; Taniguchi, Toshiyasu

2017-01-01

ABSTRACT Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51. PMID:27892797

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection.

PubMed

Abeyta, Antonio; Castella, Maria; Jacquemont, Celine; Taniguchi, Toshiyasu

2017-02-16

Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51.

A pre-protective strategy for precise tumor targeting and efficient photodynamic therapy with a switchable DNA/upconversion nanocomposite.

PubMed

Yu, Zhengze; Ge, Yegang; Sun, Qiaoqiao; Pan, Wei; Wan, Xiuyan; Li, Na; Tang, Bo

2018-04-14

Tumor-specific targeting based on folic acid (FA) is one of the most common and significant approaches in cancer therapy. However, the expression of folate receptors (FRs) in normal tissues will lead to unexpected targeting and unsatisfactory therapeutic effect. To address this issue, we develop a pre-protective strategy for precise tumor targeting and efficient photodynamic therapy (PDT) using a switchable DNA/upconversion nanocomposite, which can be triggered in the acidic tumor microenvironment. The DNA/upconversion nanocomposite is composed of polyacrylic acid (PAA) coated upconversion nanoparticles (UCNPs), the surface of which is modified using FA and chlorin e6 (Ce6) functionalized DNA sequences with different lengths. Initially, FA on the shorter DNA was protected by a longer DNA to prevent the bonding to FRs on normal cells. Once reaching the acidic tumor microenvironment, C base-rich longer DNA forms a C-quadruplex, resulting in the exposure of the FA groups and the bonding of FA and FRs on cancer cell membranes to achieve precise targeting. Simultaneously, the photosensitizer chlorin e6 (Ce6) gets close to the surface of UCNPs, enabling the excitation of Ce6 to generate singlet oxygen ( 1 O 2 ) under near infrared light via Förster resonance energy transfer (FRET). In vivo experiments indicated that higher tumor targeting efficiency was achieved and the tumor growth was greatly inhibited through the pre-protective strategy.

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. Copyright © 2012 Elsevier Ltd. All rights reserved.

Protective effect of DNA-mediated immunization with liposome-encapsulated GRA4 against infection of Toxoplasma gondii *

PubMed Central

Chen, Rui; Lu, Shao-hong; Tong, Qun-bo; Lou, Di; Shi, Dong-yan; Jia, Bing-bing; Huang, Guo-ping; Wang, Jin-fu

2009-01-01

The dense granule protein 4 (GRA4) is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3.1-GRA4 (pGRA4), encoding for the GRA4 antigen, was incorporated by the dehydration-rehydration method into liposomes composed of 16 mmol/L egg phosphatidylcholine (PC), 8 mmol/L dioleoyl phosphatidylethanolamine (DOPE), and 4 mmol/L 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP). C57BL/6 mice and BALB/c mice were immunized intramuscularly three times with liposome-encapsulated pGRA4 to determine whether DNA immunization could elicit a protective immune response to T. gondii. Enzyme-linked immunosorbent assay (ELISA) of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4. Production of primary interferon (IFN)-γ and interleukin (IL)-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th1-type response. 72.7% of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived the challenge with 80 tissue cysts of ME49 strain, whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%. When immunized BALB/c mice were intraperitoneally challenged with 103 tachyzoites of the highly virulent RH strain, the survival time of mice immunized with liposome-encapsulated pGRA4 was markedly longer than that of other groups. Our observations show that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T. gondii. PMID:19585669

A DNA Vaccine That Targets Hemagglutinin to Antigen-Presenting Cells Protects Mice against H7 Influenza

PubMed Central

Andersen, Tor Kristian; Zhou, Fan; Cox, Rebecca; Bogen, Bjarne

2017-01-01

ABSTRACT Zoonotic influenza H7 viral infections have a case fatality rate of about 40%. Currently, no or limited human to human spread has occurred, but we may be facing a severe pandemic threat if the virus acquires the ability to transmit between humans. Novel vaccines that can be rapidly produced for global distribution are urgently needed, and DNA vaccines may be the only type of vaccine that allows for the speed necessary to quench an emerging pandemic. Here, we constructed DNA vaccines encoding the hemagglutinin (HA) from influenza A/chicken/Italy/13474/99 (H7N1). In order to increase the efficacy of DNA vaccination, HA was targeted to either major histocompatibility complex class II molecules or chemokine receptors 1, 3, and 5 (CCR1/3/5) that are expressed on antigen-presenting cells (APC). A single DNA vaccination with APC-targeted HA significantly increased antibody levels in sera compared to nontargeted control vaccines. The antibodies were confirmed neutralizing in an H7 pseudotype-based neutralization assay. Furthermore, the APC-targeted vaccines increased the levels of antigen-specific cytotoxic T cells, and a single DNA vaccination could confer protection against a lethal challenge with influenza A/turkey/Italy/3889/1999 (H7N1) in mice. In conclusion, we have developed a vaccine that rapidly could contribute protection against a pandemic threat from avian influenza. IMPORTANCE Highly pathogenic avian influenza H7 constitute a pandemic threat that can cause severe illness and death in infected individuals. Vaccination is the main method of prophylaxis against influenza, but current vaccine strategies fall short in a pandemic situation due to a prolonged production time and insufficient production capabilities. In contrast, a DNA vaccine can be rapidly produced and deployed to prevent the potential escalation of a highly pathogenic influenza pandemic. We here demonstrate that a single DNA delivery of hemagglutinin from an H7 influenza could mediate full

DNA vaccines elicit durable protective immunity against individual or simultaneous infections with Lassa and Ebola viruses in guinea pigs.

PubMed

Cashman, Kathleen A; Wilkinson, Eric R; Wollen, Suzanne E; Shamblin, Joshua D; Zelko, Justine M; Bearss, Jeremy J; Zeng, Xiankun; Broderick, Kate E; Schmaljohn, Connie S

2017-12-02

We previously developed optimized DNA vaccines against both Lassa fever and Ebola hemorrhagic fever viruses and demonstrated that they were protective individually in guinea pig and nonhuman primate models. In this study, we vaccinated groups of strain 13 guinea pigs two times, four weeks apart with 50 µg of each DNA vaccine or a mock vaccine at discrete sites by intradermal electroporation. Five weeks following the second vaccinations, guinea pigs were exposed to lethal doses of Lassa virus, Ebola virus, or a combination of both viruses simultaneously. None of the vaccinated guinea pigs, regardless of challenge virus and including the coinfected group, displayed weight loss, fever or other disease signs, and all survived to the study endpoint. All of the mock-vaccinated guinea pigs that were infected with Lassa virus, and all but one of the EBOV-infected mock-vaccinated guinea pigs succumbed. In order to determine if the dual-agent vaccination strategy could protect against both viruses if exposures were temporally separated, we held the surviving vaccinates in BSL-4 for approximately 120 days to perform a cross-challenge experiment in which guinea pigs originally infected with Lassa virus received a lethal dose of Ebola virus and those originally infected with Ebola virus were infected with a lethal dose of Lassa virus. All guinea pigs remained healthy and survived to the study endpoint. This study clearly demonstrates that DNA vaccines against Lassa and Ebola viruses can elicit protective immunity against both individual virus exposures as well as in a mixed-infection environment.

DNA vaccines elicit durable protective immunity against individual or simultaneous infections with Lassa and Ebola viruses in guinea pigs

PubMed Central

Cashman, Kathleen A.; Wilkinson, Eric R.; Wollen, Suzanne E.; Shamblin, Joshua D.; Zelko, Justine M.; Bearss, Jeremy J.; Zeng, Xiankun; Broderick, Kate E.; Schmaljohn, Connie S.

2017-01-01

ABSTRACT We previously developed optimized DNA vaccines against both Lassa fever and Ebola hemorrhagic fever viruses and demonstrated that they were protective individually in guinea pig and nonhuman primate models. In this study, we vaccinated groups of strain 13 guinea pigs two times, four weeks apart with 50 µg of each DNA vaccine or a mock vaccine at discrete sites by intradermal electroporation. Five weeks following the second vaccinations, guinea pigs were exposed to lethal doses of Lassa virus, Ebola virus, or a combination of both viruses simultaneously. None of the vaccinated guinea pigs, regardless of challenge virus and including the coinfected group, displayed weight loss, fever or other disease signs, and all survived to the study endpoint. All of the mock-vaccinated guinea pigs that were infected with Lassa virus, and all but one of the EBOV-infected mock-vaccinated guinea pigs succumbed. In order to determine if the dual-agent vaccination strategy could protect against both viruses if exposures were temporally separated, we held the surviving vaccinates in BSL-4 for approximately 120 days to perform a cross-challenge experiment in which guinea pigs originally infected with Lassa virus received a lethal dose of Ebola virus and those originally infected with Ebola virus were infected with a lethal dose of Lassa virus. All guinea pigs remained healthy and survived to the study endpoint. This study clearly demonstrates that DNA vaccines against Lassa and Ebola viruses can elicit protective immunity against both individual virus exposures as well as in a mixed-infection environment. PMID:29135337

Plasmid DNA Delivery: Nanotopography Matters.

PubMed

Song, Hao; Yu, Meihua; Lu, Yao; Gu, Zhengying; Yang, Yannan; Zhang, Min; Fu, Jianye; Yu, Chengzhong

2017-12-20

Plasmid DNA molecules with unique loop structures have widespread bioapplications, in many cases relying heavily on delivery vehicles to introduce them into cells and achieve their functions. Herein, we demonstrate that control over delicate nanotopography of silica nanoparticles as plasmid DNA vectors has significant impact on the transfection efficacy. For silica nanoparticles with rambutan-, raspberry-, and flower-like morphologies composed of spike-, hemisphere-, and bowl-type subunit nanotopographies, respectively, the rambutan-like nanoparticles with spiky surfaces demonstrate the highest plasmid DNA binding capability and transfection efficacy of 88%, higher than those reported for silica-based nanovectors. Moreover, it is shown that the surface spikes of rambutan nanoparticles provide a continuous open space to bind DNA chains via multivalent interactions and protect the gene molecules sheltered in the spiky layer against nuclease degradation, exhibiting no significant transfection decay. This unique protection feature is in great contrast to a commercial transfection agent with similar transfection performance but poor protection capability against enzymatic cleavage. Our study provides new understandings in the rational design of nonviral vectors for efficient gene delivery.

Population connectivity and the effectiveness of marine protected areas to protect vulnerable, exploited and endemic coral reef fishes at an endemic hotspot

NASA Astrophysics Data System (ADS)

van der Meer, M. H.; Berumen, M. L.; Hobbs, J.-P. A.; van Herwerden, L.

2015-06-01

Marine protected areas (MPAs) aim to mitigate anthropogenic impacts by conserving biodiversity and preventing overfishing. The effectiveness of MPAs depends on population connectivity patterns between protected and non-protected areas. Remote islands are endemism hotspots for coral reef fishes and provide rare examples of coral reefs with limited fishing pressure. This study explored population genetic connectivity across a network of protected and non-protected areas for the endemic wrasse, Coris bulbifrons, which is listed as "vulnerable" by the IUCN due to its small, decreasing geographic range and declining abundance. Mitochondrial DNA (mtDNA) and microsatellite DNA (msatDNA) markers were used to estimate historic and contemporary gene flow to determine the level of population self-replenishment and to measure genetic and genotypic diversity among all four locations in the species range (south-west Pacific Ocean)—Middleton Reef (MR), Elizabeth Reef (ER), Lord Howe Island (LHI) and Norfolk Island (NI). MPAs exist at MR and LHI and are limited or non-existent at ER and NI, respectively. There was no obvious differentiation in mtDNA among locations, however, msatDNA revealed differentiation between the most peripheral (NI) and all remaining locations (MR, ER and LHI). Despite high mtDNA connectivity ( M = 259-1,144), msatDNA connectivity was limited ( M = 3-9) with high self-replenishment (68-93 %) at all locations. NI is the least connected and heavily reliant on self-replenishment, and the absence of MPAs at NI needs to be rectified to ensure the persistence of endemic species at this location. Other endemic fishes exhibit similar patterns of high self-replenishment across the four locations, indicating that a single spatial management approach consisting of a MPA network protecting part of each location could provide reasonable protection for these species. Thus, the existing network of MPAs at this endemic hotspot appears adequate at some locations, but not

Chemotherapy-induced bystander effect in response to several chloroethylnitrosoureas: an origin independent of DNA damage?

PubMed

Merle, Patrick; Morvan, Daniel; Caillaud, Denis; Demidem, Aicha

2008-01-01

Chloroethylnitrosourea (CENU) chemotherapy is used for the treatment of melanoma tumors. The main mechanism of action of this anticancer agent is via DNA damage. We recently showed in murine experiments using a parental double B16 melanoma tumor model that, after treatment of primary tumors with cystemustine (CENU agent), untreated secondary tumors exhibited growth inhibition and metabolism disorders. The response of secondary untreated tumor was called the chemotherapy-induced bystander effect. To see whether chemotherapy-induced bystander effects were induced with other members of the CENU family, we compared three CENU(s) used in melanoma treatment: cystemustine, carmustine and fotemustine. Our results demonstrate that fotemustine, like cystemustine, but not carmustine induced a protective effect against secondary untreated tumors including alterations in phospholipid derivative and glutathione which are the metabolic signature of the bystander effect. From these data we may conclude that DNA damage to the primary tumor is not sufficient to explain chemotherapy-induced bystander effects.

Protective effects of pulmonary epithelial lining fluid on oxidative stress and DNA single-strand breaks caused by ultrafine carbon black, ferrous sulphate and organic extract of diesel exhaust particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, Hsiao-Chi; Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan; Cheng, Yi-Ling

Pulmonary epithelial lining fluid (ELF) is the first substance to make contact with inhaled particulate matter (PM) and interacts chemically with PM components. The objective of this study was to determine the role of ELF in oxidative stress, DNA damage and the production of proinflammatory cytokines following physicochemical exposure to PM. Ultrafine carbon black (ufCB, 15 nm; a model carbonaceous core), ferrous sulphate (FeSO{sub 4}; a model transition metal) and a diesel exhaust particle (DEP) extract (a model organic compound) were used to examine the acellular oxidative potential of synthetic ELF and non-ELF systems. We compared the effects of exposuremore » to ufCB, FeSO{sub 4} and DEP extract on human alveolar epithelial Type II (A549) cells to determine the levels of oxidative stress, DNA single-strand breaks and interleukin-8 (IL-8) production in ELF and non-ELF systems. The effects of ufCB and FeSO{sub 4} on the acellular oxidative potential, cellular oxidative stress and DNA single-strand breakage were mitigated significantly by the addition of ELF, whereas there was no decrease following treatment with the DEP extract. There was no significant effect on IL-8 production following exposure to samples that were suspended in ELF/non-ELF systems. The results of the present study indicate that ELF plays an important role in the initial defence against PM in the pulmonary environment. Experimental components, such as ufCB and FeSO{sub 4}, induced the production of oxidative stress and led to DNA single-strand breaks, which were moderately prevented by the addition of ELF. These findings suggest that ELF plays a protective role against PM-driven oxidative stress and DNA damage. -- Highlights: ► To determine the role of ELF in ROS, DNA damage and IL-8 after exposure to PM. ► ufCB, FeSO{sub 4} and DEP extract were used to examine the protective effects of ELF. ► PM-driven oxidative stress and DNA single-strand breakage were mitigated by ELF. ► The

Light scattering on PHA granules protects bacterial cells against the harmful effects of UV radiation.

PubMed

Slaninova, Eva; Sedlacek, Petr; Mravec, Filip; Mullerova, Lucie; Samek, Ota; Koller, Martin; Hesko, Ondrej; Kucera, Dan; Marova, Ivana; Obruca, Stanislav

2018-02-01

Numerous prokaryotes accumulate polyhydroxyalkanoates (PHA) in the form of intracellular granules. The primary function of PHA is the storage of carbon and energy. Nevertheless, there are numerous reports that the presence of PHA granules in microbial cells enhances their stress resistance and fitness when exposed to various stress factors. In this work, we studied the protective mechanism of PHA granules against UV irradiation employing Cupriavidus necator as a model bacterial strain. The PHA-accumulating wild type strain showed substantially higher UV radiation resistance than the PHA non-accumulating mutant. Furthermore, the differences in UV-Vis radiation interactions with both cell types were studied using various spectroscopic approaches (turbidimetry, absorption spectroscopy, and nephelometry). Our results clearly demonstrate that intracellular PHA granules efficiently scatter UV radiation, which provides a substantial UV-protective effect for bacterial cells and, moreover, decreases the intracellular level of reactive oxygen species in UV-challenged cells. The protective properties of the PHA granules are enhanced by the fact that granules specifically bind to DNA, which in turn provides shield-like protection of DNA as the most UV-sensitive molecule. To conclude, the UV-protective action of PHA granules adds considerable value to their primary storage function, which can be beneficial in numerous environments.

Immunogenicity and Protective Efficacy of a Plasmodium yoelii Hsp60 DNA Vaccine in BALB/c Mice

PubMed Central

Sanchez, Gloria I.; Sedegah, Martha; Rogers, William O.; Jones, Trevor R.; Sacci, John; Witney, Adam; Carucci, Daniel J.; Kumar, Nirbhay; Hoffman, Stephen L.

2001-01-01

The gene encoding the 60-kDa heat shock protein of Plasmodium yoelii (PyHsp60) was cloned into the VR1012 and VR1020 mammalian expression vectors. Groups of 10 BALB/c mice were immunized intramuscularly at 0, 3, and 9 weeks with 100 μg of PyHsp60 DNA vaccine alone or in combination with 30 μg of pmurGMCSF. Sera from immunized mice but not from vector control groups recognized P. yoelii sporozoites, liver stages, and infected erythrocytes in an indirect fluorescent antibody test. Two weeks after the last immunization, mice were challenged with 50 P. yoelii sporozoites. In one experiment the vaccine pPyHsp60-VR1012 used in combination with pmurGMCSF gave 40% protection (Fisher's exact test; P = 0.03, vaccinated versus control groups). In a second experiment this vaccine did not protect any of the immunized mice but induced a delay in the onset of parasitemia. In neither experiment was there any evidence of a protective effect against the asexual erythrocytic stage of the life cycle. In a third experiment mice were primed with PyHsp60 DNA, were boosted 2 weeks later with 2 × 103 irradiated P. yoelii sporozoites, and were challenged several weeks later. The presence of PyHsp60 in the immunization regimen did not lead to reduced blood-stage infection or development of parasites in hepatocytes. PyHsp60 DNA vaccines were immunogenic in BALB/c mice but did not consistently, completely protect against sporozoite challenge. The observation that in some of the PyHsp60 DNA vaccine-immunized mice there was protection against infection or a delay in the onset of parasitemia after sporozoite challenge deserves further evaluation. PMID:11349057

Silymarin Protects Epidermal Keratinocytes from Ultraviolet Radiation-Induced Apoptosis and DNA Damage by Nucleotide Excision Repair Mechanism

PubMed Central

Katiyar, Santosh K.; Mantena, Sudheer K.; Meeran, Syed M.

2011-01-01

Solar ultraviolet (UV) radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum), inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK) with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs) and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER) genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells. PMID:21731736

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.

Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.

PubMed

Yang, XinChao; Li, MengHui; Liu, JianHua; Ji, YiHong; Li, XiangRui; Xu, LiXin; Yan, RuoFeng; Song, XiaoKai

2017-02-16

Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines. With the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed. Adopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 10 6 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 10 7 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite. Our

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 themore » 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.« less

Off-Target Effects of Drugs that Disrupt Human Mitochondrial DNA Maintenance

PubMed Central

Young, Matthew J.

2017-01-01

Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus (HIV) the cause of acquired immunodeficiency syndrome. Development of severe mitochondrial toxicity has been well documented in patients infected with HIV and administered NRTIs. In vitro biochemical experiments have demonstrated that the replicative mitochondrial DNA (mtDNA) polymerase gamma, Polg, is a sensitive target for inhibition by metabolically active forms of NRTIs, nucleotide reverse transcriptase inhibitors (NtRTIs). Once incorporated into newly synthesized daughter strands NtRTIs block further DNA polymerization reactions. Human cell culture and animal studies have demonstrated that cell lines and mice exposed to NRTIs display mtDNA depletion. Further complicating NRTI off-target effects on mtDNA maintenance, two additional DNA polymerases, Pol beta and PrimPol, were recently reported to localize to mitochondria as well as the nucleus. Similar to Polg, in vitro work has demonstrated both Pol beta and PrimPol incorporate NtRTIs into nascent DNA. Cell culture and biochemical experiments have also demonstrated that antiviral ribonucleoside drugs developed to treat hepatitis C infection act as off-target substrates for POLRMT, the mitochondrial RNA polymerase and primase. Accompanying the above-mentioned topics, this review examines: (1) mtDNA maintenance in human health and disease, (2) reports of DNA polymerases theta and zeta (Rev3) localizing to mitochondria, and (3) additional drugs with off-target effects on mitochondrial function. Lastly, mtDNA damage may induce cell death; therefore, the possibility of utilizing compounds that disrupt mtDNA maintenance to kill cancer cells is discussed. PMID:29214156

DNA vaccination elicits protective immune responses against pandemic and classic swine influenza viruses in pigs.

PubMed

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

2011-11-01

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.

Cross-protection among lethal H5N2 influenza viruses induced by DNA vaccine to the hemagglutinin.

PubMed Central

Kodihalli, S; Haynes, J R; Robinson, H L; Webster, R G

1997-01-01

Inoculation of mice with hemagglutinin (HA)-expressing DNA affords reliable protection against lethal influenza virus infection, while in chickens the same strategy has yielded variable results. Here we show that gene gun delivery of DNA encoding an H5 HA protein confers complete immune protection to chickens challenged with lethal H5 viruses. In tests of the influence of promoter selection on vaccine efficacy, close correlations were obtained between immune responses and the dose of DNA administered, whether a cytomegalovirus (CMV) immediate-early promoter or a chicken beta-actin promoter was used. Perhaps most important, the HA-DNA vaccine conferred 95% cross-protection against challenge with lethal antigenic variants that differed from the primary antigen by 11 to 13% (HA1 amino acid sequence homology). Overall, the high levels of protection seen with gene gun delivery of HA-DNA were as good as, if not better than, those achieved with a conventional whole-virus vaccine, with fewer instances of morbidity and death. The absence of detectable antibody titers after primary immunization, together with the rapid appearance of high titers immediately after challenge, implicates efficient B-cell priming as the principal mechanism of DNA-mediated immune protection. Our results suggest that the efficacy of HA-DNA influenza virus vaccine in mice extends to chickens and probably to other avian species as well. Indeed, the H5 preparation we describe offers an attractive means to protect the domestic poultry industry in the United States from lethal H5N2 viruses, which continue to circulate in Mexico. PMID:9094608

DNA Dosimetry Assessment for Sunscreen Genotoxic Photoprotection

PubMed Central

Schuch, André Passaglia; Lago, Juliana Carvalhães; Yagura, Teiti; Menck, Carlos Frederico Martins

2012-01-01

Background Due to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter. Methodology/Principal Findings The Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations. Conclusions/Significance The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage. PMID:22768281

Zoledronate Attenuates Accumulation of DNA Damage in Mesenchymal Stem Cells and Protects Their Function

PubMed Central

Misra, Juhi; Mohanty, Sindhu T.; Madan, Sanjeev; Fernandes, James A.; Hal Ebetino, F.; Russell, R. Graham G.

2015-01-01

Abstract Mesenchymal stem cells (MSCs) undergo a decline in function following ex vivo expansion and exposure to irradiation. This has been associated with accumulation of DNA damage and has important implications for tissue engineering approaches or in patients receiving radiotherapy. Therefore, interventions, which limit accumulation of DNA damage in MSC, are of clinical significance. We were intrigued by findings showing that zoledronate (ZOL), an anti‐resorptive nitrogen containing bisphosphonate, significantly extended survival in patients affected by osteoporosis. The effect was too large to be simply due to the prevention of fractures. Moreover, in combination with statins, it extended the lifespan in a mouse model of Hutchinson Gilford Progeria Syndrome. Therefore, we asked whether ZOL was able to extend the lifespan of human MSC and whether this was due to reduced accumulation of DNA damage, one of the important mechanisms of aging. Here, we show that this was the case both following expansion and irradiation, preserving their ability to proliferate and differentiate in vitro. In addition, administration of ZOL before irradiation protected the survival of mesenchymal progenitors in mice. Through mechanistic studies, we were able to show that inhibition of mTOR signaling, a pathway involved in longevity and cancer, was responsible for these effects. Our data open up new opportunities to protect MSC from the side effects of radiotherapy in cancer patients and during ex vivo expansion for regenerative medicine approaches. Given that ZOL is already in clinical use with a good safety profile, these opportunities can be readily translated for patient benefit. Stem Cells 2016;34:756–767 PMID:26679354

Zoledronate Attenuates Accumulation of DNA Damage in Mesenchymal Stem Cells and Protects Their Function.

PubMed

Misra, Juhi; Mohanty, Sindhu T; Madan, Sanjeev; Fernandes, James A; Hal Ebetino, F; Russell, R Graham G; Bellantuono, Ilaria

2016-03-01

Mesenchymal stem cells (MSCs) undergo a decline in function following ex vivo expansion and exposure to irradiation. This has been associated with accumulation of DNA damage and has important implications for tissue engineering approaches or in patients receiving radiotherapy. Therefore, interventions, which limit accumulation of DNA damage in MSC, are of clinical significance. We were intrigued by findings showing that zoledronate (ZOL), an anti-resorptive nitrogen containing bisphosphonate, significantly extended survival in patients affected by osteoporosis. The effect was too large to be simply due to the prevention of fractures. Moreover, in combination with statins, it extended the lifespan in a mouse model of Hutchinson Gilford Progeria Syndrome. Therefore, we asked whether ZOL was able to extend the lifespan of human MSC and whether this was due to reduced accumulation of DNA damage, one of the important mechanisms of aging. Here, we show that this was the case both following expansion and irradiation, preserving their ability to proliferate and differentiate in vitro. In addition, administration of ZOL before irradiation protected the survival of mesenchymal progenitors in mice. Through mechanistic studies, we were able to show that inhibition of mTOR signaling, a pathway involved in longevity and cancer, was responsible for these effects. Our data open up new opportunities to protect MSC from the side effects of radiotherapy in cancer patients and during ex vivo expansion for regenerative medicine approaches. Given that ZOL is already in clinical use with a good safety profile, these opportunities can be readily translated for patient benefit. © 2015 AlphaMed Press.

Evaluation of cytotoxicity and oxidative DNA damaging effects of di(2-ethylhexyl)-phthalate (DEHP) and mono(2-ethylhexyl)-phthalate (MEHP) on MA-10 Leydig cells and protection by selenium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erkekoglu, Pinar; Hacettepe University, Faculty of Pharmacy, Department of Toxicology, 06100 Ankara; Rachidi, Walid

2010-10-01

Di(2-ethylhexyl)-phthalate (DEHP) is the most abundantly used phthalate derivative, inevitable environmental exposure of which is suspected to contribute to the increasing incidence of testicular dysgenesis syndrome in humans. Oxidative stress and mitochondrial dysfunction in germ cells are suggested to contribute to phthalate-induced disruption of spermatogenesis in rodents, and Leydig cells are one of the main targets of phthalates' testicular toxicity. Selenium is known to be involved in the modulation of intracellular redox equilibrium, and plays a critical role in testis, sperm, and reproduction. This study was aimed to investigate the oxidative stress potential of DEHP and its consequences in testicularmore » cells, and examine the possible protective effects of selenium using the MA-10 mouse Leydig tumor cell line as a model. In the presence and absence of selenium compounds [30 nM sodium selenite (SS), and 10 {mu}M selenomethionine (SM)], the effects of exposure to DEHP and its main metabolite mono(2-ethylhexyl)-phthalate (MEHP) on the cell viability, enzymatic and non-enzymatic antioxidant status, ROS production, p53 expression, and DNA damage by alkaline Comet assay were investigated. The overall results of this study demonstrated the cytotoxicity and genotoxicity potential of DEHP, where MEHP was found to be more potent than the parent compound. SS and SM produced almost the same level of protection against antioxidant status modifying effects, ROS and p53 inducing potentials, and DNA damaging effects of the two phthalate derivatives. It was thus shown that DEHP produced oxidative stress in MA-10 cells, and selenium supplementation appeared to be an effective redox regulator in the experimental conditions used in this study, emphasizing the critical importance of the appropriate selenium status.« less

DNA vaccines encoding proteins from wild-type and attenuated canine distemper virus protect equally well against wild-type virus challenge.

PubMed

Nielsen, Line; Jensen, Trine Hammer; Kristensen, Birte; Jensen, Tove Dannemann; Karlskov-Mortensen, Peter; Lund, Morten; Aasted, Bent; Blixenkrone-Møller, Merete

2012-10-01

Immunity induced by DNA vaccines containing the hemagglutinin (H) and nucleoprotein (N) genes of wild-type and attenuated canine distemper virus (CDV) was investigated in mink (Mustela vison), a highly susceptible natural host of CDV. All DNA-immunized mink seroconverted, and significant levels of virus-neutralizing (VN) antibodies were present on the day of challenge with wild-type CDV. The DNA vaccines also primed the cell-mediated memory responses, as indicated by an early increase in the number of interferon-gamma (IFN-γ)-producing lymphocytes after challenge. Importantly, the wild-type and attenuated CDV DNA vaccines had a long-term protective effect against wild-type CDV challenge. The vaccine-induced immunity induced by the H and N genes from wild-type CDV and those from attenuated CDV was comparable. Because these two DNA vaccines were shown to protect equally well against wild-type virus challenge, it is suggested that the genetic/antigenic heterogeneity between vaccine strains and contemporary wild-type strains are unlikely to cause vaccine failure.

New insight into multifunctional role of peroxiredoxin family protein: Determination of DNA protection properties of bacterioferritin comigratory protein under hyperthermal and oxidative stresses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Sangmin, E-mail: taeinlee2011@kangwon.ac.kr; Chung, Jeong Min; Yun, Hyung Joong

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 stressmore » 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. - Highlights: • Bacterioferritin comigratory protein (BCP) protects DNA from oxidative stress by reducing ROS. • TkBCP does not only scavenge ROS, but also protect DNA from hyperthermal stress. • BCP potentially adopts the multi-functional role in DNA binding activities and anti-oxidant functions.« less

DNA labelling of varieties covered by patent protection: a new solution for managing intellectual property rights in the seed industry.

PubMed

Fister, Karin; Fister, Iztok; Murovec, Jana; Bohanec, Borut

2017-02-01

Plant breeders' rights are undergoing dramatic changes due to changes in patent rights in terms of plant variety rights protection. Although differences in the interpretation of »breeder's exemption«, termed research exemption in the 1991 UPOV, did exist in the past in some countries, allowing breeders to use protected varieties as parents in the creation of new varieties of plants, current developments brought about by patenting conventionally bred varieties with the European Patent Office (such as EP2140023B1) have opened new challenges. Legal restrictions on germplasm availability are therefore imposed on breeders while, at the same time, no practical information on how to distinguish protected from non-protected varieties is given. We propose here a novel approach that would solve this problem by the insertion of short DNA stretches (labels) into protected plant varieties by genetic transformation. This information will then be available to breeders by a simple and standardized procedure. We propose that such a procedure should consist of using a pair of universal primers that will generate a sequence in a PCR reaction, which can be read and translated into ordinary text by a computer application. To demonstrate the feasibility of such approach, we conducted a case study. Using the Agrobacterium tumefaciens transformation protocol, we inserted a stretch of DNA code into Nicotiana benthamiana. We also developed an on-line application that enables coding of any text message into DNA nucleotide code and, on sequencing, decoding it back into text. In the presented case study, a short command line coding the phrase »Hello world« was transformed into a DNA sequence that was inserted in the plant genome. The encoded message was reconstructed from the resulting T1 seedlings with 100 % accuracy. The feasibility and possible other applications of this approach are discussed.

Parallel In Vivo DNA Assembly by Recombination: Experimental Demonstration and Theoretical Approaches

PubMed Central

Shi, Zhenyu; Wedd, Anthony G.; Gras, Sally L.

2013-01-01

The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration. PMID:23468883

Evaluation of genotoxicity and DNA protective effects of mangiferin, a glucosylxanthone isolated from Mangifera indica L. stem bark extract.

PubMed

Rodeiro, I; Hernandez, S; Morffi, J; Herrera, J A; Gómez-Lechón, M J; Delgado, R; Espinosa-Aguirre, J J

2012-09-01

Mangiferin is a glucosylxantone isolated from Mangifera indica L. stem bark. Several studies have shown its pharmacological properties which make it a promising candidate for putative therapeutic use. This study was focused to investigate the in vitro genotoxic effects of mangiferin in the Ames test, SOS Chromotest and Comet assay. The genotoxic effects in bone marrow erythrocytes from NMRI mice orally treated with mangiferin (2000 mg/kg) were also evaluated. Additionally, its potential antimutagenic activity against several mutagens in the Ames test and its effects on CYP1A1 activity were assessed. Mangiferin (50-5000 μg/plate) did not increased the frequency of reverse mutations in the Ames test, nor induced primary DNA damage (5-1000 μg/mL) to Escherichia coli PQ37 cells under the SOS Chromotest. It was observed neither single strand breaks nor alkali-labile sites in blood peripheral lymphocytes or hepatocytes after 1h exposition to 10-500 μg/mL of mangiferin under the Comet assay. Furthermore, micronucleus studies showed mangiferin neither induced cytotoxic activity nor increased the frequency of micronucleated/binucleated cells in mice bone marrow. In short, mangiferin did not induce cytotoxic or genotoxic effects but it protect against DNA damage which would be associated with its antioxidant properties and its capacity to inhibit CYP enzymes. Copyright © 2012 Elsevier Ltd. All rights reserved.

DNA condensing effects and sequence selectivity of DNA binding of antitumor noncovalent polynuclear platinum complexes.

PubMed

Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

2014-02-03

The noncovalent analogues of antitumor polynuclear platinum complexes represent a structurally discrete class of platinum drugs. Their chemical and biological properties differ significantly from those of most platinum chemotherapeutics, which bind to DNA in a covalent manner by formation of Pt-DNA adducts. In spite of the fact that these noncovalent polynuclear platinum complexes contain no leaving groups, they have been shown to bind to DNA with high affinity. We report here on the DNA condensation properties of a series of noncovalent analogues of antitumor polynuclear platinum complexes described by biophysical and biochemical methods. The results demonstrate that these polynuclear platinum compounds are capable of inducing DNA condensation at more than 1 order of magnitude lower concentrations than conventional spermine. Atomic force microscopy studies of DNA condensation confined to a mica substrate have revealed that the DNA morphologies become more compact with increasing concentration of the platinum complexes. Moreover, we also found that the noncovalent polynuclear platinum complex [{Pt(NH3)3}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](6+) (TriplatinNC-A) binds to DNA in a sequence-dependent manner, namely, to A/T-rich sequences and A-tract regions, and that noncovalent polynuclear platinum complexes protect DNA from enzymatic cleavage by DNase I. The results suggest that mechanisms of antitumor and cytotoxic activities of these complexes may be associated with their unique ability to condense DNA along with their sequence-specific DNA binding. Owing to their high cellular accumulation, it is also reasonable to suggest that their mechanism of action is based on the competition with naturally occurring DNA condensing agents, such as polyamines spermine, spermidine, and putrescine, for intracellular binding sites, resulting in the disturbance of the correct binding of regulatory proteins initiating the onset of apoptosis.

Prime-boost bacillus Calmette-Guérin vaccination with lentivirus-vectored and DNA-based vaccines expressing antigens Ag85B and Rv3425 improves protective efficacy against Mycobacterium tuberculosis in mice.

PubMed

Xu, Ying; Yang, Enzhuo; Wang, Jianguang; Li, Rui; Li, Guanghua; Liu, Guoyuan; Song, Na; Huang, Qi; Kong, Cong; Wang, Honghai

2014-10-01

To prevent the global spread of tuberculosis (TB), more effective vaccines and vaccination strategies are urgently needed. As a result of the success of bacillus Calmette-Guérin (BCG) in protecting children against miliary and meningeal TB, the majority of individuals will have been vaccinated with BCG; hence, boosting BCG-primed immunity will probably be a key component of future vaccine strategies. In this study, we compared the ability of DNA-, protein- and lentiviral vector-based vaccines that express the antigens Ag85B and Rv3425 to boost the effects of BCG in the context of immunity and protection against Mycobacterium tuberculosis in C57BL/6 mice. Our results demonstrated that prime-boost BCG vaccination with a lentiviral vector expressing the antigens Ag85B and Rv3425 significantly enhanced immune responses, including T helper type 1 and CD8(+) cytotoxic T lymphocyte responses, compared with DNA- and protein-based vaccines. However, lentivirus-vectored and DNA-based vaccines greatly improved the protective efficacy of BCG against M. tuberculosis, as indicated by a lack of weight loss and significantly reduced bacterial loads and histological damage in the lung. Our study suggests that the use of lentiviral or DNA vaccines containing the antigens Ag85B and Rv3425 to boost BCG is a good choice for the rational design of an efficient vaccination strategy against TB. © 2014 John Wiley & Sons Ltd.

A novel "in-feed" delivery platform applied for oral DNA vaccination against IPNV enables high protection in Atlantic salmon (Salmon salar).

PubMed

Reyes, Miguel; Ramírez, Cesar; Ñancucheo, Ivan; Villegas, Ricardo; Schaffeld, Guillermo; Kriman, Luis; Gonzalez, Javier; Oyarzun, Patricio

2017-01-23

DNA vaccination has emerged as a promising tool against infectious diseases of farmed fish. Oral delivery allows stress-free administration that is ideal for mass immunization and of paramount importance for infectious pancreatic necrosis (IPN) and other viral disease that affect young salmonids and cause economic losses in aquaculture worldwide. We describe the development and in vivo assessment of an "in-feed" formulation strategy for oral immunization with liposomal DNA vaccines, by delivering a vaccine construct coding for an immunogenic region of the VP2 capsid protein. A challenge against IPNV was carried out to determine the vaccine efficacy, by comparing the mortality of pre-smolt Atlantic salmons immunized and non-immunized with the oral vaccine. The antibody response (ELISA) and hematological parameters after immunization were examined, as well as the vaccine effect on the growth and internal structures of fry salmons (histological analysis). The vaccine distribution in the experimental tank after oral administration was investigated by HPLC and PCR amplification. The oral vaccine induced detectable levels of VP2-specific antibodies and conferred significant protection following IPNV challenge, with relative percent survivals (RPS) of 58.2%, for single dose (1mg pDNA /kg fish ⋅d), and 66% for double dose (2mg pDNA /kg fish ⋅d). We further provide evidence in favour of the vaccine safety to fish and demonstrated absence of pDNA in the tank water, but presence of vaccine residues in faeces and unconsumed feed sediments (solid wastes). The delivery platform for liposomal DNA vaccination via feed was successfully proved against IPNV in Atlantic salmon, showing the oral vaccine to be immunogenic and safe for fish, and providing significant protection after oral administration. The "in-feed" technology for oral DNA vaccination holds potential to be applied against IPNV and other pathogens that currently threaten the aquaculture worldwide. Copyright © 2016

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.

Protein- and DNA-based anthrax toxin vaccines confer protection in guinea pigs against inhalational challenge with Bacillus cereus G9241.

PubMed

Palmer, John; Bell, Matt; Darko, Christian; Barnewall, Roy; Keane-Myers, Andrea

2014-11-01

In the past decade, several Bacillus cereus strains have been isolated from otherwise healthy individuals who succumbed to bacterial pneumonia presenting symptoms resembling inhalational anthrax. One strain was indistinguishable from B. cereus G9241, previously cultured from an individual who survived a similar pneumonia-like illness and which was shown to possess a complete set of plasmid-borne anthrax toxin-encoding homologs. The finding that B. cereus G9241 pathogenesis in mice is dependent on pagA1-derived protective antigen (PA) synthesis suggests that an anthrax toxin-based vaccine may be effective against this toxin-encoding B. cereus strain. Dunkin Hartley guinea pigs were immunized with protein- and DNA-based anthrax toxin-based vaccines, immune responses were evaluated and survival rates were calculated after lethal aerosol exposure with B. cereus G9241 spores. Each vaccine induced seroconversion with the protein immunization regimen eliciting significantly higher serum levels of antigen-specific antibodies at the prechallenge time-point compared with the DNA-protein prime-boost immunization schedule. Complete protection against lethal challenge was observed in all groups with a detectable prechallenge serum titer of toxin neutralizing antibodies. For the first time, we demonstrated that the efficacy of fully defined anthrax toxin-based vaccines was protective against lethal B. cereus G9241 aerosol challenge in the guinea pig animal model. Published 2014. This article is a US Government work and is in the public domain in the USA.

Dual-degradable disulfide-containing PEI–Pluronic/DNA polyplexes: transfection efficiency and balancing protection and DNA release

PubMed Central

Zhang, Lifen; Chen, Zhenzhen; Li, Yanfeng

2013-01-01

Polymeric gene-delivery vectors to achieve lack of toxicity and a balance between protection and DNA release remains a formidable challenge. Incorporating intracellular environment-responsive degradable bonds is an appreciable step toward developing safer transfection agents. In this study, novel, dual-degradable polycation copolymers (Pluronic-diacrylate [PA]–polyethyleneimine [PEI]–SS) were synthesized through the addition of low molecular weight (800 Da) PEI cross-linked with SS (PEI-SS) to PA. Three PA-PEI-SS copolymers (PA-PEI-SS1, 2, and 3) with different PEI-SS to Pluronic molar ratios were investigated and found to strongly condense plasmid DNA into positively charged nanoparticles with an average particle size of approximately 200 nm and to possess higher stability against DNase I digestion and sodium heparin. Disulfide and ester bonds of the copolymers were susceptible to intracellular redox conditions. In vitro experiments demonstrated that the PA-PEI-SS copolymers had significantly lower cytotoxicity and higher transfection efficiency in both BGC-823 and 293T cell lines than the controls of degradable PEI-SS and nondegradable 25 kDa PEI. Transfection activity was influenced by the PEI-SS content in the polymers and PA-PEI-SS1 showed the highest efficiency of the three copolymers. These studies suggest that these dual-degradable copolymers could be used as potential biocompatible gene delivery carriers. PMID:24109182

Prime-boost vaccination using DNA and whole inactivated virus vaccines provides limited protection against virulent feline immunodeficiency virus.

PubMed

Dunham, Stephen P; Bruce, Jennifer; Klein, Dieter; Flynn, J Norman; Golder, Matthew C; MacDonald, Susan; Jarrett, Oswald; Neil, James C

2006-11-30

Protection against feline immunodeficiency virus (FIV) has been achieved using a variety of vaccines notably whole inactivated virus (WIV) and DNA. However protection against more virulent isolates, typical of those encountered in natural infections, has been difficult to achieve. In an attempt to improve protection against virulent FIV(GL8), we combined both DNA and WIV vaccines in a "prime-boost" approach. Thirty cats were divided into four groups receiving vaccinations and one unvaccinated control group. Following viral challenge, two vaccinated animals, one receiving DNA alone and one the prime-boost vaccine remained free of viraemia, whilst all controls became viraemic. Animals vaccinated with WIV showed apparent early enhancement of infection at 2 weeks post challenge (pc) with higher plasma viral RNA loads than control animals or cats immunised with DNA alone. Despite this, animals vaccinated with WIV or DNA alone showed significantly lower proviral loads in peripheral blood mononuclear cells and mesenteric lymph node cells, whilst those receiving the DNA-WIV prime-boost vaccine showed significantly lower proviral loads in PBMC, than control animals, at 35 weeks pc. Therefore both DNA and WIV vaccines conferred limited protection against viral challenge but the combination of WIV and DNA in a prime-boost approach appeared to offer no significant advantage over either vaccine alone.

Enhanced immune response and protective efficacy of a Treponema pallidum Tp92 DNA vaccine vectored by chitosan nanoparticles and adjuvanted with IL-2.

PubMed

Zhao, Feijun; Wu, Yimou; Zhang, Xiaohong; Yu, Jian; Gu, Weiming; Liu, Shuangquan; Zeng, Tiebing; Zhang, Yuejun; Wang, Shiping

2011-10-01

In this study, the immune-modulatory and protective efficacy of using an interleukin-2 (IL-2) expression plasmid as a genetic adjuvant and chitosan (CS) nanoparticles as vectors to enhance a Tp92 DNA vaccine candidate were investigated in a Treponema pallidum (Tp) rabbit challenge model. CS vectoring of pTp92 or pIL-2 were both demonstrated to augment anti-Tp92 antibody levels induced by pTp92 DNA vaccines. Interestingly, the combination of CS vectored Tp92 and pIL-2 led to the greatest enhancements of anti-Tp92 antibodies and T-cell proliferation (p < 0.05). At week 10 after the first immunization, 15 of the 18 rabbits in each group were challenged with Tp Nichols strain and monitored for skin lesions and ulcer lesions. Ratios of positive skin lesions and ratios of ulcer lesions in groups immunized with pTp92 were significantly lower than those of the empty vector or PBS groups (p < 0.05), demonstrating that pTp92 immunization elicited significant protective efficacy against the Tp Nichols strain challenge. CS vectored and pIL-2 adjuvanted pTp92 immunized animals exhibited the lowest rates of positive skin and ulcer lesions. Male New Zealand white rabbits were randomly assigned to groups (n = 18/group) and immunized intramuscularly with pTp92 based plasmid DNA constructs (100 μg of DNA/rabbit/immunization). Two weeks before Tp challenge (Week 8), three rabbits from each group were used to determine cytokine measurements and fifteen rabbits from each group were used for Tp challenge studies. Intramuscular injection of pTp92 induced strong humoral and cellular immune responses and conferred protection from Tp challenge in rabbits. The use of CS as a pTp92 vector or pIL-2 as an adjuvant achieved a superior level of protective efficacy against Tp challenge, however CS vectored, IL-2 adjuvanted pTp92 immunization conferred the highest level of protective efficacy.

An Interleukin 12 Adjuvanted Herpes Simplex Virus 2 DNA Vaccine Is More Protective Than a Glycoprotein D Subunit Vaccine in a High-Dose Murine Challenge Model.

PubMed

Bagley, Kenneth C; Schwartz, Jennifer A; Andersen, Hanne; Eldridge, John H; Xu, Rong; Ota-Setlik, Ayuko; Geltz, Joshua J; Halford, William P; Fouts, Timothy R

2017-04-01

Vaccination is a proven intervention against human viral diseases; however, success against Herpes Simplex Virus 2 (HSV-2) remains elusive. Most HSV-2 vaccines tested in humans to date contained just one or two immunogens, such as the virion attachment receptor glycoprotein D (gD) and/or the envelope fusion protein, glycoprotein B (gB). At least three factors may have contributed to the failures of subunit-based HSV-2 vaccines. First, immune responses directed against one or two viral antigens may lack sufficient antigenic breadth for efficacy. Second, the antibody responses elicited by these vaccines may have lacked necessary Fc-mediated effector functions. Third, these subunit vaccines may not have generated necessary protective cellular immune responses. We hypothesized that a polyvalent combination of HSV-2 antigens expressed from a DNA vaccine with an adjuvant that polarizes immune responses toward a T helper 1 (Th1) phenotype would compose a more effective vaccine. We demonstrate that delivery of DNA expressing full-length HSV-2 glycoprotein immunogens by electroporation with the adjuvant interleukin 12 (IL-12) generates substantially greater protection against a high-dose HSV-2 vaginal challenge than a recombinant gD subunit vaccine adjuvanted with alum and monophosphoryl lipid A (MPL). Our results further show that DNA vaccines targeting optimal combinations of surface glycoproteins provide better protection than gD alone and provide similar survival benefits and disease symptom reductions compared with a potent live attenuated HSV-2 0ΔNLS vaccine, but that mice vaccinated with HSV-2 0ΔNLS clear the virus much faster. Together, our data indicate that adjuvanted multivalent DNA vaccines hold promise for an effective HSV-2 vaccine, but that further improvements may be required.

A randomised trial to demonstrate the effectiveness of electronic messages on sun protection behaviours.

PubMed

Szabó, Csanád; Ócsai, Henriette; Csabai, Márta; Kemény, Lajos

2015-08-01

Message exposure is effective at changing a variety of health behaviours. Our aim was to improve sun protection habits of a volunteer sample. We conducted a randomised, non-blinded, investigator-initiated trial (from 1st June to 31st August in 2011) on the effect of an electronic text-message system on sun protection behaviours. The assessments of 149 healthy volunteer participants took place at the Clinical Department of Dermatology and Allergology at the University of Szeged in Hungary. Psychological and medical assessments were also made. Total motivation scores for adherence to sunscreen use improved at a nearly significant level (t=-1.954, p=0.054). The intervention group used sunscreens more often than the other groups according to their sun exposure diaries (F=8.173, p<0.05) and their interview results (F=3.44, p<0.05). Using electronic messages offers an effective method to improve sun protection behaviours. Our intervention is a cost-effective method and it can easily be implemented at worksites. Copyright © 2015 Elsevier B.V. All rights reserved.

TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA) enhances CD8+ T Cell responses providing protection against Leishmania (Viannia).

PubMed

Jayakumar, Asha; Castilho, Tiago M; Park, Esther; Goldsmith-Pestana, Karen; Blackwell, Jenefer M; McMahon-Pratt, Diane

2011-06-01

Leishmania (Viannia) parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective. Using a newly developed mouse model of chronic L. (Viannia) panamensis infection and the heterologous DNA prime - modified vaccinia virus Ankara (MVA) boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP) could provide protection against infection/disease. Heterologous prime - boost (DNA/MVA) vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V.) panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses. Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia) to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that CD8 T

The mitochondrially targeted antioxidant MitoQ protects the intestinal barrier by ameliorating mitochondrial DNA damage via the Nrf2/ARE signaling pathway.

PubMed

Hu, Qiongyuan; Ren, Jianan; Li, Guanwei; Wu, Jie; Wu, Xiuwen; Wang, Gefei; Gu, Guosheng; Ren, Huajian; Hong, Zhiwu; Li, Jieshou

2018-03-14

Disruption of the mucosal barrier following intestinal ischemia reperfusion (I/R) is life threatening in clinical practice. Mitochondrial dysfunction and oxidative stress significantly contribute to the early phase of I/R injury and amplify the inflammatory response. MitoQ is a mitochondrially targeted antioxidant that exerts protective effects following I/R injury. In the present study, we aimed to determine whether and how MitoQ protects intestinal epithelial cells (IECs) from I/R injury. In both in vivo and in vitro studies, we found that MitoQ pretreatment downregulated I/R-induced oxidative stress and stabilized the intestinal barrier, as evidenced by MitoQ-treated I/R mice exhibiting attenuated intestinal hyperpermeability, inflammatory response, epithelial apoptosis, and tight junction damage compared to controls. Mechanistically, I/R elevated mitochondrial 8-hydroxyguanine content, reduced mitochondrial DNA (mtDNA) copy number and mRNA transcription levels, and induced mitochondrial disruption in IECs. However, MitoQ pretreatment dramatically inhibited these deleterious effects. mtDNA depletion alone was sufficient to induce apoptosis and mitochondrial dysfunction of IECs. Mitochondrial transcription factor A (TFAM), a key activator of mitochondrial transcription, was significantly reduced during I/R injury, a phenomenon that was prevented by MitoQ treatment. Furthermore, we observed that thee protective properties of MitoQ were affected by upregulation of cellular antioxidant genes, including HO-1, NQO-1, and γ-GCLC. Transfection with Nrf2 siRNA in IECs exposed to hypoxia/reperfusion conditions partially blocked the effects of MitoQ on mtDNA damage and mitochondrial oxidative stress. In conclusion, our data suggest that MitoQ exerts protective effect on I/R-induced intestinal barrier dysfunction.

DNA vaccine expressing herpes simplex virus 1 glycoprotein C and D protects mice against herpes simplex keratitis

PubMed Central

Dong, Li-Li; Tang, Ru; Zhai, Yu-Jia; Malla, Tejsu; Hu, Kai

2017-01-01

AIM To investigate whether DNA vaccine encoding herpes simplex virus 1 (HSV-1) glycoprotein C (gC) and glycoprotein D (gD) will achieve better protective effect against herpes simplex keratitis (HSK) than DNA vaccine encoding gD alone. METHODS DNA vaccine expressing gD or gC combined gD (gD.gC) were constructed and carried by chitosan nanoparticle. The expression of fusion protein gD and gC were detected in DNA/nanoparticle transfected 293T cells by Western-blot. For immunization, mice were inoculated with DNA/nanoparticle for 3 times with 2wk interval, and two weeks after the final immunization, the specific immune responses and clinical degrees of primary HSK were evaluated. RESULTS Fusion protein gD.gC could be expressed successfully in cultured 293T cells. And, pRSC-gC.gD-IL21 DNA/chitosan nanoparticle could effectively elicit strongest humoral and cellular immune response in primary HSK mice evidenced by higher levels of specific neutralizing antibody and sIgA production, enhanced cytotoxicities of splenocytes and nature killer cells (NK), when compared with those of gD alone or mocked vaccine immunized mice. As a result, gC-based vaccine immunized mice showed least HSK disease. CONCLUSION gC-based DNA vaccine could effectively prevent the progress of primary HSK, suggesting that this DNA vaccine could be a promising vaccine for HSK treatment in the future. PMID:29181304

Raman microspectroscopic study of effects of Na(I) and Mg(II) ions on low pH induced DNA structural changes.

PubMed

Muntean, C M; Segers-Nolten, G M J

2003-01-01

In this work a confocal Raman microspectrometer is used to investigate the influence of Na(+) and Mg(2+) ions on the DNA structural changes induced by low pH. Measurements are carried out on calf thymus DNA at neutral pH (7) and pH 3 in the presence of low and high concentrations of Na(+) and Mg(2+) ions, respectively. It is found that low concentrations of Na(+) ions do not protect DNA against binding of H(+). High concentrations of monovalent ions can prevent protonation of the DNA double helix. Our Raman spectra show that low concentrations of Mg(2+) ions partly protect DNA against protonation of cytosine (line at 1262 cm(-1)) but do not protect adenine and guanine N(7) against binding of H(+) (characteristic lines at 1304 and 1488 cm(-1), respectively). High concentrations of Mg(2+) can prevent protonation of cytosine and protonation of adenine (disruption of AT pairs). By analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high magnesium salt protect the N(7) of guanine against protonation. A high salt concentration can prevent protonation of guanine, cytosine, and adenine in DNA. Higher salt concentrations cause less DNA protonation than lower salt concentrations. Magnesium ions are found to be more effective in protecting DNA against binding of H(+) as compared with calcium ions presented in a previous study. Divalent metal cations (Mg(2+), Ca(2+)) are more effective in protecting DNA against protonation than monovalent ions (Na(+)). Copyright 2003 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 72: 000-000, 2003

Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection

PubMed Central

Tian, Maopeng; Zhou, Zijie; Tan, Songwei; Fan, Xionglin; Li, Longmeng; Ullah, Nadeem

2018-01-01

Despite the vaccine Mycobacterium bovis Bacillus Calmette–Guérin is used worldwide, tuberculosis (TB) remains the first killer among infectious diseases. An effective vaccine is urgently required. DNA vaccine has shown prophylactic as well as therapeutic effects against TB, while its weak immunogenicity hinders the application. As a strong inducer of Th1-biased immune response, DMT, consisting of dimethyldioctadecylammonium (DDA) and two pattern recognition receptor agonists monophosphoryl lipid A and trehalose 6,6′-dibehenate (TDB), was a newly developed liposomal adjuvant. To elucidate the action mechanism of DMT and improve immunological effects induced by DNA vaccine, a new recombinant eukaryotic expression plasmid pCMFO that secretes the fusion of four multistage antigens (Rv2875, Rv3044, Rv2073c, and Rv0577) of Mycobacterium tuberculosis was constructed. pCMFO/DDA and pCMFO/DMT complexes were then prepared and their physicochemical properties were analyzed. The immunogenicity and protection against M. tuberculosis infection in vaccinated C57BL/6 mice were compared. Formulation of DNA and two agonists into the DDA liposome decreased zeta potential but increased the stability of storage, which resulted in a slower and longer-lasting release of DNA from the DNA–DMT complex than the DNA–DDA liposome. Besides Th1-biased responses, pCMFO/DMT vaccinated mice elicited more significantly CFMO-specific IL2+ TCM cell responses in the spleen and provided an enhanced and persistent protection against M. tuberculosis aerosol infection, compared to pCMFO/DDA and pCMFO groups. Therefore, the adjuvant DMT can release DNA and agonists slowly, which might attribute to the improved protection of DMT adjuvanted vaccines. pCMFO/DMT, a very promising TB vaccine, warrants for further preclinical and clinical trials. PMID:29535714

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

2014-04-07

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

EFFECTS OF INGESTED ARSENIC ON DNA AND CHROMOSOME IN HUMAN EXFOLIATED EPITHELIA

EPA Science Inventory

Effects of Ingested Arsenic on DNA and Chromosome in Human Exfoliated Epithelia

Judy L. Mumford, Human Studies Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711

Arsenic...

Protective effect of alpha glucosyl hesperidin (G-hesperidin) on chronic vanadium induced testicular toxicity and sperm nuclear DNA damage in male Sprague Dawley rats.

PubMed

Vijaya Bharathi, B; Jaya Prakash, G; Krishna, K M; Ravi Krishna, C H; Sivanarayana, T; Madan, K; Rama Raju, G A; Annapurna, A

2015-06-01

The study was conducted to evaluate the vanadium-induced testicular toxicity and its effect on sperm parameters, sperm nuclear DNA damage and histological alterations in Sprague Dawley rats and to assess the protective effect of G-hesperidin against this damage. Treatment of rats with vanadium at a dose of 1 mg kg bw(-1) for 90 days resulted in significant reduction in serum testosterone levels, sperm count and motility. Further, a parallel increase in abnormal sperm morphology and adverse histopathological changes in testis was also associated with vanadium administration when compared to normal control. Moreover, sperm chromatin dispersion assay revealed that vanadium induces sperm nuclear DNA fragmentation. A marked increase in testicular malondialdehyde levels and decreased activity of antioxidant enzymes such as superoxide dismutase and catalase indicates vanadium-induced oxidative stress. Co-administration of G-hesperidin at a dose of 25 and 50 mg kg bw(-1) significantly attenuated the sperm parameters and histological changes by restoring the antioxidant levels in rat testis. These results suggested that vanadium exposure caused reduced bioavailability of androgens to the tissue and increased free radical formation, thereby causing structural and functional changes in spermatozoa. G-hesperidin exhibited antioxidant effect by protecting the rat testis against vanadium-induced oxidative damage, further ensures antioxidant potential of bioflavonoids. © 2014 Blackwell Verlag GmbH.

The effect of 2-[(aminopropyl)amino] ethanethiol on fission-neutron-induced DNA damage and repair.

PubMed Central

Grdina, D. J.; Sigdestad, C. P.; Dale, P. J.; Perrin, J. M.

1989-01-01

The effect(s) of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR 1065) on fission-neutron-induced DNA damage and repair in V79 Chinese hamster cells was determined by using a neutral filter elution procedure (pH 7.2). When required, WR1065, at a final working concentration of 4 mM, was added to the culture medium, either 30 min before and during irradiation with fission spectrum neutrons (beam energy of 0.85 MeV) from the JANUS research reactor, or for selected intervals of time following exposure. The frequency of neutron-induced DNA strand breaks as measured by neutral elution as a function of dose equalled that observed for 60Co gamma-ray-induced damage (relative biological effectiveness of one). In contrast to the protective effect exhibited by WR1065 in reducing 60Co-induced DNA damage, WR1065 was ineffective in reducing or protecting against induction of DNA strand breaks by JANUS neutrons. The kinetics of DNA double-strand rejoining were measured following neutron irradiation. In the absence of WR1065, considerable DNA degradation by cellular enzymes was observed. This process was inhibited when WR1065 was present. These results indicate that, under the conditions used, the quality (i.e. nature), rather than quantity, of DNA lesions (measured by neutral elution) formed by neutrons was significantly different from that formed by gamma-rays. PMID:2667608

Intra-muscular and oral vaccination using a Koi Herpesvirus ORF25 DNA vaccine does not confer protection in common carp (Cyprinus carpio L.).

PubMed

Embregts, Carmen W E; Tadmor-Levi, Roni; Veselý, Tomáš; Pokorová, Dagmar; David, Lior; Wiegertjes, Geert F; Forlenza, Maria

2018-03-19

Koi Herpes Virus (KHV or Cyprinid Herpesvirus 3, CyHV-3) is among the most threatening pathogens affecting common carp production as well as the highly valuable ornamental koi carp. To date, no effective commercial vaccine is available for worldwide use. A previous study reported that three intramuscular injections with an ORF25-based DNA vaccine, led to the generation of neutralizing antibodies and conferred significant protection against an intraperitoneal challenge with KHV. In the present study, we set out to optimize an ORF25-based DNA vaccination protocol that required fewer injections and would confer protection upon a challenge that better resembled the natural route of infection. To this end, ORF25 was cloned in pcDNA3 either as a soluble protein or as a full-length transmembrane GFP-fusion protein. We tested our ORF25-based DNA vaccines in multiple vaccination trials using different doses, vaccination routes (i.m. injection and oral gavage) and challenge methods (bath and cohabitation). Furthermore, we analysed local and systemic responses to the i.m. injected DNA vaccine through histological and RT-qPCR analysis. We observed a strong protection when fish received three injections of either of the two DNA vaccines. However, this protection was observed only after bath challenge and not after cohabitation challenge. Furthermore, protection was insufficient when fish received one injection only, or received the plasmid orally. The importance of choosing a challenge model that best reflects the natural route of infection and the possibility to include additional antigens in future DNA vaccination strategies against KHV will be discussed. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Antioxidant Potential and DNA Damage Protection by the Slate Grey Saddle Mushroom, Helvella lacunosa (Ascomycetes), from Kashmir Himalaya (India).

PubMed

Shameem, Nowsheen; Kamili, Azra N; Ahmad, Mushtaq; Masoodi, F A; Parray, Javid A

2016-01-01

This study pertains to the radical scavenging potential of and DNA protection by Helvella lacunosa, an edible mushroom from Kashmir Himalaya (India). Different solvents, on the basis of their polarities, were used to extract all solvent-soluble bioactive compounds. Seven different antioxidant methods were also used to determine extensive radical scavenging activity. The mushroom ethanol extract and butanol extract showed effective scavenging activity of radicals at 95% and 89%, respectively. At 800 µg/mg, the ethanol extract was potent enough to protect DNA from degradation by hydroxyl radicals. It is evident from these findings that the presence of antioxidant substances signifies the use of H. lacunosa as food in the mountainous valleys of the Himalayan region.

Positive immunomodulatory effects of heterologous DNA vaccine- modified live vaccine, prime-boost immunization, against the highly-pathogenic PRRSV infection.

PubMed

Sirisereewan, Chaitawat; Nedumpun, Teerawut; Kesdangsakonwut, Sawang; Woonwong, Yonlayong; Kedkovid, Roongtham; Arunorat, Jirapat; Thanawongnuwech, Roongroje; Suradhat, Sanipa

2017-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) infection is one of the most important swine pathogens, and causes a major economic impact worldwide. Recently, a new variant type 2 PRRSV, highly pathogenic PRRSV (HP-PRRSV) has emerged and continued to circulate in Southeast Asia region. Currently, commercially available PRRSV vaccines, modified live PRRS vaccines (MLV) are not able to provide complete protection against HP-PRRSV and been reported to induce negative immunomodulatory effects. Interestingly, a novel DNA vaccine was developed and successfully used to improve PRRSV-specific immune responses following MLV vaccination. To investigate the efficacy of a heterologous DNA-MLV prime-boost immunization against the HP-PRRSV infection, an experimental vaccinated-challenged study was conducted. Two-week-old, PRRSV-seronegative, crossbred pigs (5-8 pigs/group) were allocated into 5 groups. At day -14 (D-14), the treatment group (DNA-MLV) was immunized with a DNA vaccine encoding PRRSV-truncated nucleocapsid protein (pORF7t), followed by a commercial modified live type 2 PRRS vaccine (MLV) at D0. The other groups included the group that received PBS at D-14 followed by MLV at D0 (MLV), pORF7t at D-14 (DNA), PBS at D0 (PBS) and the negative control group. At D42, all groups, except the negative control group, were challenged with HP-PRRSV (strain 10PL1). The results demonstrated that pigs that received MLV, regardless of the DNA priming, exhibited less clinical signs and faster viral clearance. Following HP-PRRSV challenge, the DNA-MLV group exhibited improved PRRSV-specific immunity, as observed by increased neutralizing antibody titers and PRRSV-specific IFN-γ production, and reduced IL-10 and PRRSV-specific Treg productions. However, neither the prime-boost immunization nor the MLV was able to induce complete clinical protection against HP-PRRSV infection. In conclusion, improved immunological responses, but not clinical protection, were achieved by

Identification of conserved pathways of DNA-damage response and radiation protection by genome-wide RNAi.

PubMed

van Haaften, Gijs; Romeijn, Ron; Pothof, Joris; Koole, Wouter; Mullenders, Leon H F; Pastink, Albert; Plasterk, Ronald H A; Tijsterman, Marcel

2006-07-11

Ionizing radiation is extremely harmful for human cells, and DNA double-strand breaks (DSBs) are considered to be the main cytotoxic lesions induced. Improper processing of DSBs contributes to tumorigenesis, and mutations in DSB response genes underlie several inherited disorders characterized by cancer predisposition. Here, we performed a comprehensive screen for genes that protect animal cells against ionizing radiation. A total of 45 C. elegans genes were identified in a genome-wide RNA interference screen for increased sensitivity to ionizing radiation in germ cells. These genes include orthologs of well-known human cancer predisposition genes as well as novel genes, including human disease genes not previously linked to defective DNA-damage responses. Knockdown of eleven genes also impaired radiation-induced cell-cycle arrest, and seven genes were essential for apoptosis upon exposure to irradiation. The gene set was further clustered on the basis of increased sensitivity to DNA-damaging cancer drugs cisplatin and camptothecin. Almost all genes are conserved across animal phylogeny, and their relevance for humans was directly demonstrated by showing that their knockdown in human cells results in radiation sensitivity, indicating that this set of genes is important for future cancer profiling and drug development.

Resveratrol protects mouse embryonic stem cells from ionizing radiation by accelerating recovery from DNA strand breakage.

PubMed

Denissova, Natalia G; Nasello, Cara M; Yeung, Percy L; Tischfield, Jay A; Brenneman, Mark A

2012-01-01

Resveratrol has elicited many provocative anticancer effects in laboratory animals and cultured cells, including reduced levels of oxidative DNA damage, inhibition of tumor initiation and progression and induction of apoptosis in tumor cells. Use of resveratrol as a cancer-preventive agent in humans will require that its anticancer effects not be accompanied by damage to normal tissue stem or progenitor cells. In mouse embryonic stem cells (mESC) or early mouse embryos exposed to ethanol, resveratrol has been shown to suppress apoptosis and promote survival. However, in cells exposed to genotoxic stress, survival may come at the expense of genome stability. To learn whether resveratrol can protect stem cells from DNA damage and to study its effects on genomic integrity, we exposed mESC pretreated with resveratrol to ionizing radiation (IR). Forty-eight hours pretreatment with a comparatively low concentration of resveratrol (10 μM) improved survival of mESC >2-fold after exposure to 5 Gy of X-rays. Cells pretreated with resveratrol sustained the same levels of reactive oxygen species and DNA strand breakage after IR as mock-treated controls, but repaired DNA damage more rapidly and resumed cell division sooner. Frequencies of IR-induced mutation at a chromosomal reporter locus were not increased in cells pretreated with resveratrol as compared with controls, indicating that resveratrol can improve viability in mESC after DNA damage without compromising genomic integrity.

Effects of melatonin on DNA damage induced by cyclophosphamide in rats

PubMed Central

Ferreira, S.G.; Peliciari-Garcia, R.A.; Takahashi-Hyodo, S.A.; Rodrigues, A.C.; Amaral, F.G.; Berra, C.M.; Bordin, S.; Curi, R.; Cipolla-Neto, J.

2013-01-01

The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and of Xpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy. PMID:23471360

A demonstrator for an integrated subway protection system

NASA Astrophysics Data System (ADS)

Detoma, E.; Capetti, P.; Casati, G.; Billington, S.

2008-04-01

In 2006 SEPA has carried on the installation and tests of a demonstrator for an integrated subway protection system at a new subway station in the Naples, Italy) metropolitan area. Protection of a subway system is a difficult task given the amount of passengers transported every day. The demonstrator has been limited to non-intrusive detection techniques not to impair the passenger flow into the station. The demonstrator integrates several technologies and products that have been developed by SEPA or are already available on the market (MKS Instruments,...). The main purpose is to provide detection capabilities for attempts to introduce radioactive substances in the subway station, in order to foil possible attempts to place a dirty bomb, and threat detection and identification following release of chemical agents. The system integrates additional sensors such as video surveillance cameras and air flow sensing to complement the basic sensors suite. The need to protect sensitive installations such as subway stations has been highlighted by the series of terroristics actions carried out in recent years in the subway in London. However, given the number of passengers of a metro system, it is impossible to propose security techniques operating in ways similar to the screening of passengers in airports. Passengers screening and threat detection and identification must be quick, non-intrusive and capable of screening a large number of passengers to be applicable to mass transit systems. In 2005 SEPA, a small company operating in the field of trains video-surveillance systems and radiation detectors, started developing an integrated system to provide a comprehensive protection to subway stations, based on ready available or off-the-shelf components in order to quickly develop a reliable system with available technology. We ruled out at the beginning any new development in order to speed up the fielding of the system in less than one year. The system was developed with

Experimental iron-inactivated Pasteurella multocida A: 1 vaccine adjuvanted with bacterial DNA is safe and protects chickens from fowl cholera.

PubMed

Herath, Chitra; Kumar, Pankaj; Singh, Mithilesh; Kumar, Devender; Ramakrishnan, Saravanan; Goswami, Tapas Kumar; Singh, Ajit; Ram, G C

2010-03-08

Fowl cholera is a serious problem in large and small scale poultry production. The present study describes the development and testing of an inactivated whole-cell, low-cost, safe, and effective vaccine for fowl cholera based on a previous work (Vaccine 23:5590-5598). Pasteurella multocida A: 1 grown in the presence of low FeCl(3) concentrations, inactivated with higher concentrations of FeCl(3), and adjuvanted with bacterial DNA from P. multocida B: 2 containing immunostimulatory CpG motifs protect chickens with a lethal P. multocida A: 1 challenge. Chickens were immunized with two whole-cell inactivated vaccine doses at 4 weeks apart and challenged 4 weeks after booster immunization. Experimental vaccines were pure, easy injectable, and caused very little distress in chickens due to their aqueous consistency. Vaccines and bacterial DNA (bDNA) posed no safety problems when chickens were injected subcutaneously (s.c.) with a single, double, and overdose of these preparations. Immunized chickens produced systemic IgY antibodies (Ab) responses and vaccine adjuvanted with bDNA protected 100% chickens from lethal intrapertoneal (i.p.) P. multocida A: 1 challenge. This work suggests that use of bDNA as an adjuvant can improve the cost-effectiveness of inactivated veterinary vaccines for their use in developing countries. Our future studies will focus on safety and potency evaluation of experimental and current vaccines using bDNA as an adjuvant. Copyright 2010 Elsevier Ltd. All rights reserved.

Mutagenic effect of freezing on nuclear DNA of Saccharomyces cerevisiae.

PubMed

Todorova, T; Pesheva, M; Stamenova, R; Dimitrov, M; Venkov, P

2012-05-01

Although fragmentation of DNA has been observed in cells undergoing freezing procedures, a mutagenic effect of sub-zero temperature treatment has not been proved by induction and isolation of mutants in nuclear DNA (nDNA). In this communication we supply evidence for mutagenicity of freezing on nDNA of Saccharomyces cerevisiae cells. In the absence of cryoprotectors, cooling for 2 h at +4°C and freezing for 1 h at -10°C and 16 h at -20°C, with a cooling rate of 3°C/min, resulted in induction of frame-shift and reverse mutations in microsatellite and coding regions of nDNA. The sub-zero temperature exposure also has a strong recombinogenic effect, evidenced by induction of gene-conversion and crossing-over events. Freezing induces mutations and enhances recombination with a frequency equal to or higher than that of methylmethanesulphonate at comparable survival rates. The signals for the appearance of nDNA lesions induced by freezing are detected and transduced by the DNA damage pathway. Extracellular cryoprotectors did not prevent the mutagenic effect of freezing, while accumulation of trehalose inside cells reduced nDNA cryodamage. Freezing of cells is accompanied by generation of high ROS levels, and the oxidative stress raised during the freeze-thaw process is the most likely reason for the DNA damaging effect. Experiments with mitochondrial rho⁻ mutants or scavengers of ROS indicated that mutagenic and recombinogenic effects of sub-zero temperatures can be decreased but not eliminated by reduction of ROS level. The complete protection against cryodamage in nDNA required simultaneous usage of intracellular cryoprotector and ROS scavenger during the freeze-thaw process. Copyright © 2012 John Wiley & Sons, Ltd.

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.

Effects of a Mangifera indica L. stem bark extract and mangiferin on radiation-induced DNA damage in human lymphocytes and lymphoblastoid cells.

PubMed

Rodeiro, I; Delgado, R; Garrido, G

2014-02-01

Mangifera indica L. (mango) stem bark aqueous extract (MSBE) that has antioxidant, anti-inflammatory and immunomodulatory properties, can be obtained in Cuba. It is rich in polyphenols, where mangiferin is the main component. In this study, we have tested DNA damage and protection effects of MSBE and mangiferin on primary human lymphocytes and lymphoblastoid cells. Cell suspensions were incubated with the products (50-1000 μg/ml) for experiments on damage induction, and evaluation of any potential protective effects (5-100 μg/ml) for 60 min at 37 °C. Irradiation was performed using a γ-ray source, absorbed dose 5 Gy. At the end of exposure, DNA damage, protection and repair processes were evaluated using the comet assay. MSBE (100-1000 μg/ml) induced DNA damage in a concentration dependent manner in both cell types tested, primary cells being more sensitive. Mangiferin (200 μg/ml) only induced light DNA damage at higher concentrations. DNA repair capacity was not affected after MSBE or mangiferin exposure. On the other hand, MSBE (25 and 50 μg/ml) and mangiferin (5-25 ug/ml) protected against gamma radiation-induced DNA damage. These results show MSBE has protector or harmful effects on DNA in vitro depending on the experimental conditions, which suggest that the extract could be acting as an antioxidant or pro-oxidant product. Mangiferin was involved in protective effects of the extract. © 2013 John Wiley & Sons Ltd.

Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

PubMed Central

Warleta, Fernando; Quesada, Cristina Sánchez; Campos, María; Allouche, Yosra; Beltrán, Gabriel; Gaforio, José J.

2011-01-01

Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells. PMID:22254082

DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions

PubMed Central

Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

2016-01-01

Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. PMID:27604871

A Demonstration Project in New York and Virginia: Retrofitting Cost-Effective Roll-over Protective Structures (CROPS) on Tractors.

PubMed

Hard, D L; McKenzie, E A; Cantis, D; May, J; Sorensen, J; Bayes, B; Madden, E; Wyckoff, S; Stone, B; Maass, J

2015-07-01

The NIOSH cost-effective roll-over protective structure (CROPS) demonstration project sought to determine whether three prototype roll-over protective structures (ROPS) designed to be retrofitted on Ford 8N, Ford 3000, Ford 4000, and Massey Ferguson 135 tractors could be installed in the field and whether they would be acceptable by the intended end users (farmers). There were a total of 50 CROPS. demonstrators (25 in New York and 25 in Virginia), with 45 observers attending the New York CROPS demonstrations and 36 observers attending the Virginia CROPS demonstrations, for a total of 70 participants in New York and 61 in Virginia. The oldest retrofitted tractors were 77 to 62 years old, while the newest retrofitted tractors were 40 to 37 years old. The most frequently retrofitted tractor in the CROPS demonstration project was a Ford 3000 series tractor (n = 19; 38%), followed by Ford 4000 (n = 11; 22%), Massey Ferguson 135 (n = 11; 22%), and Ford 8N (n = 9; 18%). A major issue of CROPS retrofitting was the rear wheel fenders. The effort involved in disassembling the fenders (removing the old bolts was often faster by cutting them with a torch), modifying the fender mounting brackets, and then reinstalling the fenders with the CROPS generally required the most time. In addition, various other semi-permanent equipment attachments, such as front-end loaders, required additional time and effort to fit with the CROPS. Demonstrators were asked to rank the reasons why they had not retrofitted their tractors with ROPS until they had enrolled in the CROPS demonstration program. ROPS "cost too much" was ranked as the primary reason for participants in both states (80% for New York and 88% for Virginia). The second highest ranked reasons were "ROPS wasn't available" for Virginia (80%) and "hassle to find ROPS" for New York (69%). The third highest ranked reasons were "not enough time to find ROPS" for New York (67%) and "hassle to find ROPS" for Virginia (79%). All

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

PubMed Central

Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

2009-01-01

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

Protective effect of grape seed extracts on human lymphocytes: a preliminary study.

PubMed

Szeto, Yim Tong; Lee, Kit Yee; Kalle, Wouter; Pak, Sok Cheon

2013-03-01

Grape seed extracts (GSEs) possess a broad spectrum of antioxidative properties that protects various cells from free radicals and oxidative stress. In this study, the genoprotective effect of GSE on human lymphocytic DNA was studied using standard and lysed cell comet assays. Lymphocytes from 5 healthy subjects were pretreated with GSE in different concentrations. The standard and lysed cell comet assays were performed on treated, untreated, challenged, and unchallenged cells in parallel. Cells were then subjected to an oxidant challenge induced with 5-min exposures to hydrogen peroxide. In the standard comet assay, GSE significantly diminished hydrogen-peroxide-induced DNA damage in a dose-dependent manner. In the lysed cell assay, however, the antioxidant effect was diminished at a higher GSE concentration. Data indicate that the cell membrane might play a role in limiting cellular access to antioxidants, which directly affects the genoprotective or potential pro-oxidant effect of antioxidants on human DNA. Using both standard and lysed cell comet assays in parallel could be a useful way to elucidate the mechanism of protection or damage by antioxidants.

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.

Chemical Composition, Antioxidant, DNA Damage Protective, Cytotoxic and Antibacterial Activities of Cyperus rotundus Rhizomes Essential Oil against Foodborne Pathogens

PubMed Central

Hu, Qing-Ping; Cao, Xin-Ming; Hao, Dong-Lin; Zhang, Liang-Liang

2017-01-01

Cyperus rotundus L. (Cyperaceae) is a medicinal herb traditionally used to treat various clinical conditions at home. In this study, chemical composition of Cyperus rotundus rhizomes essential oil, and in vitro antioxidant, DNA damage protective and cytotoxic activities as well as antibacterial activity against foodborne pathogens were investigated. Results showed that α-cyperone (38.46%), cyperene (12.84%) and α-selinene (11.66%) were the major components of the essential oil. The essential oil had an excellent antioxidant activity, the protective effect against DNA damage, and cytotoxic effects on the human neuroblastoma SH-SY5Y cell, as well as antibacterial activity against several foodborne pathogens. These biological activities were dose-dependent, increasing with higher dosage in a certain concentration range. The antibacterial effects of essential oil were greater against Gram-positive bacteria as compared to Gram-negative bacteria, and the antibacterial effects were significantly influenced by incubation time and concentration. These results may provide biological evidence for the practical application of the C. rotundus rhizomes essential oil in food and pharmaceutical industries. PMID:28338066

Protection against avian influenza H9N2 virus challenge by immunization with hemagglutinin- or neuraminidase-expressing DNA in BALB/c mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu Meizhen; Fang Fang; Chen Yan

2006-05-19

Avian influenza viruses of H9N2 subtype are widely spread in avian species. The viruses have recently been transmitted to mammalian species, including humans, accelerating the efforts to devise protective strategies against them. In this study, an avian influenza H9N2 virus strain (A/Chicken/Jiangsu/7/2002), isolated in Jiangsu Province, China, was used to infect BALB/c mice for adaptation. After five lung-to-lung passages, the virus was stably proliferated in a large quantity in the murine lung and caused the deaths of mice. In addition, we explored the protection induced by H9N2 virus hemagglutinin (HA)- and neuraminidase (NA)-expressing DNAs in BALB/c mice. Female BALB/c micemore » aged 6-8 weeks were immunized once or twice at a 3-week interval with HA-DNA and NA-DNA by electroporation, respectively, each at a dose of 3, 10 or 30 {mu}g. The mice were challenged with a lethal dose (40x LD{sub 5}) of influenza H9N2 virus four weeks after immunization once or one week after immunization twice. The protections of DNA vaccines were evaluated by the serum antibody titers, residual lung virus titers, and survival rates of the mice. The result showed that immunization once with not less than 10 {mu}g or twice with 3 {mu}g HA-DNA or NA-DNA provided effective protection against homologous avian influenza H9N2 virus.« less

Chemical composition and inhibitory effects of water extract of Henna leaves on reactive oxygen species, DNA scission and proliferation of cancer cells

PubMed Central

Kumar, Manish; Chandel, Madhu; Kaur, Paramjeet; Pandit, Kritika; Kaur, Varinder; Kaur, Sandeep; Kaur, Satwinderjeet

2016-01-01

From the centuries, Lawsonia inermis L. (Henna) is utilized in traditional health care system as a medicinal and cosmetic agent. The present study was intended to assess antiradical, DNA protective and antiproliferative activity of water extract of Lawsonia inermis L. leaves (W-LI). Antioxidant activity was estimated using various in vitro assays such as DPPH, ABTS, superoxide anion radical scavenging, FRAP, deoxyribose degradation and DNA protection assay. Growth inhibitory effects of W-LI were assessed using MTT assay against different cancer cell lines viz. HeLa, MCF-7, A549, C6 and COLO-205. From the results of antioxidant assays, it was found that W-LI quenched DPPH and ABTS cation radicals with IC50 value of 352.77 µg/ml and 380.87 µg/ml respectively. It demonstrated hydroxyl radical scavenging potential of 59.75 % at highest test dose of 1000 µg/ml in deoxyribose degradation assay. The results of FRAP assay showed that W-LI also possesses significant reducing activity. Extract inhibited hydroxyl radical induced pBR322 plasmid DNA strand scission, thus conferring DNA protection. Growth inhibition of various cancer cell lines was achieved to the varying extent on treatment with W-LI. Further, it was observed that activity was quite promising against colon cancer COLO-205 cells (GI50 121.03 µg/ml). HPLC profiling of W-LI revealed the presence of different polyphenolic compounds such as ellagic acid, catechin, quercetin, kaempferol etc. which might be contributing towards antioxidant and cytotoxic activity. The present study demonstrated that polyphenols rich W-LI extract from leaves of L. inermis possesses ability to inhibit oxidative radicals and cancer cells proliferation. PMID:28337113

DNA damage by various radiations

NASA Astrophysics Data System (ADS)

Hasegawa, K.; Yoshioka, H.; Yoshioka, H.

1997-01-01

In an attempt to shed light on the influence of tritiated water on DNA we have investigated the post-irradiation damage with a simple plasmid DNA, pBR322 and pUC18. The survival of covalently closed circular (CCC) DNA form was directly followed by agarose gel electrophoresis. The survival percentage of DNA in tritiated water was almost the same as with the irradiation with X-rays at the same absorbed dose. For irradiation with γ-rays, on the other hand, the decay rate was larger than those observed with both tritiated water and X-rays. The percentages of breakage for DNA in tritiated water, X-rays and γ-rays were found to be 34, 38 and 33% at 100 Gy of absorbed dose. The effect of dose rate was not observed for irradiation with tritiated water, X-rays and γ-rays. In order to study protection of DNA against radiation, we investigated the protecting effect of tea catechin which is the main component of (-)-epigallocatechin gallate (EGCg). The protection mechanism for DNA against radiation-induced scission has been studied using ESR spin-trapping method.

Curcumin stably interacts with DNA hairpin through minor groove binding and demonstrates enhanced cytotoxicity in combination with FdU nucleotides.

PubMed

Ghosh, Supratim; Mallick, Sumana; Das, Upasana; Verma, Ajay; Pal, Uttam; Chatterjee, Sabyasachi; Nandy, Abhishek; Saha, Krishna D; Maiti, Nakul Chandra; Baishya, Bikash; Suresh Kumar, G; Gmeiner, William H

2018-03-01

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV-Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two ɣ H of curcumin heptadiene chain are closely positioned to the A 16 -H8 and A 17 -H8, while G 12 -H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Protection of Mice against Plasmodium yoelii Sporozoite Challenge with P. yoelii Merozoite Surface Protein 1 DNA Vaccines

PubMed Central

Becker, Sylvia I.; Wang, Ruobing; Hedstrom, Richard C.; Aguiar, Joao C.; Jones, Trevor R.; Hoffman, Stephen L.; Gardner, Malcolm J.

1998-01-01

Immunization of mice with DNA vaccines encoding the full-length form and C and N termini of Plasmodium yoelii merozoite surface protein 1 provided partial protection against sporozoite challenge and resulted in boosting of antibody titers after challenge. In C57BL/6 mice, two DNA vaccines provided protection comparable to that of recombinant protein consisting of the C terminus in Freund’s adjuvant. PMID:9632624

DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions.

PubMed

Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

2016-11-02

Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

ESWT and alendronate sodium demonstrate equal protective effects in osteoarthritis of the knee

NASA Astrophysics Data System (ADS)

Wang, Ching-Jen; Chou, Wen-Yi; Hsu, Shan-Ling; Huang, Chien-Yiu; Cheng, Jai-Hong

2016-01-01

This study compared the effects of extracorporeal shock wave therapy (ESWT) and alendronate sodium (alendronate) in osteoarthritis (OA) of rat knees. The control group was subjected to a sham surgery and did not receive either ESWT or alendronate treatment. The OA group underwent anterior cruciate ligament transection (ACLT) and medial meniscectomy (MM) surgery and did not receive either ESWT or alendronate. The ESWT group underwent ACLT and MM surgery and received ESWT after the surgery. The alendronate group received alendronate after ACLT and MM surgery. The evaluations included radiograph, bone mineral density (BMD), serum C-telopeptide collagen II (CTX-II), cartilage oligomeric protein (COMP), alkaline phosphatase and osteocalcin, histopathological examination and immunohistochemical analysis. Radiographs at 12 weeks showed pronounced OA changes in the OA group. The BMD values, CTX-II, COMP, alkaline phosphatase and osteocalcin showed no significant difference between ESWT and alendronate groups. In histopathology, the Mankin and Safranin O scores significantly increased in the OA, ESWT and alendronate groups, but without any significant difference between the ESWT and alendronate groups. In immunohistochemical analysis, the von Willebrand factor (vWF), vascular endothelial factor (VEGF), soluble vascular cell adhesion molecule (sVCAM), proliferating cell nuclear antigen (PCNA), bone morphogenetic protein 2 (BMP-2), and osteocalcin expressions in articular cartilage and subchondral bone showed a significant decrease in the OA group, but no difference was noted between the ESWT and alendronate groups. In conclusion, ESWT and alendronate sodium demonstrate equal protective effects from developing osteoarthritis of the knee in rats.

Helminth Coinfection Does Not Affect Therapeutic Effect of a DNA Vaccine in Mice Harboring Tuberculosis

PubMed Central

Frantz, Fabiani G.; Rosada, Rogério S.; Peres-Buzalaf, Camila; Perusso, Franciele R. T.; Rodrigues, Vanderlei; Ramos, Simone G.; Kunkel, Steven L.; Silva, Célio L.; Faccioli, Lúcia H.

2010-01-01

Background Helminthiasis and tuberculosis (TB) coincide geographically and there is much interest in exploring how concurrent worm infections might alter immune responses against bacilli and might necessitate altered therapeutic approaches. A DNA vaccine that codifies heat shock protein Hsp65 from M. leprae (DNAhsp65) has been used in therapy during experimental tuberculosis. This study focused on the impact of the co-existence of worms and TB on the therapeutic effects of DNAhsp65. Methodology/Principal Findings Mice were infected with Toxocara canis or with Schistosoma mansoni, followed by coinfection with M. tuberculosis and treatment with DNAhsp65. While T. canis infection did not increase vulnerability to pulmonary TB, S. mansoni enhanced susceptibility to TB as shown by higher numbers of bacteria in the lungs and spleen, which was associated with an increase in Th2 and regulatory cytokines. However, in coinfected mice, the therapeutic effect of DNAhsp65 was not abrogated, as indicated by colony forming units and analysis of histopathological changes. In vitro studies indicated that Hsp65-specific IFN-γ production was correlated with vaccine-induced protection in coinfected mice. Moreover, in S. mansoni-coinfected mice, DNA treatment inhibited in vivo TGF-β and IL-10 production, which could be associated with long-term protection. Conclusions/Significance We have demonstrated that the therapeutic effects of DNAhsp65 in experimental TB infection are persistent in the presence of an unrelated Th2 immune response induced by helminth infections. PMID:20544012

The use of suction blisters to measure sunscreen protection against UVR-induced DNA damage.

PubMed

Josse, Gwendal; Douki, Thierry; Le Digabel, Jimmy; Gravier, Eleonore; Questel, Emmanuel

2018-02-01

The formation of DNA photoproducts caused by solar UVR exposure needs to be investigated in-vivo and in particular in order to assess sunscreens' level of protection against solar genotoxicity. The study's purposes were: i) to evaluate if the roof of suction blisters is an appropriate sampling method for measuring photoproducts, and ii) to measure in-vivo sunscreen protection against cyclobutane pyrimidine dimers. Skin areas on the interior forearms of eight healthy volunteers were exposed in-vivo to 2 MED of simulated solar radiation (SSR) and to 15 MED on a sunscreen protected area. After irradiation, six suction blisters were induced and the blister roofs were collected. Analysis of SSR-induced CPDs was performed by two independent methods: a chromatography coupled to mass spectroscopy (HPLC-MS/MS) approach and a 3D-imaging of CPD immunostaining by multiphoton microscopy on floating epidermal sheets. HPLC-MS/MS analyses showed that SSR-unexposed skin presented no CPD dimers, whereas 2 MED SSR-exposed skin showed a significant number of TT-CPD. The sunscreen covered skin exposed to 15 MED appeared highly protected from DNA damage, as the amount of CPD-dimers remained below the detection limit. The multiphoton-immunostaining analysis consistently showed that no CPD staining was observed on the non-SSR-exposed skin. A significant increase of CPD staining intensity and number of CPD-positive cells were observed on the 2 MED SSR-exposed skin. Sunscreen protected skin presented a very low staining intensity and the number of CPD-positive cells remained very close to non-SSR-exposed skin. This study showed that suction blister samples are very appropriate for measuring CPD dimers in-vivo, and that sunscreens provide high protection against UVR-induced DNA damage. Copyright © 2017 Elsevier B.V. All rights reserved.

Environmental DNA for freshwater fish monitoring: insights for conservation within a protected area

PubMed Central

Sandin, Miguel M.; Beaulieu, Paul G.; Clusa, Laura; Martinez, Jose L.; Ardura, Alba; García-Vázquez, Eva

2018-01-01

Background Many fish species have been introduced in wild ecosystems around the world to provide food or leisure, deliberately or from farm escapes. Some of those introductions have had large ecological effects. The north American native rainbow trout (Oncorhynchus mykiss Walbaum, 1792) is one of the most widely farmed fish species in the world. It was first introduced in Spain in the late 19th century for sport fishing (Elvira 1995) and nowadays is used there for both fishing and aquaculture. On the other hand, the European native brown trout (Salmo trutta L.) is catalogued as vulnerable in Spain. Detecting native and invasive fish populations in ecosystem monitoring is crucial, but it may be difficult from conventional sampling methods such as electrofishing. These techniques encompass some mortality, thus are not adequate for some ecosystems as the case of protected areas. Environmental DNA (eDNA) analysis is a sensitive and non-invasive method that can be especially useful for rare and low-density species detection and inventory in water bodies. Methods In this study we employed two eDNA based methods (qPCR and nested PCR-RFLP) to detect salmonid species from mountain streams within a protected area, The Biosphere Reserve and Natural Park of Redes (Upper Nalón Basin, Asturias, Northern Spain), where brown trout is the only native salmonid. We also measured some habitat variables to see how appropriate for salmonids the area is. The sampling area is located upstream impassable dams and contains one rainbow trout fish farm. Results Employing qPCR methodology, brown trout eDNA was detected in all the nine sampling sites surveyed, while nested PCR-RFLP method failed to detect it in two sampling points. Rainbow trout eDNA was detected with both techniques at all sites in the Nalón River’ (n1, n2 and n3). Salmonid habitat units and water quality were high from the area studied. Discussion In this study, a high quantity of rainbow trout eDNA was found upstream and

Environmental DNA for freshwater fish monitoring: insights for conservation within a protected area.

PubMed

Fernandez, Sara; Sandin, Miguel M; Beaulieu, Paul G; Clusa, Laura; Martinez, Jose L; Ardura, Alba; García-Vázquez, Eva

2018-01-01

Many fish species have been introduced in wild ecosystems around the world to provide food or leisure, deliberately or from farm escapes. Some of those introductions have had large ecological effects. The north American native rainbow trout ( Oncorhynchus mykiss Walbaum, 1792) is one of the most widely farmed fish species in the world. It was first introduced in Spain in the late 19th century for sport fishing (Elvira 1995) and nowadays is used there for both fishing and aquaculture. On the other hand, the European native brown trout ( Salmo trutta L.) is catalogued as vulnerable in Spain. Detecting native and invasive fish populations in ecosystem monitoring is crucial, but it may be difficult from conventional sampling methods such as electrofishing. These techniques encompass some mortality, thus are not adequate for some ecosystems as the case of protected areas. Environmental DNA (eDNA) analysis is a sensitive and non-invasive method that can be especially useful for rare and low-density species detection and inventory in water bodies. In this study we employed two eDNA based methods (qPCR and nested PCR-RFLP) to detect salmonid species from mountain streams within a protected area, The Biosphere Reserve and Natural Park of Redes (Upper Nalón Basin, Asturias, Northern Spain), where brown trout is the only native salmonid. We also measured some habitat variables to see how appropriate for salmonids the area is. The sampling area is located upstream impassable dams and contains one rainbow trout fish farm. Employing qPCR methodology, brown trout eDNA was detected in all the nine sampling sites surveyed, while nested PCR-RFLP method failed to detect it in two sampling points. Rainbow trout eDNA was detected with both techniques at all sites in the Nalón River' (n1, n2 and n3). Salmonid habitat units and water quality were high from the area studied. In this study, a high quantity of rainbow trout eDNA was found upstream and downstream of a fish farm located

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.

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.

Immunization with plasmid DNA encoding the hemagglutinin and the nucleoprotein confers robust protection against a lethal canine distemper virus challenge.

PubMed

Dahl, Lotte; Jensen, Trine Hammer; Gottschalck, Elisabeth; Karlskov-Mortensen, Peter; Jensen, Tove Dannemann; Nielsen, Line; Andersen, Mads Klindt; Buckland, Robin; Wild, T Fabian; Blixenkrone-Møller, Merete

2004-09-09

We have investigated the protective effect of immunization of a highly susceptible natural host of canine distemper virus (CDV) with DNA plasmids encoding the viral nucleoprotein (N) and hemagglutinin (H). The combined intradermal and intramuscular routes of immunization elicited high virus-neutralizing serum antibody titres in mink (Mustela vison). To mimic natural exposure, we also conducted challenge infection by horizontal transmission from infected contact animals. Other groups received a lethal challenge infection by administration to the mucosae of the respiratory tract and into the muscle. One of the mink vaccinated with N plasmid alone developed severe disease after challenge. In contrast, vaccination with the H plasmid together with the N plasmid conferred solid protection against disease and we were unable to detect CDV infection in PBMCs or in different tissues after challenge. Our findings show that DNA immunization by the combined intradermal and intramuscular routes can confer solid protective immunity against naturally transmitted morbillivirus infection and disease.

A DNA vaccine for Crimean-Congo hemorrhagic fever protects against disease and death in two lethal mouse models

PubMed Central

Fitzpatrick, Collin J.; Suschak, John J.; Richards, Michelle J.; Badger, Catherine V.; Six, Carolyn M.; Martin, Jacqueline D.; Hannaman, Drew; Zivcec, Marko; Bergeron, Eric; Koehler, Jeffrey W.; Schmaljohn, Connie S.

2017-01-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus capable of causing a severe hemorrhagic fever disease in humans. There are currently no licensed vaccines to prevent CCHFV-associated disease. We developed a DNA vaccine expressing the M-segment glycoprotein precursor gene of CCHFV and assessed its immunogenicity and protective efficacy in two lethal mouse models of disease: type I interferon receptor knockout (IFNAR-/-) mice; and a novel transiently immune suppressed (IS) mouse model. Vaccination of mice by muscle electroporation of the M-segment DNA vaccine elicited strong antigen-specific humoral immune responses with neutralizing titers after three vaccinations in both IFNAR-/- and IS mouse models. To compare the protective efficacy of the vaccine in the two models, groups of vaccinated mice (7–10 per group) were intraperitoneally (IP) challenged with a lethal dose of CCHFV strain IbAr 10200. Weight loss was markedly reduced in CCHFV DNA-vaccinated mice as compared to controls. Furthermore, whereas all vector-control vaccinated mice succumbed to disease by day 5, the DNA vaccine protected >60% of the animals from lethal disease. Mice from both models developed comparable levels of antibodies, but the IS mice had a more balanced Th1/Th2 response to vaccination. There were no statistical differences in the protective efficacies of the vaccine in the two models. Our results provide the first comparison of these two mouse models for assessing a vaccine against CCHFV and offer supportive data indicating that a DNA vaccine expressing the glycoprotein genes of CCHFV elicits protective immunity against CCHFV. PMID:28922426

A DNA vaccine for Crimean-Congo hemorrhagic fever protects against disease and death in two lethal mouse models.

PubMed

Garrison, Aura R; Shoemaker, Charles J; Golden, Joseph W; Fitzpatrick, Collin J; Suschak, John J; Richards, Michelle J; Badger, Catherine V; Six, Carolyn M; Martin, Jacqueline D; Hannaman, Drew; Zivcec, Marko; Bergeron, Eric; Koehler, Jeffrey W; Schmaljohn, Connie S

2017-09-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus capable of causing a severe hemorrhagic fever disease in humans. There are currently no licensed vaccines to prevent CCHFV-associated disease. We developed a DNA vaccine expressing the M-segment glycoprotein precursor gene of CCHFV and assessed its immunogenicity and protective efficacy in two lethal mouse models of disease: type I interferon receptor knockout (IFNAR-/-) mice; and a novel transiently immune suppressed (IS) mouse model. Vaccination of mice by muscle electroporation of the M-segment DNA vaccine elicited strong antigen-specific humoral immune responses with neutralizing titers after three vaccinations in both IFNAR-/- and IS mouse models. To compare the protective efficacy of the vaccine in the two models, groups of vaccinated mice (7-10 per group) were intraperitoneally (IP) challenged with a lethal dose of CCHFV strain IbAr 10200. Weight loss was markedly reduced in CCHFV DNA-vaccinated mice as compared to controls. Furthermore, whereas all vector-control vaccinated mice succumbed to disease by day 5, the DNA vaccine protected >60% of the animals from lethal disease. Mice from both models developed comparable levels of antibodies, but the IS mice had a more balanced Th1/Th2 response to vaccination. There were no statistical differences in the protective efficacies of the vaccine in the two models. Our results provide the first comparison of these two mouse models for assessing a vaccine against CCHFV and offer supportive data indicating that a DNA vaccine expressing the glycoprotein genes of CCHFV elicits protective immunity against CCHFV.

DNA Damage Protecting Activity and Antioxidant Potential of Launaea taraxacifolia Leaves Extract.

PubMed

Adinortey, Michael Buenor; Ansah, Charles; Weremfo, Alexander; Adinortey, Cynthia Ayefoumi; Adukpo, Genevieve Etornam; Ameyaw, Elvis Ofori; Nyarko, Alexander Kwadwo

2018-01-01

The leaf extract of Launaea taraxacifolia commonly known as African Lettuce is used locally to treat dyslipidemia and liver diseases, which are associated with oxidative stress. Methanol extract from L. taraxacifolia leaves was tested for its antioxidant activity and its ability to protect DNA from oxidative damage. In vitro antioxidant potential of the leaf extract was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide (NO), and hydroxyl (OH) radical scavenging assays. Ferric reducing power, total antioxidant capacity (TAC), metal chelating, and anti-lipid peroxidation ability of the extract were also examined using gallic acid, ascorbic acid, citric acid, and ethylenediaminetetraacetic acid as standards. L. taraxacifolia leaves extract showed antioxidant activity with IC 50 values of 16.18 μg/ml (DPPH), 123.3 μg/ml (NO), 128.2 μg/ml (OH radical), 97.94 μg/ml (metal chelating), 80.28 μg/ml (TAC), and 23 μg/ml (anti-lipid peroxidation activity). L. taraxacifolia leaves extract exhibited a strong capability for DNA damage protection at 20 mg/ml concentration. These findings suggest that the methanolic leaf extract of L. taraxacifolia could be used as a natural antioxidant and also as a preventive therapy against diseases such as arteriosclerosis associated with DNA damage.

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.

DNA vaccination protects mice against Zika virus-induced damage to the testes

PubMed Central

Griffin, Bryan D.; Muthumani, Kar; Warner, Bryce M.; Majer, Anna; Hagan, Mable; Audet, Jonathan; Stein, Derek R.; Ranadheera, Charlene; Racine, Trina; De La Vega, Marc-Antoine; Piret, Jocelyne; Kucas, Stephanie; Tran, Kaylie N.; Frost, Kathy L.; De Graff, Christine; Soule, Geoff; Scharikow, Leanne; Scott, Jennifer; McTavish, Gordon; Smid, Valerie; Park, Young K.; Maslow, Joel N.; Sardesai, Niranjan Y.; Kim, J. Joseph; Yao, Xiao-jian; Bello, Alexander; Lindsay, Robbin; Boivin, Guy; Booth, Stephanie A.; Kobasa, Darwyn; Embury-Hyatt, Carissa; Safronetz, David; Weiner, David B.; Kobinger, Gary P.

2017-01-01

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract. PMID:28589934

Lithium promotes DNA stability and survival of ischemic retinal neurocytes by upregulating DNA ligase IV.

PubMed

Yang, Ying; Wu, Nandan; Tian, Sijia; Li, Fan; Hu, Huan; Chen, Pei; Cai, Xiaoxiao; Xu, Lijun; Zhang, Jing; Chen, Zhao; Ge, Jian; Yu, Keming; Zhuang, Jing

2016-11-17

Neurons display genomic fragility and show fragmented DNA in pathological degeneration. A failure to repair DNA breaks may result in cell death or apoptosis. Lithium protects retinal neurocytes following nutrient deprivation or partial nerve crush, but the underlying mechanisms are not well defined. Here we demonstrate that pretreatment with lithium protects retinal neurocytes from ischemia-induced damage and enhances light response in rat retina following ischemia-reperfusion injury. Moreover, we found that DNA nonhomologous end-joining (NHEJ) repair is implicated in this process because in ischemic retinal neurocytes, lithium significantly reduces the number of γ-H2AX foci (well-characterized markers of DNA double-strand breaks in situ) and increases the DNA ligase IV expression level. Furthermore, we also demonstrate that nuclear respiratory factor 1 (Nrf-1) and phosphorylated cyclic AMP-response element binding protein-1 (P-CREB1) bind to ligase IV promoter to cause upregulation of ligase IV in neurocytes. The ischemic upregulation of Nrf-1 and lithium-induced increase of P-CREB1 cooperate to promote transcription of ligase IV. Short hairpin RNAs against Nrf-1 and CREB1 could significantly inhibit the increase in promoter activity and expression of ligase IV observed in the control oligos following lithium treatment in retinal neurocytes. More importantly, ischemic stimulation triggers the expression of ligase IV. Taken together, our results thus reveal a novel mechanism that lithium offers neuroprotection from ischemia-induced damage by enhancing DNA NHEJ repair.

Lithium promotes DNA stability and survival of ischemic retinal neurocytes by upregulating DNA ligase IV

PubMed Central

Yang, Ying; Wu, Nandan; Tian, Sijia; Li, Fan; Hu, Huan; Chen, Pei; Cai, Xiaoxiao; Xu, Lijun; Zhang, Jing; Chen, Zhao; Ge, Jian; Yu, Keming; Zhuang, Jing

2016-01-01

Neurons display genomic fragility and show fragmented DNA in pathological degeneration. A failure to repair DNA breaks may result in cell death or apoptosis. Lithium protects retinal neurocytes following nutrient deprivation or partial nerve crush, but the underlying mechanisms are not well defined. Here we demonstrate that pretreatment with lithium protects retinal neurocytes from ischemia-induced damage and enhances light response in rat retina following ischemia–reperfusion injury. Moreover, we found that DNA nonhomologous end-joining (NHEJ) repair is implicated in this process because in ischemic retinal neurocytes, lithium significantly reduces the number of γ-H2AX foci (well-characterized markers of DNA double-strand breaks in situ) and increases the DNA ligase IV expression level. Furthermore, we also demonstrate that nuclear respiratory factor 1 (Nrf-1) and phosphorylated cyclic AMP-response element binding protein-1 (P-CREB1) bind to ligase IV promoter to cause upregulation of ligase IV in neurocytes. The ischemic upregulation of Nrf-1 and lithium-induced increase of P-CREB1 cooperate to promote transcription of ligase IV. Short hairpin RNAs against Nrf-1 and CREB1 could significantly inhibit the increase in promoter activity and expression of ligase IV observed in the control oligos following lithium treatment in retinal neurocytes. More importantly, ischemic stimulation triggers the expression of ligase IV. Taken together, our results thus reveal a novel mechanism that lithium offers neuroprotection from ischemia-induced damage by enhancing DNA NHEJ repair. PMID:27853172

Persistence of Protective Immunity to Malaria Induced by DNA Priming and Poxvirus Boosting: Characterization of Effector and Memory CD8+-T-Cell Populations

PubMed Central

Sedegah, Martha; Brice, Gary T.; Rogers, William O.; Doolan, Denise L.; Charoenvit, Yupin; Jones, Trevor R.; Majam, Victoria F.; Belmonte, Arnel; Lu, Minh; Belmonte, Maria; Carucci, Daniel J.; Hoffman, Stephen L.

2002-01-01

The persistence of immunity to malaria induced in mice by a heterologous DNA priming and poxvirus boosting regimen was characterized. Mice were immunized by priming with DNA vaccine plasmids encoding the Plasmodium yoelii circumsporozoite protein (PyCSP) and murine granulocyte-macrophage colony-stimulating factor and boosting with recombinant vaccinia encoding PyCSP. BALB/c mice immunized with either high-dose (100 μg of p PyCSP plus 30 μg of pGM-CSF) or low-dose (1 μg of p PyCSP plus 1 μg of pGM-CSF DNA) priming were protected against challenge with 50 P. yoelii sporozoites. Protection 2 weeks after immunization was 70 to 100%, persisted at this level for at least 20 weeks, and declined to 30 to 40% by 28 weeks. Eight of eight mice protected at 20 weeks were still protected when rechallenged at 40 weeks. The antigen (Ag)-specific effector CD8+-T-cell population present 2 weeks after boosting had ex vivo Ag-specific cytolytic activity, expressed both gamma interferon (IFN-γ) and tumor necrosis factor alpha, and constituted 12 to 20% of splenic CD8+ T cells. In contrast, the memory CD8+-Ag-specific-cell population at 28 weeks lacked cytolytic activity and constituted only 6% of splenic CD8+ T cells, but at the single-cell level it produced significantly higher levels of IFN-γ than the effectors. High levels of Ag- or parasite-specific antibodies present 2 weeks after boosting had declined three- to sevenfold by 28 weeks. Low-dose priming was similarly immunogenic and as protective as high-dose priming against a 50-, but not a 250-, sporozoite challenge. These results demonstrate that a heterologous priming and boosting vaccination can provide lasting protection against malaria in this model system. PMID:12065488

Salmonella DNA Adenine Methylase Mutants Confer Cross-Protective Immunity

PubMed Central

Heithoff, Douglas M.; Enioutina, Elena Y.; Daynes, Raymond A.; Sinsheimer, Robert L.; Low, David A.; Mahan, Michael J.

2001-01-01

Salmonella isolates that lack or overproduce DNA adenine methylase (Dam) elicited a cross-protective immune response to different Salmonella serovars. The protection afforded by the Salmonella enterica serovar Typhimurium Dam vaccine was greater than that elicited in mice that survived a virulent infection. S. enterica serovar Typhimurium Dam mutant strains exhibited enhanced sensitivity to mediators of innate immunity such as antimicrobial peptides, bile salts, and hydrogen peroxide. Also, S. enterica serovar Typhimurium Dam− vaccines were not immunosuppressive; unlike wild-type vaccines, they failed to induce increased nitric oxide levels and permitted a subsequent robust humoral response to diptheria toxoid antigen in infected mice. Dam mutant strains exhibited a low-grade persistence which, coupled with the nonimmunosuppression and the ectopic protein expression caused by altered levels of Dam, may provide an expanded source of potential antigens in vaccinated hosts. PMID:11598044

Quantitative analysis of the flexibility effect of cisplatin on circular DNA

NASA Astrophysics Data System (ADS)

Ji, Chao; Zhang, Lingyun; Wang, Peng-Ye

2013-10-01

We study the effects of cisplatin on the circular configuration of DNA using atomic force microscopy (AFM) and observe that the DNA gradually transforms to a complex configuration with an intersection and interwound structures from a circlelike structure. An algorithm is developed to extract the configuration profiles of circular DNA from AFM images and the radius of gyration is used to describe the flexibility of circular DNA. The quantitative analysis of the circular DNA demonstrates that the radius of gyration gradually decreases and two processes on the change of flexibility of circular DNA are found as the cisplatin concentration increases. Furthermore, a model is proposed and discussed to explain the mechanism for understanding the complicated interaction between DNA and cisplatin.

An improved DNA force field for ssDNA interactions with gold nanoparticles

NASA Astrophysics Data System (ADS)

Jiang, Xiankai; Gao, Jun; Huynh, Tien; Huai, Ping; Fan, Chunhai; Zhou, Ruhong; Song, Bo

2014-06-01

The widespread applications of single-stranded DNA (ssDNA) conjugated gold nanoparticles (AuNPs) have spurred an increasing interest in the interactions between ssDNA and AuNPs. Despite extensive studies using the most sophisticated experimental techniques, the detailed molecular mechanisms still remain largely unknown. Large scale molecular dynamics (MD) simulations can thus be used to supplement experiments by providing complementary information about ssDNA-AuNP interactions. However, up to now, all modern force fields for DNA were developed based on the properties of double-stranded DNA (dsDNA) molecules, which have hydrophilic outer backbones "protecting" hydrophobic inner nucleobases from water. Without the double-helix structure of dsDNA and thus the "protection" by the outer backbone, the nucleobases of ssDNA are directly exposed to solvent, and their behavior in water is very different from that of dsDNA, especially at the interface with nanoparticles. In this work, we have improved the force field of ssDNA for use with nanoparticles, such as AuNPs, based on recent experimental results and quantum mechanics calculations. With the new improved force field, we demonstrated that a poly(A) sequence adsorbed on a AuNP surface is much more stable than a poly(T) sequence, which is consistent with recent experimental observations. On the contrary, the current standard force fields, including AMBER03, CHARMM27, and OPLSAA, all gave erroneous results as compared to experiments. The current improved force field is expected to have wide applications in the study of ssDNA with nanomaterials including AuNPs, which might help promote the development of ssDNA-based biosensors and other bionano-devices.

An improved DNA force field for ssDNA interactions with gold nanoparticles.

PubMed

Jiang, Xiankai; Gao, Jun; Huynh, Tien; Huai, Ping; Fan, Chunhai; Zhou, Ruhong; Song, Bo

2014-06-21

The widespread applications of single-stranded DNA (ssDNA) conjugated gold nanoparticles (AuNPs) have spurred an increasing interest in the interactions between ssDNA and AuNPs. Despite extensive studies using the most sophisticated experimental techniques, the detailed molecular mechanisms still remain largely unknown. Large scale molecular dynamics (MD) simulations can thus be used to supplement experiments by providing complementary information about ssDNA-AuNP interactions. However, up to now, all modern force fields for DNA were developed based on the properties of double-stranded DNA (dsDNA) molecules, which have hydrophilic outer backbones "protecting" hydrophobic inner nucleobases from water. Without the double-helix structure of dsDNA and thus the "protection" by the outer backbone, the nucleobases of ssDNA are directly exposed to solvent, and their behavior in water is very different from that of dsDNA, especially at the interface with nanoparticles. In this work, we have improved the force field of ssDNA for use with nanoparticles, such as AuNPs, based on recent experimental results and quantum mechanics calculations. With the new improved force field, we demonstrated that a poly(A) sequence adsorbed on a AuNP surface is much more stable than a poly(T) sequence, which is consistent with recent experimental observations. On the contrary, the current standard force fields, including AMBER03, CHARMM27, and OPLSAA, all gave erroneous results as compared to experiments. The current improved force field is expected to have wide applications in the study of ssDNA with nanomaterials including AuNPs, which might help promote the development of ssDNA-based biosensors and other bionano-devices.

Involvement of CD8+ T cell-mediated immune responses in LcrV DNA vaccine induced protection against lethal Yersinia pestis challenge.

PubMed

Wang, Shixia; Goguen, Jon D; Li, Fusheng; Lu, Shan

2011-09-09

Yersinia pestis (Y. pestis) is the causative pathogen of plague, a highly fatal disease for which an effective vaccine, especially against mucosal transmission, is still not available. Like many bacterial infections, antigen-specific antibody responses have been traditionally considered critical, if not solely responsible, for vaccine-induced protection against Y. pestis. Studies in recent years have suggested the importance of T cell immune responses against Y. pestis infection but information is still limited about the details of Y. pestis antigen-specific T cell immune responses. In current report, studies are conducted to identify the presence of CD8+ T cell epitopes in LcrV protein, the leading antigen of plague vaccine development. Furthermore, depletion of CD8+ T cells in LcrV DNA vaccinated Balb/C mice led to reduced protection against lethal intranasal challenge of Y. pestis. These findings establish that an LcrV DNA vaccine is able to elicit CD8+ T cell immune responses against specific epitopes of this key plague antigen and that a CD8+ T cell immune response is involved in LcrV DNA vaccine-elicited protection. Future studies in plague vaccine development will need to examine if the presence of detectable T cell immune responses, in particular CD8+ T-cell immune responses, will enhance the protection against Y. pestis in higher animal species or humans. Copyright © 2010 Elsevier Ltd. All rights reserved.

DNA damage and lipid peroxidation vs. protection responses in the gill of Dicentrarchus labrax L. from a contaminated coastal lagoon (Ria de Aveiro, Portugal).

PubMed

Ahmad, I; Maria, V L; Oliveira, M; Serafim, A; Bebianno, M J; Pacheco, M; Santos, M A

2008-11-15

The present research work aimed to investigate the damage vs. protection responses in gill of European sea bass (Dicentrarchus labrax) captured at a polluted coastal lagoon, Ria de Aveiro (Portugal), as a tool to evaluate the human impacts on environmental health. Damage was assessed as DNA strand breakage and lipid peroxidation (LPO) whereas protection was evaluated by measuring catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione S-transferase (GST), total glutathione (GSHt), thiols and metallothioneins (MT). Fish were caught at five locations: Gafanha (GAF), Rio Novo do Príncipe (RIO), Laranjo (LAR) and Vagos (VAG) presenting each a different recognized source of contamination, and Torreira (TOR), assumed as reference site. Among the surveyed sites, gill damage was observed only at GAF, as measured by DNA integrity loss and LPO increase. An overall induction in enzymatic antioxidant protection was perceptible in fish from GAF and VAG, expressed as higher GPX, GR and GST activities. In addition, LAR fish showed elevated GST and CAT activities. Thiols content was higher in all study sites irrespective to the pollution spectrum, whereas GSHt increase was only observed at GAF and VAG. The highest MT level was detected in fish from VAG and the lowest level from RIO suggesting a low contamination degree on this particular site. Globally, the results expressed site-specific response patterns, signalling two critical areas - GAF and VAG. Additionally, a joint analysis (damage vs. protection) allowed the following ordering of surveyed sites according to the contamination degree and risk to fish health; GAF>VAG>LAR>RIO>TOR. D. labrax gill responses demonstrated their efficacy as early warning signals of the contaminants presence. Moreover, the adopted approach, considering simultaneously protection responses and damaging effects, also revealed its usefulness on the pollution extent assessment.

Alum adjuvanted rabies DNA vaccine confers 80% protection against lethal 50 LD50 rabies challenge virus standard strain.

PubMed

Garg, Rajni; Kaur, Manpreet; Saxena, Ankur; Prasad, Rajendra; Bhatnagar, Rakesh

2017-05-01

Rabies is a serious concern world-wide. Despite availability of rabies vaccines for long; their efficacy, safety, availability and cost effectiveness has been a tremendous issue. This calls for improvement of rabies vaccination strategies. DNA vaccination has immense potential in this regard. The DNA vaccine pgp.LAMP-1 conferred 60% protection to BALB/c mice against 20 LD 50 rabies challenge virus standard (CVS) strain challenge. Upon supplementation with Emulsigen-D, the vaccine formulation conferred complete protection against lethal challenge. To assess the feasibility of this vaccine formulation for human use, it was tested along with other FDA approved adjuvants, namely, Alum, Immuvac, Montanide ISA720 VG. Enhanced immune response correlated with high IgG antibody titer, Th2 biased response with a high level of rabies virus neutralizing antibodies (RVNAs) and IgG1/IgG2a ratio >1, observed upon alum supplementation of the rabies DNA vaccine. The total IgG antibody titer was 2IU/ml and total RVNA titer was observed to be 4IU/ml which is eight times higher than the minimum protective titer recommended by WHO. Furthermore, it conferred 80% protection against challenge with 50 LD 50 of the rabies CVS strain, conducted in compliance with the potency test for rabies recommended by the National Institutes of Health (NIH), USA. Previously, we have established pre-clinical safety of this vaccine as per the guidelines of Schedule Y, FDA as well as The European Agency for evaluation of Medicinal Products. The vaccine showed no observable toxicity at the site of injection as well as at systemic level in Wistar rats when administered with 10X recommended dose. Therefore, supplementation of rabies DNA vaccine, pgp.LAMP-1 with alum would lead to development of a non-toxic, efficacious, stable and affordable vaccine that can be used to combat high numbers of fatal rabies infections tormenting developing countries. Copyright © 2017 Elsevier Ltd. All rights reserved.

Measuring DNA hybridization using fluorescent DNA-stabilized silver clusters to investigate mismatch effects on therapeutic oligonucleotides.

PubMed

de Bruin, Donny; Bossert, Nelli; Aartsma-Rus, Annemieke; Bouwmeester, Dirk

2018-04-06

Short nucleic acid oligomers have found a wide range of applications in experimental physics, biology and medicine, and show potential for the treatment of acquired and genetic diseases. These applications rely heavily on the predictability of hybridization through Watson-Crick base pairing to allow positioning on a nanometer scale, as well as binding to the target transcripts, but also off-target binding to transcripts with partial homology. These effects are of particular importance in the development of therapeutic oligonucleotides, where off-target effects caused by the binding of mismatched sequences need to be avoided. We employ a novel method of probing DNA hybridization using optically active DNA-stabilized silver clusters (Ag-DNA) to measure binding efficiencies through a change in fluorescence intensity. In this way we can determine their location-specific sensitivity to individual mismatches in the sequence. The results reveal a strong dependence of the hybridization on the location of the mismatch, whereby mismatches close to the edges and center show a relatively minor impact. In parallel, we propose a simple model for calculating the annealing ratios of mismatched DNA sequences, which supports our experimental results. The primary result shown in this work is a demonstration of a novel technique to measure DNA hybridization using fluorescent Ag-DNA. With this technique, we investigated the effect of mismatches on the hybridization efficiency, and found a significant dependence on the location of individual mismatches. These effects are strongly influenced by the length of the used oligonucleotides. The novel probe method based on fluorescent Ag-DNA functions as a reliable tool in measuring this behavior. As a secondary result, we formulated a simple model that is consistent with the experimental data.

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.

Protective efficacy of a Mycoplasma pneumoniae P1C DNA vaccine fused with the B subunit of Escherichia coli heat-labile enterotoxin.

PubMed

Zhu, Cuiming; Wang, Shiping; Hu, Shihai; Yu, Minjun; Zeng, Yanhua; You, Xiaoxing; Xiao, Jinhong; Wu, Yimou

2012-06-01

In the present study, we investigated the immunomodulatory responses of a DNA vaccine constructed by fusing Mycoplasma pneumoniae P1 protein carboxy terminal region (P1C) with the Escherichia coli heat-labile toxin B subunit (LTB). BALB/c mice were immunized by intranasal inoculation with control DNAs, the P1C DNA vaccine or the LTB-P1C fusion DNA vaccine. Levels of the anti-M. pneumoniae antibodies and levels of interferon-γ and IL-4 in mice were increased significantly upon inoculation of the LTB-P1C fusion DNA vaccine when compared with the inoculation with P1C DNA vaccine. The LTB-P1C fusion DNA vaccine efficiently enhanced the M. pneumoniae-specific IgA and IgG levels. The IgG2a/IgG1 ratio was significantly higher in bronchoalveolar lavages fluid and sera from mice fusion with LTB and P1C than mice receiving P1C alone. When the mice were challenged intranasally with 10(7) CFU M. pneumoniae strain (M129), the LTB-P1C fusion DNA vaccine conferred significantly better protection than P1C DNA vaccine (P < 0.05), as suggested by the results, such as less inflammation, lower histopathological score values, lower detectable number of M. pneumoniae strain, and lower mortality of challenging from 5 × 10(8) CFU M. pneumoniae. These results indicated that the LTB-P1C fusion DNA vaccine efficiently improved protective efficacy against M. pneumoniae infection and effectively attenuated development of M. pneumoniae in mice.

The synergistic effect of combined immunization with a DNA vaccine and chimeric yellow fever/dengue virus leads to strong protection against dengue.

PubMed

Azevedo, Adriana S; Gonçalves, Antônio J S; Archer, Marcia; Freire, Marcos S; Galler, Ricardo; Alves, Ada M B

2013-01-01

The dengue envelope glycoprotein (E) is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2) and a chimeric yellow fever/dengue 2 virus (YF17D-D2). The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.

Capturing a DNA duplex under near-physiological conditions

NASA Astrophysics Data System (ADS)

Zhang, Huijuan; Xu, Wei; Liu, Xiaogang; Stellacci, Francesco; Thong, John T. L.

2010-10-01

We report in situ trapping of a thiolated DNA duplex with eight base pairs into a polymer-protected gold nanogap device under near-physiological conditions. The double-stranded DNA was captured by electrophoresis and covalently attached to the nanogap electrodes through sulfur-gold bonding interaction. The immobilization of the DNA duplex was confirmed by direct electrical measurements under near-physiological conditions. The conductance of the DNA duplex was estimated to be 0.09 μS. We also demonstrate the control of DNA dehybridization by heating the device to temperatures above the melting point of the DNA.

Folate, colorectal cancer and the involvement of DNA methylation.

PubMed

Williams, Elizabeth A

2012-11-01

Diet is a major factor in the aetiology of colorectal cancer (CRC). Epidemiological evidence suggests that folate confers a modest protection against CRC risk. However, the relationship is complex, and evidence from human intervention trials and animal studies suggests that a high-dose of folic acid supplementation may enhance the risk of colorectal carcinogenesis in certain circumstances. The molecular mechanisms underlying the apparent dual modulatory effect of folate on colorectal carcinogenesis are not fully understood. Folate is central to C1 metabolism and is needed for both DNA synthesis and DNA methylation, providing plausible biological mechanisms through which folate could modulate cancer risk. Aberrant DNA methylation is an early event in colorectal carcinogenesis and is typically associated with the transcriptional silencing of tumour suppressor genes. Folate is required for the production of S-adenosyl methionine, which serves as a methyl donor for DNA methylation events; thereby folate availability is proposed to modulate DNA methylation status. The evidence for an effect of folate on DNA methylation in the human colon is limited, but a modulation of DNA methylation in response to folate has been demonstrated. More research is required to clarify the optimum intake of folate for CRC prevention and to elucidate the effect of folate availability on DNA methylation and the associated impact on CRC biology.

Pregnane X receptor regulates the AhR/Cyp1A1 pathway and protects liver cells from benzo-[α]-pyrene-induced DNA damage.

PubMed

Cui, Hongmei; Gu, Xinsheng; Chen, Jingshu; Xie, Ying; Ke, Sui; Wu, Jing; Golovko, Andrei; Morpurgo, Benjamin; Yan, Chunhong; Phillips, Timothy D; Xie, Wen; Luo, Jianyuan; Zhou, Zhijun; Tian, Yanan

2017-06-05

Pregnane X receptor (PXR) plays an important role in protecting cells from mutagenic DNA damages induced by endogenous and exogenous toxicants. This protective function is often attributed to the PXR-regulated metabolic detoxification. Here we report a novel potential mechanism that PXR reduces benzo-[α]-pyrene(BaP)-induced DNA damage through inhibiting the transcriptional activity of aryl hydrocarbon receptor (AhR) which plays a pivotal role in the bioactivation of BaP. We have utilized three well-characterized cell lines, i.e. Hepa1c1c7, AhR +/+; Bpr lacks AhR obligatory partner ARNT; Tao, lacks AhR, to analyze pivotal role of AhR/ARNT complex in mediating the BaP-induced DNA damages using comet assay (single-cell gel electrophoresis). We found that PXR activation could significantly inhibit BaP-induced DNA damage in the HepG2 cells as well as mouse hepatocytes. Using PXR-null and wild type mouse hepatocytes we showed that PXR activation by pregnenolone 16α-carbonitrile (PCN) significantly inhibited BaP-induced DNA damage and this protective effect was abolished in PXR-null hepatocytes. Mechanistically, PXR activation inhibited expression of AhR-target genes for CYP1A1, CYP1B1 and CYP1A2 that are required for BaP biotransformation in cultured liver cells, or in the livers of C57BL/6J mice. Using an AhR-responsive reporter assay as well as chromatin immunoprecipitation assay we found that PXR activation transcriptionally represses AhR-regulated gene expression. Furthermore, we found that PXR directly bound AhR at its DNA-binding domain, and this association may play a role in preventing of the AhR from binding to its target genes as shown in the ChIP assay. Taken together, our study has revealed a novel mechanism by which PXR protects liver cells from BaP-induced DNA damage through inhibiting the BaP biotransformation. Copyright © 2017 Elsevier B.V. All rights reserved.

Protection of DNA against low-energy electrons by amino acids: a first-principles molecular dynamics study.

PubMed

Gu, Bin; Smyth, Maeve; Kohanoff, Jorge

2014-11-28

Using first-principles molecular dynamics simulations, we have investigated the notion that amino acids can play a protective role when DNA is exposed to excess electrons produced by ionizing radiation. In this study we focus on the interaction of glycine with the DNA nucleobase thymine. We studied thymine-glycine dimers and a condensed phase model consisting of one thymine molecule solvated in amorphous glycine. Our results show that the amino acid acts as a protective agent for the nucleobase in two ways. If the excess electron is initially captured by the thymine, then a proton is transferred in a barrier-less way from a neighboring hydrogen-bonded glycine. This stabilizes the excess electron by reducing the net partial charge on the thymine. In the second mechanism the excess electron is captured by a glycine, which acts as a electron scavenger that prevents electron localization in DNA. Both these mechanisms introduce obstacles to further reactions of the excess electron within a DNA strand, e.g. by raising the free energy barrier associated with strand breaks.

Comparison of the effect of raw and blanched-frozen broccoli on DNA damage in colonocytes.

PubMed

Lynn, Anthony; Fuller, Zoë; Collins, Andrew R; Ratcliffe, Brian

2015-07-01

Consumption of cruciferous vegetables may protect against colorectal cancer. Cruciferous vegetables are rich in a number of bioactive constituents including polyphenols, vitamins and glucosinolates. Before consumption, cruciferous vegetables often undergo some form of processing that reduces their content of bioactive constituents and may determine whether they exert protective effects. The aim of this study was to compare the ability of raw and blanched-frozen broccoli to protect colonocytes against DNA damage, improve antioxidant status and induce xenobiotic metabolizing enzymes (XME). Fifteen Landrace × Large White male pigs were divided into five age-matched and weight-matched sets (79 days, SD 3, and 34·7 kg, SD 3·9, respectively). Each set consisted of siblings to minimize genetic variation. Within each set, pigs received a cereal-based diet, unsupplemented (control) or supplemented with 600 g day(-1) of raw or blanched-frozen broccoli for 12 days. The consumption of raw broccoli caused a significant 27% increase in DNA damage in colonocytes (p = 0·03) relative to the control diet, whereas blanched-frozen broccoli had no significant effect. Both broccoli diets had no significant effect on plasma antioxidant status or hepatic and colonic XME. This study is the first to report that the consumption of raw broccoli can damage DNA in porcine colonocytes. Copyright © 2015 John Wiley & Sons, Ltd.

Mitochondrial nucleoid clusters protect newly synthesized mtDNA during Doxorubicin- and Ethidium Bromide-induced mitochondrial stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alán, Lukáš, E-mail: lukas.alan@fgu.cas.cz; Špaček

Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators.more » We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (> 48 h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity. - Highlights: • The mechanism for mitochondrial nucleoid clustering is proposed. • DNA intercalators (Doxorubicin or Ethidium Bromide) prevent

Hantavirus Gc induces long-term immune protection via LAMP-targeting DNA vaccine strategy.

PubMed

Jiang, Dong-Bo; Zhang, Jin-Peng; Cheng, Lin-Feng; Zhang, Guan-Wen; Li, Yun; Li, Zi-Chao; Lu, Zhen-Hua; Zhang, Zi-Xin; Lu, Yu-Chen; Zheng, Lian-He; Zhang, Fang-Lin; Yang, Kun

2018-02-01

Hemorrhagic fever with renal syndrome (HFRS) occurs widely throughout Eurasia. Unfortunately, there is no effective treatment, and prophylaxis remains the best option against the major pathogenic agent, hantaan virus (HTNV), which is an Old World hantavirus. However, the absence of cellular immune responses and immunological memory hampers acceptance of the current inactivated HFRS vaccine. Previous studies revealed that a lysosome-associated membrane protein 1 (LAMP1)-targeting strategy involving a DNA vaccine based on the HTNV glycoprotein Gn successfully conferred long-term immunity, and indicated that further research on Gc, another HTNV antigen, was warranted. Plasmids encoding Gc and lysosome-targeted Gc, designated pVAX-Gc and pVAX-LAMP/Gc, respectively, were constructed. Proteins of interest were identified by fluorescence microscopy following cell line transfection. Five groups of 20 female BALB/c mice were subjected to the following inoculations: inactivated HTNV vaccine, pVAX-LAMP/Gc, pVAX-Gc, and, as the negative controls, pVAX-LAMP or the blank vector pVAX1. Humoral and cellular immunity were assessed by enzyme-linked immunosorbent assays (ELISAs) and 15-mer peptide enzyme-linked immunospot (ELISpot) epitope mapping assays. Repeated immunization with pVAX-LAMP/Gc enhanced adaptive immune responses, as demonstrated by the specific and neutralizing antibody titers and increased IFN-γ production. The inactivated vaccine induced a comparable humoral reaction, but the negative controls only elicited insignificant responses. Using a mouse model of HTNV challenge, the in vivo protection conferred by the inactivated vaccine and Gc-based constructs (with/without LAMP recombination) was confirmed. Evidence of pan-epitope reactions highlighted the long-term cellular response to the LAMP-targeting strategy, and histological observations indicated the safety of the LAMP-targeting vaccines. The long-term protective immune responses induced by pVAX-LAMP/Gc may be

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

PubMed Central

Lo, Pang-Kuo; Lee, Ji Shin; Sukumar, Saraswati

2011-01-01

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G1 arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G1 arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1. PMID:21964066

Evaluation of Antioxidant and DNA Damage Protection Activity of the Hydroalcoholic Extract of Desmostachya bipinnata L. Stapf

PubMed Central

Bhimathati, Solomon Sunder Raj

2014-01-01

Desmostachya bipinnata Stapf (Poaceae/Gramineae) is an official drug of ayurvedic pharmacopoeia. Various parts of this plant were used extensively in traditional and folklore medicine to cure various human ailments. The present study was aimed to evaluate the antioxidant and DNA damage protection activity of hydroalcoholic extract of Desmostachya bipinnata both in vitro and in vivo, to provide scientific basis for traditional usage of this plant. The extract showed significant antioxidant activity in a dose-dependent manner with an IC50 value of 264.18 ± 3.47 μg/mL in H2O2 scavenging assay and prevented the oxidative damage to DNA in presence of DNA damaging agent (Fenton's reagent) at a concentration of 50 μg/mL. Also, the presence of extract protected yeast cells in a dose-dependent manner against DNA damaging agent (Hydroxyurea) in spot assay. Moreover, the presence of extract exhibited significant antioxidant activity in vivo by protecting yeast cells against oxidative stressing agent (H2O2). Altogether, the results of current study revealed that Desmostachya bipinnata is a potential source of antioxidants and lends pharmacological credence to the ethnomedical use of this plant in traditional system of medicine, justifying its therapeutic application for free-radical-induced diseases. PMID:24574873

Evaluation of antioxidant and DNA damage protection activity of the hydroalcoholic extract of Desmostachya bipinnata L. Stapf.

PubMed

Golla, Upendarrao; Bhimathati, Solomon Sunder Raj

2014-01-01

Desmostachya bipinnata Stapf (Poaceae/Gramineae) is an official drug of ayurvedic pharmacopoeia. Various parts of this plant were used extensively in traditional and folklore medicine to cure various human ailments. The present study was aimed to evaluate the antioxidant and DNA damage protection activity of hydroalcoholic extract of Desmostachya bipinnata both in vitro and in vivo, to provide scientific basis for traditional usage of this plant. The extract showed significant antioxidant activity in a dose-dependent manner with an IC50 value of 264.18±3.47  μg/mL in H2O2 scavenging assay and prevented the oxidative damage to DNA in presence of DNA damaging agent (Fenton's reagent) at a concentration of 50  μg/mL. Also, the presence of extract protected yeast cells in a dose-dependent manner against DNA damaging agent (Hydroxyurea) in spot assay. Moreover, the presence of extract exhibited significant antioxidant activity in vivo by protecting yeast cells against oxidative stressing agent (H2O2). Altogether, the results of current study revealed that Desmostachya bipinnata is a potential source of antioxidants and lends pharmacological credence to the ethnomedical use of this plant in traditional system of medicine, justifying its therapeutic application for free-radical-induced diseases.

Nuclear aggregates of polyamines in a radiation-induced DNA damage model.

PubMed

Iacomino, Giuseppe; Picariello, Gianluca; Stillitano, Ilaria; D'Agostino, Luciano

2014-02-01

Polyamines (PA) are believed to protect DNA minimizing the effect of radiation damage either by inducing DNA compaction and aggregation or acting as scavengers of free radicals. Using an in vitro pDNA double strand breakage assay based on gel electrophoretic mobility, we compared the protective capability of PA against γ-radiation with that of compounds generated by the supramolecular self-assembly of nuclear polyamines and phosphates, named Nuclear Aggregates of Polyamines (NAPs). Both unassembled PA and in vitro produced NAPs (ivNAPs) were ineffective in conferring pDNA protection at the sub-mM concentration. Single PA showed an appreciable protective effect only at high (mM) concentrations. However, concentrations of spermine (4+) within a critical range (0.481 mM) induced pDNA precipitation, an event that was not observed with NAPs-pDNA interaction. We conclude that the interaction of individual PA is ineffective to assure DNA protection, simultaneously preserving the flexibility and charge density of the double strand. Furthermore, data obtained by testing polyamine and ivNAPS with the current radiation-induced DNA damage model support the concept that PA-phosphate aggregates are the only forms through which PA interact with DNA. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of trimethylamine N-oxide and urea on DNA duplex and G-quadruplex.

PubMed

Ueda, Yu-Mi; Zouzumi, Yu-Ki; Maruyama, Atsushi; Nakano, Shu-Ichi; Sugimoto, Naoki; Miyoshi, Daisuke

2016-01-01

We systematically investigated effects of molecular crowding with trimethylamine N -oxide (TMAO) as a zwitterionic and protective osmolyte and urea as a nonionic denaturing osmolyte on conformation and thermodynamics of the canonical DNA duplex and the non-canonical DNA G-quadruplex. It was found that TMAO and urea stabilized and destabilized, respectively, the G-quadruplex. On the other hand, these osmolytes generally destabilize the duplex; however, it was observed that osmolytes having the trimethylamine group stabilized the duplex at the lower concentrations because of a direct binding to a groove of the duplex. These results are useful not only to predict DNA structures and their thermodynamics under physiological environments in living cells, but also design of polymers and materials to regulate structure and stability of DNA sequences.

Effect of seven Indian plant extracts on Fenton reaction-mediated damage to DNA constituents.

PubMed

Kar, Indrani; Chattopadhyaya, Rajagopal

2017-11-01

The influences of substoichiometric amounts of seven plant extracts in the Fenton reaction-mediated damage to deoxynucleosides, deoxynucleoside monophosphates, deoxynucleoside triphosphates, and supercoiled plasmid DNA were studied to rationalize anticancer properties reported in some of these extracts. Extracts from Acacia catechu, Emblica officinalis, Spondias dulcis, Terminalia belerica, Terminalia chebula, as well as gallic acid, epicatechin, chebulagic acid and chebulinic acid enhance the extent of damage in Fenton reactions with all monomeric substrates but protect supercoiled plasmid DNA, compared to standard Fenton reactions. The damage to pyrimidine nucleosides/nucleotides is enhanced by these extracts and compounds to a greater extent than for purine ones in a concentration dependent manner. Dolichos biflorus and Hemidesmus indicus extracts generally do not show this enhancement for the monomeric substrates though they protect plasmid DNA. Compared to standard Fenton reactions for deoxynucleosides with ethanol, the presence of these five plant extracts render ethanol scavenging less effective as the radical is generated in the vicinity of the target. Since substoichiometric amounts of these extracts and the four compounds produce this effect, a catalytic mechanism involving the presence of a ternary complex of the nucleoside/nucleotide substrate, a plant compound and the hydroxyl radical is proposed. Such a mechanism cannot operate for plasmid DNA as the planar rings in the extract compounds cannot stack with the duplex DNA bases. These plant extracts, by enhancing Fenton reaction-mediated damage to deoxynucleoside triphosphates, slow down DNA replication in rapidly dividing cancer cells, thus contributing to their anticancer properties.

Recovery Based Nanowire Field-Effect Transistor Detection of Pathogenic Avian Influenza DNA

NASA Astrophysics Data System (ADS)

Lin, Chih-Heng; Chu, Chia-Jung; Teng, Kang-Ning; Su, Yi-Jr; Chen, Chii-Dong; Tsai, Li-Chu; Yang, Yuh-Shyong

2012-02-01

Fast and accurate diagnosis is critical in infectious disease surveillance and management. We proposed a DNA recovery system that can easily be adapted to DNA chip or DNA biosensor for fast identification and confirmation of target DNA. This method was based on the re-hybridization of DNA target with a recovery DNA to free the DNA probe. Functionalized silicon nanowire field-effect transistor (SiNW FET) was demonstrated to monitor such specific DNA-DNA interaction using high pathogenic strain virus hemagglutinin 1 (H1) DNA of avian influenza (AI) as target. Specific electric changes were observed in real-time for AI virus DNA sensing and device recovery when nanowire surface of SiNW FET was modified with complementary captured DNA probe. The recovery based SiNW FET biosensor can be further developed for fast identification and further confirmation of a variety of influenza virus strains and other infectious diseases.

In Vitro Protective Effects of Lycium barbarum Berries Cultivated in Umbria (Italy) on Human Hepatocellular Carcinoma Cells.

PubMed

Ceccarini, M R; Vannini, S; Cataldi, S; Moretti, M; Villarini, M; Fioretti, B; Albi, E; Beccari, T; Codini, M

2016-01-01

Lycium barbarum is a famous plant in the traditional Chinese medicine. The plant is known to have health-promoting bioactive components. The properties of Lycium barbarum berries cultivated in Umbria (Italy) and their effect on human hepatocellular carcinoma cells (HepG2) have been investigated in this work. The obtained results demonstrated that the Lycium barbarum berries from Umbria region display high antioxidant properties evaluated by total phenolic content and ORAC method, on hydrophilic and lipophilic fractions. Moreover, on HepG2 cell line Lycium barbarum berries extract did not change cell viability analyzed by MTT and Trypan blue exclusion assay and did not induce genotoxic effect analyzed by comet assay. Furthermore, it was demonstrated, for the first time, that the berries extract showed a protective effect on DNA damage, expressed as antigenotoxic activity in vitro . Finally, Lycium barbarum berries extract was able to modulate the expression of genes involved in oxidative stress, proliferation, apoptosis, and cancer. In particular, downexpression of genes involved in tumor migration and invasion (CCL5), in increased risk of metastasis and antiapoptotic signal (DUSP1), and in carcinogenesis (GPx-3 and PTGS1), together with overexpression of tumor suppressor gene (MT3), suggested that Umbrian Lycium barbarum berries could play a protective role against hepatocellular carcinoma.

Anti-apoptotic and myocardial protective effects of ethyl pyruvate after regional ischaemia/reperfusion myocardial damage in an in vivo rat model.

PubMed

Shim, Haeng Seon; Lee, Wang Gyu; Kim, Yeon A; Han, Jeong Yeol; Park, Miyeong; Song, Yun Gyu; Kim, Joon Soo; Shin, Il-Woo

2017-09-01

The integration of reactive oxygen species is strongly associated with important pathophysiological mechanisms that mediate myocardial ischaemia/reperfusion (I/R) damage. Pyruvate is an efficacious scavenger of reactive oxygen species and a previous study has shown that ethyl pyruvate (EP) has a myocardial protective effect against regional I/R damage in an in vivo rat model. The purpose of this study was to determine whether the myocardial protective effect of EP is associated with anti-apoptosis. Rats were allocated to receive EP dissolved in lactated Ringer's solution or lactated Ringer's solution alone, via intraperitoneal infusion one hour before ischaemia. They were exposed to 30 minutes of ischaemia followed by reperfusion of the left coronary artery territory over two hours. Anti-apoptotic effects were checked using several biochemical parameters after two hours of reperfusion. Apoptosis was analysed using measured caspase-3 activity, Western blotting of B-cell lymphoma 2 (Bcl-2) family protein cleaved by caspase-3, and assessment of DNA laddering patterns and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining test. In ischaemic myocardium, EP increased Bcl-2 expression, but reduced Bcl-2-associated X protein and cleaved caspase-3 expressions. EP reduced the expression of DNA laddering and the number of myocardial I/R-damaged TUNEL-positive cells. This study demonstrated that EP has an anti-apoptotic effect after regional I/R damage in an in vivo rat heart model. The myocardial protective effect of EP may be related to its anti-apoptotic effect. Copyright: © Singapore Medical Association

Human DNA polymerase θ grasps the primer terminus to mediate DNA repair

DOE PAGES

Zahn, Karl E.; Averill, April M.; Aller, Pierre; ...

2015-03-16

DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break–inducing agents, including ionizing radiation. Reported in this paper are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contactsmore » to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. Finally, these observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.« less

The UK National DNA Database: Implementation of the Protection of Freedoms Act 2012.

PubMed

Amankwaa, Aaron Opoku; McCartney, Carole

2018-03-01

In 2008, the European Court of Human Rights, in S and Marper v the United Kingdom, ruled that a retention regime that permits the indefinite retention of DNA records of both convicted and non-convicted ("innocent") individuals is disproportionate. The court noted that there was inadequate evidence to justify the retention of DNA records of the innocent. Since the Marper ruling, the laws governing the taking, use, and retention of forensic DNA in England and Wales have changed with the enactment of the Protection of Freedoms Act 2012 (PoFA). This Act, put briefly, permits the indefinite retention of DNA profiles of most convicted individuals and temporal retention for some first-time convicted minors and innocent individuals on the National DNA Database (NDNAD). The PoFA regime was implemented in October 2013. This paper examines ten post-implementation reports of the NDNAD Strategy Board (3), the NDNAD Ethics Group (3) and the Office of the Biometrics Commissioner (OBC) (4). Overall, the reports highlight a considerable improvement in the performance of the database, with a current match rate of 63.3%. Further, the new regime has strengthened the genetic privacy protection of UK citizens. The OBC reports detail implementation challenges ranging from technical, legal and procedural issues to sufficient understanding of the requirements of PoFA by police forces. Risks highlighted in these reports include the deletion of some "retainable" profiles, which could potentially lead to future crimes going undetected. A further risk is the illegal retention of some profiles from innocent individuals, which may lead to privacy issues and legal challenges. In conclusion, the PoFA regime appears to be working well, however, critical research is still needed to evaluate its overall efficacy compared to other retention regimes. Copyright © 2018 Elsevier B.V. All rights reserved.

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. Copyright © 2014. Published by Elsevier B.V.

A private DNA motif finding algorithm.

PubMed

Chen, Rui; Peng, Yun; Choi, Byron; Xu, Jianliang; Hu, Haibo

2014-08-01

With the increasing availability of genomic sequence data, numerous methods have been proposed for finding DNA motifs. The discovery of DNA motifs serves a critical step in many biological applications. However, the privacy implication of DNA analysis is normally neglected in the existing methods. In this work, we propose a private DNA motif finding algorithm in which a DNA owner's privacy is protected by a rigorous privacy model, known as ∊-differential privacy. It provides provable privacy guarantees that are independent of adversaries' background knowledge. Our algorithm makes use of the n-gram model and is optimized for processing large-scale DNA sequences. We evaluate the performance of our algorithm over real-life genomic data and demonstrate the promise of integrating privacy into DNA motif finding. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of Noise on DNA Sequencing via Transverse Electronic Transport

PubMed Central

Krems, Matt; Zwolak, Michael; Pershin, Yuriy V.; Di Ventra, Massimiliano

2009-01-01

Abstract Previous theoretical studies have shown that measuring the transverse current across DNA strands while they translocate through a nanopore or channel may provide a statistically distinguishable signature of the DNA bases, and may thus allow for rapid DNA sequencing. However, fluctuations of the environment, such as ionic and DNA motion, introduce important scattering processes that may affect the viability of this approach to sequencing. To understand this issue, we have analyzed a simple model that captures the role of this complex environment in electronic dephasing and its ability to remove charge carriers from current-carrying states. We find that these effects do not strongly influence the current distributions due to the off-resonant nature of tunneling through the nucleotides—a result we expect to be a common feature of transport in molecular junctions. In particular, only large scattering strengths, as compared to the energetic gap between the molecular states and the Fermi level, significantly alter the form of the current distributions. Since this gap itself is quite large, the current distributions remain protected from this type of noise, further supporting the possibility of using transverse electronic transport measurements for DNA sequencing. PMID:19804730

Ultraviolet-B Protective Effect of Flavonoids from Eugenia caryophylata on Human Dermal Fibroblast Cells.

PubMed

Patwardhan, Juilee; Bhatt, Purvi

2015-10-01

The exposure of skin to ultraviolet-B (UV-B) radiations leads to deoxyribonucleic acid (DNA) damage and can induce production of free radicals which imbalance the redox status of the cell and lead to increased oxidative stress. Clove has been traditionally used for its analgesic, anti-inflammatory, anti-microbial, anti-viral, and antiseptic effects. To evaluate the UV-B protective activity of flavonoids from Eugenia caryophylata (clove) buds on human dermal fibroblast cells. Protective ability of flavonoid-enriched (FE) fraction of clove was studied against UV-B induced cytotoxicity, anti-oxidant regulation, oxidative DNA damage, intracellular reactive oxygen species (ROS) generation, apoptotic morphological changes, and regulation of heme oxygenase-1 (HO-1) gene through nuclear factor E2-related factor 2 antioxidant response element (Nrf2 ARE) pathway. FE fraction showed a significant antioxidant potential. Pretreatment of cells with FE fraction (10-40 μg/ml) reversed the effects of UV-B induced cytotoxicity, depletion of endogenous enzymatic antioxidants, oxidative DNA damage, intracellular ROS production, apoptotic changes, and overexpression of Nrf2 and HO-1. The present study demonstrated for the first time that the FE fraction from clove could confer UV-B protection probably through the Nrf2-ARE pathway, which included the down-regulation of Nrf2 and HO-1. These findings suggested that the flavonoids from clove could potentially be considered as UV-B protectants and can be explored further for its topical application to the area of the skin requiring protection. Pretreatment of human dermal fibroblast with flavonoid-enriched fraction of Eugenia caryophylata attenuated effects of ultraviolet-B radiationsIt also conferred protection through nuclear factor E2-related factor 2-antioxidant response pathway and increased tolerance of cells against oxidative stressFlavonoid-enriched fraction can be explored further for topical application to the skin as a

Induction of protective immunity against Eimeria tenella, Eimeria necatrix, Eimeria maxima and Eimeria acervulina infections using multivalent epitope DNA vaccines.

PubMed

Song, Xiaokai; Ren, Zhe; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

2015-06-04

Avian coccidiosis is mostly caused by mixed infection of several Eimeria species under natural conditions and immunity to avian coccidiosis is largely dependent on T-cell immune response. In this study, 14 T-cell epitope fragments from eight antigens of Eimeria tenella (E. tenella), Eimeria necatrix (E. necatrix), Eimeria maxima (E. maxima) and Eimeria acervulina (E. acervulina) were ligated with pVAX1 producing 14 monovalent DNA vaccines, respectively. Protective immunity of the monovalent DNA vaccines was assessed by in vivo challenge experiments and then four most protective fragments of each species were chosen to construct multivalent epitope DNA vaccines with or without chicken IL-2 as genetic adjuvant. Protective efficacies of the epitope DNA vaccines on chickens against E. tenella, E. necatrix, E. maxima and E. acervulina were evaluated. The results showed that the constructed multivalent epitope DNA vaccines significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds. Especially, the multivalent epitope DNA vaccines of pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1 and pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 not only significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds, but also resulted in anti-coccidial index (ACI) more than 170 against E. tenella, E. necatrix, E. maxima and E. acervulina, which indicated they could induce protective immunity against E. tenella, E. necatrix, E. maxima and E. acervulina. Our findings suggest the constructed multivalent epitope DNA vaccines are the potential candidate multivalent vaccines against mixed infection of Eimeria. Copyright © 2015 Elsevier Ltd. All rights reserved.

Moricandia arvensis extracts protect against DNA damage, mutagenesis in bacteria system and scavenge the superoxide anion.

PubMed

Skandrani, Ines; Bouhlel, Ines; Limem, Ilef; Boubaker, Jihed; Bhouri, Wissem; Neffati, Aicha; Ben Sghaier, Mohamed; Kilani, Soumaya; Ghedira, Kamel; Ghedira-Chekir, Leila

2009-02-01

The mutagenic potential of total aqueous, total oligomers flavonoids (TOF), ethyl acetate (EA), chloroform (Chl), petroleum ether (PE) and methanol (MeOH) extracts from aerial parts of Moricandia arvensis was assessed using Ames Salmonella tester strains TA100 and TA1535 with and without metabolic activation (S9), and using plasmid pBluescript DNA assay. None of the different extracts produced a mutagenic effect, except aqueous extract when incubated with Salmonella typhimurium TA100 after metabolic activation. Likewise, the antimutagenicity of the same extracts was tested using the "Ames test". Our results showed that M. arvensis extracts possess antimutagenic effects against sodium azide (SA) in the two tested Salmonella assay systems, except metabolized aqueous and PE extracts when tested with S. typhimurium TA100 assay system. Different extracts were also found to be effective in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals, except PE and aqueous extracts. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) (X/XOD) and the non enzymatic (NBT/Riboflavine assay) systems. TOF extract was the more effective one in inhibiting both xanthine oxidase activity and NBT reduction.

Protective effect of boric acid on lead- and cadmium-induced genotoxicity in V79 cells.

PubMed

Ustündağ, Aylin; Behm, Claudia; Föllmann, Wolfram; Duydu, Yalçin; Degen, Gisela H

2014-06-01

The toxic heavy metals cadmium (Cd) and lead (Pb) are important environmental pollutants which can cause serious damage to human health. As the metal ions (Cd(2+) and Pb(2+)) accumulate in the organism, there is special concern regarding chronic toxicity and damage to the genetic material. Metal-induced genotoxicity has been attributed to indirect mechanisms, such as induction of oxidative stress and interference with DNA repair. Boron is a naturally occurring element and considered to be an essential micronutrient, although the cellular activities of boron compounds remain largely unexplored. The present study has been conducted to evaluate potential protective effects of boric acid (BA) against genotoxicity induced by cadmium chloride (CdCl2) and lead chloride (PbCl2) in V79 cell cultures. Cytotoxicity assays (neutral red uptake and cell titer blue assay) served to determine suitable concentrations for subsequent genotoxicity assays. Chromosomal damage and DNA strand breaks were assessed by micronucleus tests and comet assays. Both PbCl2 and CdCl2 (at 3, 5 and 10 µM) were shown to induce concentration-dependent increases in micronucleus frequencies and DNA strand breaks in V79 cells. BA itself was not cytotoxic (up to 300 µM) and showed no genotoxic effects. Pretreatment of cells with low levels of BA (2.5 and 10 µM) was found to strongly reduce the genotoxic effects of the tested metals. Based on the findings of this in vitro study, it can be suggested that boron provides an efficient protection against the induction of DNA strand breaks and micronuclei by lead and cadmium. Further studies on the underlying mechanisms for the protective effect of boron are needed.

Effective Induction of Simian Immunodeficiency Virus-Specific Cytotoxic T Lymphocytes in Macaques by Using a Multiepitope Gene and DNA Prime-Modified Vaccinia Virus Ankara Boost Vaccination Regimen

PubMed Central

Hanke, Tomas; Samuel, Rachel V.; Blanchard, Tom J.; Neumann, Veronica C.; Allen, Todd M.; Boyson, Jon E.; Sharpe, Sally A.; Cook, Nicola; Smith, Geoffrey L.; Watkins, David I.; Cranage, Martin P.; McMichael, Andrew J.

1999-01-01

DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8+ lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed. PMID:10438842

DNA vaccine encoding central conserved region of G protein induces Th1 predominant immune response and protection from RSV infection in mice.

PubMed

Hua, Ying; Jiao, Yue-Ying; Ma, Yao; Peng, Xiang-Lei; Fu, Yuan-Hui; Zheng, Yan-Peng; Hong, Tao; He, Jin-Sheng

2016-11-01

Human respiratory syncytial virus (RSV) can cause serious infection in the lower respiratory tract, especially in infants, young children, the elderly and the immunocompromised population worldwide. Previous study demonstrated the polypeptide (amino acids 148-198) of RSV attachment (G) glycoprotein, corresponding to the central conserved region and encompassing CX3C chemokine motif, could induce antibodies and protection from RSV challenge in mice [1,2]. In this study, we evaluated the immune efficacy of the recombinant DNA vaccine of pVAX1/3G 148-198 encoding RSV G protein polypeptide. RSV specific serum IgG antibodies with neutralizing activity were stimulated following prime-boost immunization of pVAX1/3G 148-198 intramuscularly, and the ratio of IgG2a/IgG1 was 4.93, indicating a Th1 biased immune response. After challenged intranasally with RSV Long, the vaccinated mice showed both decreased lung RSV titers, pulmonary inflammation and body weight loss. The results suggest that pVAX1/3G 148-198 DNA vaccine may be an effective RSV vaccine candidate, and deserves further exploration. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Protection of Arabidopsis Blunt-Ended Telomeres Is Mediated by a Physical Association with the Ku Heterodimer[OPEN

PubMed Central

Valuchova, Sona; Prokop, Zbynek; Hofr, Ctirad

2017-01-01

Telomeres form specialized chromatin that protects natural chromosome termini from being recognized as DNA double-strand breaks. Plants possess unusual blunt-ended telomeres that are unable to form t-loops or complex with single-strand DNA binding proteins, raising the question of the mechanism behind their protection. We have previously suggested that blunt-ended telomeres in Arabidopsis thaliana are protected by Ku, a DNA repair factor with a high affinity for DNA ends. In nonhomologous end joining, Ku loads onto broken DNA via a channel consisting of positively charged amino acids. Here, we demonstrate that while association of Ku with plant telomeres also depends on this channel, Ku’s requirements for DNA binding differ between DNA repair and telomere protection. We show that a Ku complex proficient in DNA loading but impaired in translocation along DNA is able to protect blunt-ended telomeres but is deficient in DNA repair. This suggests that Ku physically sequesters blunt-ended telomeres within its DNA binding channel, shielding them from other DNA repair machineries. PMID:28584163

A plant based protective antigen [PA(dIV)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax.

PubMed

Gorantala, Jyotsna; Grover, Sonam; Goel, Divya; Rahi, Amit; Jayadev Magani, Sri Krishna; Chandra, Subhash; Bhatnagar, Rakesh

2011-06-15

The currently available anthrax vaccines are limited by being incompletely characterized, potentially reactogenic and have an expanded dosage schedule. Plant based vaccines offer safe alternative for vaccine production. In the present study, we expressed domain IV of Bacillus anthracis protective antigen gene [PA(dIV)] in planta (by nuclear agrobacterium and chloroplast transformation) and E. coli [rPA(dIV)]. The presence of transgene and the expression of PA(dIV) in planta was confirmed by molecular analysis. Expression levels up to 5.3% of total soluble protein (TSP) were obtained with AT rich (71.8% AT content) PA(dIV) gene in transplastomic plants while 0.8% of TSP was obtained in nuclear transformants. Further, we investigated the protective response of plant and E. coli derived PA(dIV) in mice by intraperitoneal (i.p.) and oral immunizations with or without adjuvant. Antibody titers of >10(4) were induced upon i.p. and oral immunizations with plant derived PA(dIV) and oral immunization with E. coli derived PA(dIV). Intraperitoneal injections with adjuvanted E. coli derived PA(dIV), generated highest antibody titers of >10(5). All the immunized groups demonstrated predominant IgG1 titers over IgG2a indicating a polarized Th2 type response. We also evaluated the mucosal antibody response in orally immunized groups. When fecal extracts were analyzed, low sIgA titer was demonstrated in adjuvanted plant and E. coli derived PA(dIV) groups. Further, PA(dIV) antisera enhanced B. anthracis spore uptake by macrophages in vitro and also demonstrated an anti-germinating effect suggesting a potent role at mucosal surfaces. The antibodies from various groups were efficient in neutralizing the lethal toxin in vitro. When mice were challenged with B. anthracis, mice immunized with adjuvanted plant PA(dIV) imparted 60% and 40% protection while E. coli derived PA(dIV) conferred 100% and 80% protection upon i.p. and oral immunizations. Thus, our study is the first attempt in

Effects of microbial DNA on human DNA profiles generated using the PowerPlex® 16 HS system.

PubMed

Dembinski, Gina M; Picard, Christine J

2017-11-01

Most crime scenes are not sterile and therefore may be contaminated with environmental DNA, especially if a decomposing body is found. Collecting biological evidence from this individual will yield DNA samples mixed with microbial DNA. This also becomes important if postmortem swabs are collected from sexually assaulted victims. Although genotyping kits undergo validation tests, including bacterial screens, they do not account for the diverse microbial load during decomposition. We investigated the effect of spiking human DNA samples with known concentrations of DNA from 17 microbe species associated with decomposition on DNA profiles produced using the Promega PowerPlex ® HS system. Two species, Bacillus subtilis and Mycobacterium smegmatis, produced an extraneous allele at the TPOX locus. When repeated with the PowerPlex ® Fusion kit, the extra allele no longer amplified with these two species. This experiment demonstrates that caution should be exhibited if microbial load is high and the PowerPlex ® 16HS system is used. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Protective vaccination and blood-stage malaria modify DNA methylation of gene promoters in the liver of Balb/c mice.

PubMed

Al-Quraishy, Saleh; Dkhil, Mohamed A; Abdel-Baki, Abdel-Azeem S; Ghanjati, Foued; Erichsen, Lars; Santourlidis, Simeon; Wunderlich, Frank; Araúzo-Bravo, Marcos J

2017-05-01

Epigenetic mechanisms such as DNA methylation are increasingly recognized to be critical for vaccination efficacy and outcome of different infectious diseases, but corresponding information is scarcely available for host defense against malaria. In the experimental blood-stage malaria Plasmodium chabaudi, we investigate the possible effects of a blood-stage vaccine on DNA methylation of gene promoters in the liver, known as effector against blood-stage malaria, using DNA methylation microarrays. Naturally susceptible Balb/c mice acquire, by protective vaccination, the potency to survive P. chabaudi malaria and, concomitantly, modifications of constitutive DNA methylation of promoters of numerous genes in the liver; specifically, promoters of 256 genes are hyper(=up)- and 345 genes are hypo(=down)-methylated (p < 0.05). Protective vaccination also leads to changes in promoter DNA methylation upon challenge with P. chabaudi at peak parasitemia on day 8 post infection (p.i.), when 571 and 1013 gene promoters are up- and down-methylated, respectively, in relation to constitutive DNA methylation (p < 0.05). Gene set enrichment analyses reveal that both vaccination and P. chabaudi infections mainly modify promoters of those genes which are most statistically enriched with functions relating to regulation of transcription. Genes with down-methylated promoters encompass those encoding CX3CL1, GP130, and GATA2, known to be involved in monocyte recruitment, IL-6 trans-signaling, and onset of erythropoiesis, respectively. Our data suggest that vaccination may epigenetically improve parts of several effector functions of the liver against blood-stage malaria, as, e.g., recruitment of monocyte/macrophage to the liver accelerated liver regeneration and extramedullary hepatic erythropoiesis, thus leading to self-healing of otherwise lethal P. chabaudi blood-stage malaria.

Effects of trimethylamine N-oxide and urea on DNA duplex and G-quadruplex

PubMed Central

Ueda, Yu-mi; Zouzumi, Yu-ki; Maruyama, Atsushi; Nakano, Shu-ichi; Sugimoto, Naoki; Miyoshi, Daisuke

2016-01-01

Abstract We systematically investigated effects of molecular crowding with trimethylamine N-oxide (TMAO) as a zwitterionic and protective osmolyte and urea as a nonionic denaturing osmolyte on conformation and thermodynamics of the canonical DNA duplex and the non-canonical DNA G-quadruplex. It was found that TMAO and urea stabilized and destabilized, respectively, the G-quadruplex. On the other hand, these osmolytes generally destabilize the duplex; however, it was observed that osmolytes having the trimethylamine group stabilized the duplex at the lower concentrations because of a direct binding to a groove of the duplex. These results are useful not only to predict DNA structures and their thermodynamics under physiological environments in living cells, but also design of polymers and materials to regulate structure and stability of DNA sequences. PMID:27933115

Introduction of translation stop condons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

USGS Publications Warehouse

Garver, Kyle A.; Conway, Carla M.; Kurath, Gael

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.

The protective effects of good parenting on adolescents.

PubMed

DeVore, Elise R; Ginsburg, Kenneth R

2005-08-01

To explore recent developments in the literature regarding parenting practices and adolescent development, with a focus on parenting style, parental monitoring, communication, and supervision. There have been significant recent advances in the study of the relationship between parenting and adolescent development. Several recent intervention studies with a parenting component demonstrated immediate and long-term protective effects on adolescent risk behavior. Parent-child connectedness and authoritative parenting style are protective for teens. Parental monitoring has a protective effect on many adolescent risk behaviors in both middle-class populations and poor urban environments and has been shown both to moderate the effect of peer influence and to persist into late adolescence. Whereas unsupervised time, exposure to sexual possibility situations, and out-of-home care increase sexual behavior, improved parent-child communication reduces sexual risk behaviors. Recent scholarship demonstrates the significant, enduring, and protective influence of positive parenting practices on adolescent development. In particular, parental monitoring, open parent-child communication, supervision, and high quality of the parent-child relationship deter involvement in high-risk behavior. Authoritative parenting generally leads to the best outcomes for teens. Clinicians should find opportunities to discuss evidence-based parenting practices with families. Future research should focus on the development and long-term evaluation of effective parenting interventions.

Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA.

PubMed Central

Khanna, M; Stotzky, G

1992-01-01

The equilibrium adsorption and binding of DNA from Bacillus subtilis on the clay mineral montmorillonite, the ability of bound DNA to transform competent cells, and the resistance of bound DNA to degradation by DNase I are reported. Maximum adsorption of DNA on the clay occurred after 90 min of contact and was followed by a plateau. Adsorption was pH dependent and was greatest at pH 1.0 (19.9 micrograms of DNA mg of clay-1) and least at pH 9.0 (10.7 micrograms of DNA mg of clay-1). The transformation frequency increased as the pH at which the clay-DNA complexes were prepared increased, and there was no transformation by clay-DNA complexes prepared at pH 1. After extensive washing with deionized distilled water (pH 5.5) or DNA buffer (pH 7.5), 21 and 28%, respectively, of the DNA remained bound. Bound DNA was capable of transforming competent cells (as was the desorbed DNA), indicating that adsorption, desorption, and binding did not alter the transforming ability of the DNA. Maximum transformation by bound DNA occurred at 37 degrees C (the other temperatures evaluated were 0, 25, and 45 degrees C). DNA bound on montmorillonite was protected against degradation by DNase, supporting the concept that "cryptic genes" may persist in the environment when bound on particulates. The concentration of DNase required to inhibit transformation by bound DNA was higher than that required to inhibit transformation by comparable amounts of free DNA, and considerably more bound than free DNase was required to inhibit transformation by the same amount of free DNA. Similarly, when DNA and DNase were bound on the same or separate samples of montmorillonite, the bound DNA was protected from the activity of DNase. PMID:1622268

Gata3 hypermethylation and Foxp3 hypomethylation are associated with sustained protection and bystander effect following epicutaneous immunotherapy in peanut-sensitized mice.

PubMed

Mondoulet, Lucie; Dioszeghy, Vincent; Busato, Florence; Plaquet, Camille; Dhelft, Véronique; Bethune, Kevin; Leclere, Laurence; Daviaud, Christian; Ligouis, Mélanie; Sampson, Hugh; Dupont, Christophe; Tost, Jörg

2018-05-19

Epicutaneous immunotherapy (EPIT) is a promising method for treating food allergies. In animal models, EPIT induces sustained unresponsiveness and prevents further sensitization mediated by Tregs. Here, we elucidate the mechanisms underlying the therapeutic effect of EPIT, by characterizing the kinetics of DNA methylation changes in sorted cells from spleen and blood and by evaluating its persistence and bystander effect compared to oral immunotherapy (OIT). BALB/c mice orally sensitized to peanut proteins (PPE) were treated by EPIT using a PPE-patch or by PPE-OIT. Another set of peanut-sensitized mice treated by EPIT or OIT were sacrificed following a protocol of sensitization to OVA. DNA methylation was analysed during immunotherapy and 8 weeks after the end of treatment in sorted cells from spleen and blood by pyrosequencing. Humoral and cellular responses were measured during and after immunotherapy. Analyses showed a significant hypermethylation of the Gata3 promoter detectable only in Th2 cells for EPIT from the 4 th week and a significant hypomethylation of the Foxp3 promoter in CD62L + Tregs, which was sustained only for EPIT. In addition, mice treated with EPIT were protected from subsequent sensitization and maintained the epigenetic signature characteristic for EPIT. Our study demonstrates that EPIT leads to a unique and stable epigenetic signature in specific T cell compartments with down regulation of Th2 key regulators and upregulation of Treg transcription factors, likely explaining the sustainability of protection and the observed bystander effect. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Direct DNA binding by Brca1.

PubMed

Paull, T T; Cortez, D; Bowers, B; Elledge, S J; Gellert, M

2001-05-22

The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein-DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription.

The DNA isolation method has effect on allele drop-out and on the results of fluorescent PCR and DNA fragment analysis.

PubMed

Nagy, Bálint; Bán, Zoltán; Papp, Zoltán

2005-10-01

The quality and the quantity of isolated DNA have an effect on PCR amplifications. The authors studied three DNA isolation protocols (resin binding method using fresh and frozen amniotic fluid samples, and silica adsorption method using fresh samples) on the quantity and on the quality of the isolated DNA. Amniotic fluid samples were obtained from 20 pregnant women. The isolated DNA concentrations were determined by real-time fluorimeter using SYBRGreen I method. Each sample was studied for the presence of 8 STR markers. The authors compared the number of the detected alleles, electrophoretograms and peak areas. There was a significant difference between the concentration of the obtained DNA and in the peak areas between the three isolation protocols. The numbers of detected alleles were different, we observed the most allele drop outs in the resin type DNA isolation protocol from the fresh sample (detected allele numbers 182), followed by resin binding protocol from the frozen samples (detected allele number 243) and by the silica adsorption method (detected allele number 264). The authors demonstrated that the DNA isolation method has an effect on the quantity and quality of the isolated DNA, and on further PCR amplifications.

Effects of different levels of vitamin C on UV radiation-induced DNA damage

NASA Astrophysics Data System (ADS)

Zhou, Dianfeng; Heng, Hang; Ji, Kang; Ke, Weizhong

2005-05-01

The Raman spectra of DNA in different levels of vitamin C with 10- and 30-min ultraviolet (UV) radiations were reported. The intensity of UV radiation was 18.68 W/m2. The experimental results proved that vitamin C could alone prevent UV radiation from damaging DNA, but the effects depended on the concentration of vitamin C. When the concentration of vitamin C was about 0.08-0.4 mmol/L, vitamin C decreased UV radiation-induced DNA's damage. When the concentration of vitamin C exceeded 0.4 mmol/L, vitamin C accelerated DNA's damage instead. Maybe the reason is that when DNA in aqueous solution is radiated by UV, free radicals come into being, and vitamin C can scavenge free radicals, so vitamin C in lower concentration can protect DNA. The quantity of free radicals is finite, when vitamin C is superfluous, free radicals have been scavenged absolutely and vitamin C is residual. Vitamin C is a strong reductant. When the mixture of DNA and residual vitamin C is radiated by UV, vitamin C reacts with DNA. The more residual vitamin C and the longer time of UV radiation, the more DNA is damaged.

Inhibitory effects of vitamin K3 on DNA polymerase and angiogenesis.

PubMed

Matsubara, Kiminori; Kayashima, Tomoko; Mori, Masaharu; Yoshida, Hiromi; Mizushina, Yoshiyuki

2008-09-01

Vitamins play essential roles in cellular reactions and maintain human health. Recent studies have revealed that some vitamins including D3, B6 and K2 and their derivatives have an anti-cancer effect. As a mechanism, their inhibitory effect on cancer-related angiogenesis has been demonstrated. Vitamin K2 (menaquinones) has an anti-cancer effect in particular for hepatic cancer and inhibits angiogenesis. In the current study, we demonstrated that sole vitamin K3 (menadione) selectively inhibits the in vitro activity of eukaryotic DNA polymerase gamma, which is a mitochondrial DNA polymerase, and suppresses angiogenesis in a rat aortic ring model. The anti-angiogenic effect of vitamin K3 has been shown in angiogenesis models using human umbilical vein endothelial cells (HUVECs) with regard to HUVEC growth, tube formation on reconstituted basement membrane and chemotaxis. These results suggest that vitamin K3 may be a potential anti-cancer agent like vitamin K2.

Apigenin attenuates streptozotocin-induced pancreatic β cell damage by its protective effects on cellular antioxidant defense.

PubMed

Wang, Ning; Yi, Wen Jing; Tan, Lu; Zhang, Jia Hui; Xu, Jiamin; Chen, Yi; Qin, Mengting; Yu, Shuang; Guan, Jing; Zhang, Rui

2017-06-01

Pancreatic beta cells are very sensitive to oxidative stress, which is one of the major causes of cell damages in diabetes. Growing interest has focused on the development of effective therapeutics to protect pancreatic cells from oxidative stress and searching for potentially protective antioxidants for treating diabetes. Apigenin, a plant-derived flavonoid, was investigated to determine whether it could protect rat insulinoma cell lines (RINm5F pancreatic beta cells) against streptozotocin (STZ)-induced oxidative damages and the mechanisms implicated. Our results showed that STZ treatment could induce oxidative stress and consequent cytotoxic effects in RINm5F cells. Pretreatment with apigenin effectively decreased the intracellular reactive oxygen species (ROS) production, attenuated cellular DNA damage, diminished lipid peroxidation, relieved protein carbonylation, and restored the cell apoptosis of pancreatic beta cells stressed by STZ. Our further experiments demonstrated that the beneficial effects of apigenin were related to ameliorate the loss of antioxidant enzymes of the STZ-treated cells in the level of gene transcription, protein expression, and enzyme activity. That suggested apigenin was not only a free radical scavenger but also a regulator to antioxidant defenses of pancreatic cells. Taken all together, our findings suggested that apigenin could attenuate the STZ-induced oxidative damages in pancreatic beta cells and might serve as a novel agent for the treatment of diabetes.

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.

The optical effect of a semiconductor laser on protecting wheat from UV-B radiation damage.

PubMed

Qiu, Zong-Bo; Zhu, Xin-Jun; Li, Fang-Min; Liu, Xiao; Yue, Ming

2007-07-01

Lasers have been widely used in the field of biology along with the development of laser technology, but the mechanism of the bio-effect of lasers is not explicit. The objective of this paper was to test the optical effect of a laser on protecting wheat from UV-B damage. A patent instrument was employed to emit semiconductor laser (wavelength 650 nm) and incoherent red light, which was transformed from the semiconductor laser. The wavelength, power and lightfleck diameter of the incoherent red light are the same as those of the semiconductor laser. The semiconductor laser (wavelength 650 nm, power density 3.97 mW mm(-2)) and incoherent red light (wavelength 650 nm, power density 3.97 mW mm(-2)) directly irradiated the embryo of wheat seeds for 3 min respectively, and when the seedlings were 12-day-old they were irradiated by UV-B radiation (10.08 kJ m(-2)) for 12 h in the dark. Changes in the concentration of malondialdehyde (MDA), hydrogen peroxide (H(2)O(2)), glutathione (GSH), ascorbate (AsA), carotenoids (CAR), the production rate of superoxide radical (O(2)(-)), the activities of peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and the growth parameters of seedlings (plant height, leaf area and fresh weight) were measured to test the optical effect of the laser. The results showed that the incoherent red light treatment could not enhance the activities of SOD, POD and CAT and the concentration of AsA and CAR. When the plant cells were irradiated by UV-B, the incoherent red light treatment could not eliminate active oxygen and prevent lipid peroxidation in wheat. The results also clearly demonstrate that the plant DNA was damaged by UV-B radiation and semiconductor laser irradiance had the capability to protect plants from UV-B-induced DNA damage, while the incoherent red light could not. This is the first investigation reporting the optical effect of a semiconductor laser on protecting wheat from UV-B radiation damage.

Genome defense against exogenous nucleic acids in eukaryotes by non-coding DNA occurs through CRISPR-like mechanisms in the cytosol and the bodyguard protection in the nucleus.

PubMed

Qiu, Guo-Hua

2016-01-01

In this review, the protective function of the abundant non-coding DNA in the eukaryotic genome is discussed from the perspective of genome defense against exogenous nucleic acids. Peripheral non-coding DNA has been proposed to act as a bodyguard that protects the genome and the central protein-coding sequences from ionizing radiation-induced DNA damage. In the proposed mechanism of protection, the radicals generated by water radiolysis in the cytosol and IR energy are absorbed, blocked and/or reduced by peripheral heterochromatin; then, the DNA damage sites in the heterochromatin are removed and expelled from the nucleus to the cytoplasm through nuclear pore complexes, most likely through the formation of extrachromosomal circular DNA. To strengthen this hypothesis, this review summarizes the experimental evidence supporting the protective function of non-coding DNA against exogenous nucleic acids. Based on these data, I hypothesize herein about the presence of an additional line of defense formed by small RNAs in the cytosol in addition to their bodyguard protection mechanism in the nucleus. Therefore, exogenous nucleic acids may be initially inactivated in the cytosol by small RNAs generated from non-coding DNA via mechanisms similar to the prokaryotic CRISPR-Cas system. Exogenous nucleic acids may enter the nucleus, where some are absorbed and/or blocked by heterochromatin and others integrate into chromosomes. The integrated fragments and the sites of DNA damage are removed by repetitive non-coding DNA elements in the heterochromatin and excluded from the nucleus. Therefore, the normal eukaryotic genome and the central protein-coding sequences are triply protected by non-coding DNA against invasion by exogenous nucleic acids. This review provides evidence supporting the protective role of non-coding DNA in genome defense. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of Chromatin Structure on the Extent and Distribution of DNA Double Strand Breaks Produced by Ionizing Radiation; Comparative Study of hESC and Differentiated Cells Lines.

PubMed

Venkatesh, Priyanka; Panyutin, Irina V; Remeeva, Evgenia; Neumann, Ronald D; Panyutin, Igor G

2016-01-02

Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC.

DNA condensation and size effects of DNA condensation agent

NASA Astrophysics Data System (ADS)

Liu, Yan-Hui; Jiang, Chong-Ming; Guo, Xin-Miao; Tang, Yan-Lin; Hu, Lin

2013-08-01

Based on the model of the strong correlation of counterions condensed on DNA molecule, by tailoring interaction potential, interduplex spacing and correlation spacing between condensed counterions on DNA molecule and interduplex spacing fluctuation strength, toroidal configuration, rod-like configuration and two-hole configurations are possible. The size effects of counterion structure on the toroidal structure can be detected by this model. The autocorrelation function of the tangent vectors is found as an effective way to detect the structure of toroidal conformations and the generic pathway of the process of DNA condensation. The generic pathway of all of the configurations involves an initial nucleation loop, and the next part of the DNA chain is folded on the top of the initial nucleation loop with different manners, in agreement with the recent experimental results.

Co-immunization with virus-like particle and DNA vaccines induces protection against respiratory syncytial virus infection and bronchiolitis

PubMed Central

Hwang, Hye Suk; Kwon, Young-Man; Lee, Jong Seok; Yoo, Si-Eun; Lee, Yu-Na; Ko, Eun-Ju; Kim, Min-Chul; Cho, Min-Kyoung; Lee, Young-Tae; Jung, Yu-Jin; Lee, Ji-Yun; Li, Jian Dong; Kang, Sang-Moo

2014-01-01

This study demonstrates that immunization with non-replicating virus-like particle (FFG VLP) containing RSV F and G glycoproteins together with RSV F DNA induced T helper type 1 antibody responses to RSV F similar to live RSV infection. Upon RSV challenge 21 weeks after immunization, FFG VLP vaccination induced protection against RSV infection as shown by clearance of lung viral loads, and the absence of eosinophil infiltrates, and did not cause lung pathology. In contrast, formalin-inactivated RSV (FI-RSV) vaccination showed significant pulmonary eosinophilia, severe mucus production, and extensive histopathology resulting in a hallmark of pulmonary pathology. Substantial lung pathology was also observed in mice with RSV re-infections. High levels of systemic and local inflammatory cytokine-secreting cells were induced in mice with FI-RSV but not with FFG VLP immunization after RSV challenge. Therefore, the results provide evidence that recombinant RSV FFG VLP vaccine can confer long-term protection against RSV without causing lung pathology. PMID:25110201

Jatropha curcas leaf and bark fractions protect against ultraviolet radiation-B induced DNA damage in human peripheral blood lymphocytes.

PubMed

Sundari, J; Selvaraj, R; Rajendra Prasad, N; Elumalai, R

2013-11-01

The present study is conducted to investigate the antioxidant potential of Jatropha curcas root bark extract (RB4 fraction) and leaf extract (L1 fraction), and to study their effects on UVB-radiation-induced DNA damage in cultured human blood lymphocytes. In this study, J. curcas showed strong antioxidant property in different free radical scavenging systems. Both the fractions effectively scavenged hydroxyl (OH), superoxide anion (O₂(·-)), 1,1-diphenyl-2-picrylhydrazyl (DPPH·) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical cation (ABTS(·+)) in a concentration-dependent manner. The IC₅₀ (Inhibitory Concentration 50) values of J. curcas fractions were compared to standard ascorbic acid used in this study. The antioxidant potential of a compound was directly proportional to the photoprotective effect. In this study, human peripheral blood lymphocytes (HPBL) were exposed to UVB-radiation and there was an increase in comet attributes (% tail DNA, tail length, tail movement and Olive tail moment). Jatropha curcas RB4 fraction and L1 fraction treatment before UVB-irradiation significantly decreased the % tail DNA, tail length, tail moment and Olive tail moment in irradiated HPBL. These results suggested that J. curcas exhibited strong antioxidant property and RB4 and L1 fractions protected UVB-radiation-induced DNA damage in HPBL. Copyright © 2013 Elsevier B.V. All rights reserved.

Protective immunity conferred by porcine circovirus 2 ORF2-based DNA vaccine in mice.

PubMed

Sylla, Seydou; Cong, Yan-Long; Sun, Yi-Xue; Yang, Gui-Lian; Ding, Xue-Mei; Yang, Zhan-Qing; Zhou, Yu-Long; Yang, Minnan; Wang, Chun-Feng; Ding, Zhuang

2014-07-01

Post-weaning multisystemic wasting syndrome (PMWS) associated with porcine circovirus type 2 (PCV2) has caused the swine industry significant health challenges and economic damage. Although inactivated and subunit vaccines against PMWS have been used widely, so far no DNA vaccine is available. In this study, with the aim of exploring a new route for developing a vaccine against PCV2, the immunogenicity of a DNA vaccine was evaluated in mice. The pEGFP-N1 vector was used to construct a PCV2 Cap gene recombinant vaccine. To assess the immunogenicity of pEGFP-Cap, 80 BALB/c mice were immunized three times at 2 weekly intervals with pEGFP-Cap, LG-strain vaccine, pEGFP-N1 vector or PBS and then challenged with PCV2. IgG and cytokines were assessed by indirect ELISA and ELISA, respectively. Specimens stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) techniques were examined histopathologically. It was found that vaccination of the mice with the pEGFP-Cap induced solid protection against PCV2 infection through induction of highly specific serum IgG antibodies and cytokines (IFN-γ and IL-10), and a small PCV2 viral load. The mice treated with the pEGFP-Cap and LG-strain developed no histopathologically detectable lesions (HE stain) and IHC techniques revealed only a few positive cells. Thus, this study demonstrated that recombinant pEGFP-Cap substantially alleviates PCV2 infection in mice and provides evidence that a DNA vaccine could be an alternative to PCV2 vaccines against PMWS. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

Plant Flavone Apigenin Binds to Nucleic Acid Bases and Reduces Oxidative DNA Damage in Prostate Epithelial Cells

PubMed Central

Bhaskaran, Natarajan; Gupta, Sanjay

2014-01-01

Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it’s binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2′ deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities. PMID:24614817

Plant flavone apigenin binds to nucleic acid bases and reduces oxidative DNA damage in prostate epithelial cells.

PubMed

Sharma, Haripaul; Kanwal, Rajnee; Bhaskaran, Natarajan; Gupta, Sanjay

2014-01-01

Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it's binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2' deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities.

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

Pomegranate extract protects against cerebral ischemia/reperfusion injury and preserves brain DNA integrity in rats.

PubMed

Ahmed, Maha A E; El Morsy, Engy M; Ahmed, Amany A E

2014-08-21

Interruption to blood flow causes ischemia and infarction of brain tissues with consequent neuronal damage and brain dysfunction. Pomegranate extract is well tolerated, and safely consumed all over the world. Interestingly, pomegranate extract has shown remarkable antioxidant and anti-inflammatory effects in experimental models. Many investigators consider natural extracts as novel therapies for neurodegenerative disorders. Therefore, this study was carried out to investigate the protective effects of standardized pomegranate extract against cerebral ischemia/reperfusion-induced brain injury in rats. Adult male albino rats were randomly divided into sham-operated control group, ischemia/reperfusion (I/R) group, and two other groups that received standardized pomegranate extract at two dose levels (250, 500 mg/kg) for 15 days prior to ischemia/reperfusion (PMG250+I/R, and PMG500+I/R groups). After I/R or sham operation, all rats were sacrificed and brains were harvested for subsequent biochemical analysis. Results showed reduction in brain contents of MDA (malondialdehyde), and NO (nitric oxide), in addition to enhancement of SOD (superoxide dismutase), GPX (glutathione peroxidase), and GRD (glutathione reductase) activities in rats treated with pomegranate extract prior to cerebral I/R. Moreover, pomegranate extract decreased brain levels of NF-κB p65 (nuclear factor kappa B p65), TNF-α (tumor necrosis factor-alpha), caspase-3 and increased brain levels of IL-10 (interleukin-10), and cerebral ATP (adenosine triphosphate) production. Comet assay showed less brain DNA (deoxyribonucleic acid) damage in rats protected with pomegranate extract. The present study showed, for the first time, that pre-administration of pomegranate extract to rats, can offer a significant dose-dependent neuroprotective activity against cerebral I/R brain injury and DNA damage via antioxidant, anti-inflammatory, anti-apoptotic and ATP-replenishing effects. Copyright © 2014 Elsevier Inc

Hypothermia postpones DNA damage repair in irradiated cells and protects against cell killing.

PubMed

Baird, Brandon J; Dickey, Jennifer S; Nakamura, Asako J; Redon, Christophe E; Parekh, Palak; Griko, Yuri V; Aziz, Khaled; Georgakilas, Alexandros G; Bonner, William M; Martin, Olga A

2011-06-03

Hibernation is an established strategy used by some homeothermic organisms to survive cold environments. In true hibernation, the core body temperature of an animal may drop to below 0°C and metabolic activity almost cease. The phenomenon of hibernation in humans is receiving renewed interest since several cases of victims exhibiting core body temperatures as low as 13.7°C have been revived with minimal lasting deficits. In addition, local cooling during radiotherapy has resulted in normal tissue protection. The experiments described in this paper were prompted by the results of a very limited pilot study, which showed a suppressed DNA repair response of mouse lymphocytes collected from animals subjected to 7-Gy total body irradiation under hypothermic (13°C) conditions, compared to normothermic controls. Here we report that human BJ-hTERT cells exhibited a pronounced radioprotective effect on clonogenic survival when cooled to 13°C during and 12h after irradiation. Mild hypothermia at 20 and 30°C also resulted in some radioprotection. The neutral comet assay revealed an apparent lack on double strand break (DSB) rejoining at 13°C. Extension of the mouse lymphocyte study to ex vivo-irradiated human lymphocytes confirmed lower levels of induced phosphorylated H2AX (γ-H2AX) and persistence of the lesions at hypothermia compared to the normal temperature. Parallel studies of radiation-induced oxidatively clustered DNA lesions (OCDLs) revealed partial repair at 13°C compared to the rapid repair at 37°C. For both γ-H2AX foci and OCDLs, the return of lymphocytes to 37°C resulted in the resumption of normal repair kinetics. These results, as well as observations made by others and reviewed in this study, have implications for understanding the radiobiology and protective mechanisms underlying hypothermia and potential opportunities for exploitation in terms of protecting normal tissues against radiation. 2011. Published by Elsevier B.V.

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.

Mitochondria-targeted antioxidant mitotempo protects mitochondrial function against amyloid beta toxicity in primary cultured mouse neurons.

PubMed

Hu, Hongtao; Li, Mo

2016-09-09

Mitochondrial defects including excess reactive oxygen species (ROS) production and compromised ATP generation are featured pathology in Alzheimer's disease (AD). Amyloid beta (Aβ)-mediated mitochondrial ROS overproduction disrupts intra-neuronal Redox balance, in turn exacerbating mitochondrial dysfunction leading to neuronal injury. Previous studies have found the beneficial effects of mitochondria-targeted antioxidants in preventing mitochondrial dysfunction and neuronal injury in AD animal and cell models, suggesting that mitochondrial ROS scavengers hold promise for the treatment of this neurological disorder. In this study, we have determined that mitotempo, a novel mitochondria-targeted antioxidant protects mitochondrial function from the toxicity of Aβ in primary cultured neurons. Our results showed that Aβ-promoted mitochondrial superoxide production and neuronal lipid oxidation were significantly suppressed by the application of mitotempo. Moreover, mitotempo also demonstrated protective effects on mitochondrial bioenergetics evidenced by preserved mitochondrial membrane potential, cytochrome c oxidase activity as well as ATP production. In addition, the Aβ-induced mitochondrial DNA (mtDNA) depletion and decreased expression levels of mtDNA replication-related DNA polymerase gamma (DNA pol γ) and Twinkle were substantially mitigated by mitotempo. Therefore, our study suggests that elimination of excess mitochondrial ROS rescues mitochondrial function in Aβ-insulted neruons; and mitotempo has the potential to be a promising therapeutic agent to protect mitochondrial and neuronal function in AD. Copyright © 2016 Elsevier Inc. All rights reserved.

Genoprotective effect of hyaluronic acid against benzalkonium chloride-induced DNA damage in human corneal epithelial cells

PubMed Central

Wu, Han; Zhang, Huina; Wang, Changjun; Wu, Yihua; Xie, Jiajun; Jin, Xiuming; Yang, Jun

2011-01-01

Purpose The aim of this study was to investigate hyaluronic acid (HA) protection on cultured human corneal epithelial cells (HCEs) against benzalkonium chloride (BAC)-induced DNA damage and intracellular reactive oxygen species (ROS) increase. Methods Cells were incubated with different concentrations of BAC with or without the presence of 0.2% HA for 30 min. DNA damage to HCEs was examined by alkaline comet assay and by immunofluorescence microscopic detection of the phosphorylated form of histone variant H2AX (γH2AX) foci. ROS production was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Cell apoptosis was determined with annexin V staining by flow cytometry. Results HA significantly reduced BAC-induced DNA damage as indicated by the tail length (TL) and tail moment (TM) of alkaline comet assay and by γH2AX foci formation, respectively. Moreover, HA significantly decreased BAC-induced ROS increase and cell apoptosis. However, exposure to HA alone did not produce any significant change in DNA damage, ROS generation, or cell apoptosis. Conclusions BAC could induce DNA damage and cell apoptosis in HCEs, probably through increasing oxidative stress. Furthermore, HA was an effective protective agent that had antioxidant properties and could decrease DNA damage and cell apoptosis induced by BAC. PMID:22219631

A Comparative Analysis of the Photo-Protective Effects of Soy Isoflavones in Their Aglycone and Glucoside Forms

PubMed Central

Iovine, Barbara; Iannella, Maria Luigia; Gasparri, Franco; Giannini, Valentina; Monfrecola, Giuseppe; Bevilacqua, Maria Assunta

2012-01-01

Isoflavones exist in nature predominantly as glucosides such as daidzin or genistin and are rarely found in their corresponding aglycone forms daidzein and genistein. The metabolism and absorption of isoflavones ingested with food is well documented, but little is known about their use as topical photo-protective agents. The aim of this study was to investigate in a comparative analysis the photo-protective effects of isoflavones in both their aglycone and glucoside forms. In human skin fibroblasts irradiated with 60 mJ/cm2 ultraviolet B (UVB), we measured the expression levels of COX-2 and Gadd45, which are involved in inflammation and DNA repair, respectively. We also determined the cellular response to UVB-induced DNA damage using the comet assay. Our findings suggest that both the isoflavone glucosides at a specific concentration and combination with an aglycone mixture exerted an anti-inflammatory and photo-protective effect that prevented 41% and 71% of UVB-induced DNA damage, respectively. The advantages of using either isoflavone glucosides or an aglycone mixture in applications in the field of dermatology will depend on their properties and their different potential uses. PMID:23211668

Protective efficacy of cationic-PLGA microspheres loaded with DNA vaccine encoding the sip gene of Streptococcus agalactiae in tilapia.

PubMed

Ma, Yan-Ping; Ke, Hao; Liang, Zhi-Ling; Ma, Jiang-Yao; Hao, Le; Liu, Zhen-Xing

2017-07-01

Streptococcus agalactiae (S. agalactiae) is an important fish pathogen, which has received more attention in the past decade due to the increasing economic losses in the tilapia industry worldwide. As existing effective vaccines of S. agalactiae in fish have obvious disadvantage, to select immunoprotective antigens and package materials would undoubtedly contribute to the development of novel oral vaccines. In the present study, surface immunogenic protein (sip) was selected from the S. agalactiae serovar I a genomes as immunogenic protein in DNA vaccine form with cationic chitosan and biodegradable and biocompatible PLGA. The pcSip plasmid in cationic-PLGA was successfully expressed in tissues of immunized tilapia and the immunogenicity was assessed in tilapia challenge model. A significant increase was observed in the cytokine levels of IL-1β, TNF-α, CC1, CC2 in spleen and kidney tissues. Furthermore, immunized tilapia conferred different levels of protection against challenge with a lethal dose of highly virulent serovar I a S. agalactiae. Our results indicated that the pcSip plasmid in cationic-PLGA induced high level of antibodies and protection against S. agalactiae infection, could be effective oral DNA vaccine candidates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Humoral and cell-mediated immune responses in DNA immunized mink challenged with wild-type canine distemper virus.

PubMed

Nielsen, Line; Søgaard, Mette; Karlskov-Mortensen, Peter; Jensen, Trine Hammer; Jensen, Tove Dannemann; Aasted, Bent; Blixenkrone-Møller, Merete

2009-07-30

The aim of the study was to investigate the different phases of the immune response after DNA immunization with the hemagglutinin and nucleoprotein genes from canine distemper virus (CDV). Although attenuated live CDV vaccines have effectively reduced the incidence of disease, canine distemper is still a problem worldwide. The broad host range of CDV creates a constant viral reservoir among wildlife animals. Our results demonstrated early humoral and cell-mediated immune responses (IFN-gamma) in DNA vaccinated mink compared to mock-vaccinated mink after challenge with a Danish wild-type CDV. The DNA vaccine-induced immunity protected the natural host against disease development.

Improved mitochondrial function underlies the protective effect of pirfenidone against tubulointerstitial fibrosis in 5/6 nephrectomized rats.

PubMed

Chen, Jun-Feng; Liu, Hong; Ni, Hai-Feng; Lv, Lin-Li; Zhang, Ming-Hui; Zhang, Ai-Hua; Tang, Ri-Ning; Chen, Ping-Sheng; Liu, Bi-Cheng

2013-01-01

Dysfunctional mitochondria participate in the progression of chronic kidney disease (CKD). Pirfenidone is a newly identified anti-fibrotic drug. However, its mechanism remains unclear. Mitochondrial dysfunction is an early event that occurs prior to the onset of renal fibrosis. In this context, we investigated the protective effect of pirfenidone on mitochondria and its relevance to apoptosis and oxidative stress in renal proximal tubular cells. A remnant kidney rat model was established. Human renal proximal tubular epithelial cells (HK2) using rotenone, a mitochondrial respiratory chain complex Ι inhibitor were further investigated in vitro to examine the mitochondrial protective effect of pirfenidone. Pirfenidone protected mitochondrial structures and functions by stabilizing the mitochondrial membrane potential, maintaining ATP production and improving the mitochondrial DNA (mtDNA) copy number. Pirfenidone decreased tubular cell apoptosis by inhibiting the mitochondrial apoptotic signaling pathway. Pirfenidone also reduced oxidative stress by enhancing manganese superoxide dismutase (Mn-SOD) and inhibiting intracellular reactive oxygen species (ROS) generation, which suggested that the anti-oxidant effects occurred at least partially via the mitochondrial pathway. Pirfenidone may be effective prior to the onset of renal fibrosis because this drug exerts its anti-fibrotic effect by protection of mitochondria in renal proximal tubular cells.

Improved Mitochondrial Function Underlies the Protective Effect of Pirfenidone against Tubulointerstitial Fibrosis in 5/6 Nephrectomized Rats

PubMed Central

Chen, Jun-Feng; Liu, Hong; Ni, Hai-Feng; Lv, Lin-Li; Zhang, Ming-Hui; Zhang, Ai-Hua; Tang, Ri-Ning; Chen, Ping-Sheng; Liu, Bi-Cheng

2013-01-01

Dysfunctional mitochondria participate in the progression of chronic kidney disease (CKD). Pirfenidone is a newly identified anti-fibrotic drug. However, its mechanism remains unclear. Mitochondrial dysfunction is an early event that occurs prior to the onset of renal fibrosis. In this context, we investigated the protective effect of pirfenidone on mitochondria and its relevance to apoptosis and oxidative stress in renal proximal tubular cells. A remnant kidney rat model was established. Human renal proximal tubular epithelial cells (HK2) using rotenone, a mitochondrial respiratory chain complex Ι inhibitor were further investigated in vitro to examine the mitochondrial protective effect of pirfenidone. Pirfenidone protected mitochondrial structures and functions by stabilizing the mitochondrial membrane potential, maintaining ATP production and improving the mitochondrial DNA (mtDNA) copy number. Pirfenidone decreased tubular cell apoptosis by inhibiting the mitochondrial apoptotic signaling pathway. Pirfenidone also reduced oxidative stress by enhancing manganese superoxide dismutase (Mn-SOD) and inhibiting intracellular reactive oxygen species (ROS) generation, which suggested that the anti-oxidant effects occurred at least partially via the mitochondrial pathway. Pirfenidone may be effective prior to the onset of renal fibrosis because this drug exerts its anti-fibrotic effect by protection of mitochondria in renal proximal tubular cells. PMID:24349535

Effects of a preschool staff intervention on children's sun protection: outcomes of sun protection is fun!

PubMed

Gritz, Ellen R; Tripp, Mary K; James, Aimee S; Harrist, Ronald B; Mueller, Nancy H; Chamberlain, Robert M; Parcel, Guy S

2007-08-01

The preschool is an important yet understudied setting for sun-protection interventions. This study evaluates the effects of Sun Protection is Fun! (SPF) on preschool staff behavioral and psychosocial outcomes related to protecting children from sun exposure. Twenty preschools participated in a 2-year, group-randomized trial to evaluate SPF, a behavioral intervention grounded in social cognitive theory and designed to be more extensive than previous preschool sun-protection interventions. The staff intervention included training, a video, newsletters, a curriculum, and sunscreen. Cross-sectional samples of staff completed surveys at baseline (N= 245), a 12 month intervention assessment (N = 192), and a 24-month intervention assessment (N = 225). At the 12-month and 24-month assessments, significant behavioral effects were seen for use of sunscreen, protective clothing, and shade. Knowledge, self-efficacy, and norms were among the psychosocial variables most affected by the intervention. This study demonstrates that the SPF intervention is effective in improving staff outcomes related to children's sun protection.

Probing cigarette smoke-induced DNA single-strand breaks and screening natural protective compounds by use of magnetic bead-based chemiluminescence.

PubMed

Chen, Hongjun; Yu, Zicheng; Cao, Zhijuan; Lau, Choiwan

2016-11-01

Magnetic bead (MB)-based chemiluminescence (CL) ELISA can be a sample-thrifty, time-saving tool for evaluation of cigarette smoke-induced DNA single-strand breaks (SSBs) with high specificity. This article describes a novel approach using immobilized oligonucleotide on MBs to determine cigarette smoke-induced DNA SSBs and screen some protective natural compounds. Typically, fluorescein-labeled DNA (FAM-DNA) was immobilized on the MBs and then oxidized by the smoke in the absence or presence of natural compounds, and a part of FAM-DNA was fragmented due to cigarette smoke-induced DNA SSB and then detached from MBs whereas other non-broken FAM-DNA still remained on MBs. Then, any broken FAM-DNA fragments, complex tobacco smoke matrix, and other stuff related with natural compounds were conveniently washed away by a magnetic force, and thus possible interfering substances were completely removed. Finally, those remaining non-broken FAM-DNA on MBs were reacted with HRP-labeled anti-fluorescein antibody and then detected by CL ELISA. CL signal was converted to molar concentrations of the FAM-DNA by interpolation from a pre-determined standard linear calibration curve. The level of DNA SSBs induced by cigarette smoke was thus calculated using the method. A library of 30 natural products was subsequently screened, and two among them were found to protect DNA from oxidative damage and thus may be promising compounds for the development of new drugs. The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of DNA SSBs. Graphical abstract ᅟ.

Anthocyanin-rich blackberry extract suppresses the DNA-damaging properties of topoisomerase I and II poisons in colon carcinoma cells.

PubMed

Esselen, Melanie; Boettler, Ute; Teller, Nicole; Bachler, Simone; Hutter, Melanie; Rufer, Corinna E; Skrbek, Susanne; Marko, Doris

2011-07-13

In the present study, we addressed the question whether cyanidin-3-glucoside (C3G) or complex C3G-rich blackberry extracts affect human topoisomerases with special emphasis on the contribution of the potential degradation products phloroglucinol aldehyde (PGA) and protocatechuic acid (PCA). In HT29 colon carcinoma cells a C3G-rich blackberry extract suppressed camptothecin- (CPT-) or doxorubicin- (DOX-) induced stabilization of the covalent DNA-topoisomerase intermediate, thus antagonizing the effects of these classical topoisomerase poisons on DNA integrity. As a single compound, C3G (100 μM) decreased the DNA-damaging effects of CPT as well, but did not significantly affect those induced by DOX. At the highest applied concentration (100 μM), cyanidin protected DNA from CPT- and DOX-induced damage. Earlier reports on DNA-damaging properties of cyanidin were found to result most likely from the formation of hydrogen peroxide as an artifact in the cell culture medium when the incubation was performed in the absence of catalase. The suppression of hydrogen peroxide accumulation, achieved by the addition of catalase, demonstrated that cyanidin does not exhibit DNA-damaging properties in HT29 cells (up to 100 μM). The observed effects on topoisomerase interference and DNA protection against CPT or DOX were clearly limited to the parent compound and were not observed for the potential cyanidin degradation products PGA and PCA.

Transcending epithelial and intracellular biological barriers; a prototype DNA delivery device.

PubMed

McCaffrey, Joanne; McCrudden, Cian M; Ali, Ahlam A; Massey, Ashley S; McBride, John W; McCrudden, Maelíosa T C; Vicente-Perez, Eva M; Coulter, Jonathan A; Robson, Tracy; Donnelly, Ryan F; McCarthy, Helen O

2016-03-28

Microneedle technology provides the opportunity for the delivery of DNA therapeutics by a non-invasive, patient acceptable route. To deliver DNA successfully requires consideration of both extra and intracellular biological barriers. In this study we present a novel two tier platform; i) a peptide delivery system, termed RALA, that is able to wrap the DNA into nanoparticles, protect the DNA from degradation, enter cells, disrupt endosomes and deliver the DNA to the nucleus of cells ii) a microneedle (MN) patch that will house the nanoparticles within the polymer matrix, breach the skin's stratum corneum barrier and dissolve upon contact with skin interstitial fluid thus releasing the nanoparticles into the skin. Our data demonstrates that the RALA is essential for preventing DNA degradation within the poly(vinylpyrrolidone) (PVP) polymer matrix. In fact the RALA/DNA nanoparticles (NPs) retained functionality when in the MN arrays after 28days and over a range of temperatures. Furthermore the physical strength and structure of the MNs was not compromised when loaded with the NPs. Finally we demonstrated the effectiveness of our MN-NP platform in vitro and in vivo, with systemic gene expression in highly vascularised regions. Taken together this 'smart-system' technology could be applied to a wide range of genetic therapies. Copyright © 2016. Published by Elsevier B.V.

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.

Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

PubMed

Yu, Yun-Zhou; Ma, Yao; Xu, Wen-Hui; Wang, Shuang; Sun, Zhi-Wei

2015-08-01

DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines.

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

PubMed

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

2014-08-01

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng Min; Guangxi Center for Animal Disease Control and Prevention, Nanning 530001; College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062

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-AALmore » 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.« less

The residual protective effects of enactment.

PubMed

Wammes, Jeffrey D; Fernandes, Myra A

2017-07-01

Research has demonstrated the importance of the quality of initial retrieval events (Test 1) for performance on later memory tests (Test 2). We explored whether enacting words at encoding, relative to simply reading them, provided protection against the detrimental effects of a degraded retrieval experience, through the addition of motor processing to the extant memory representation. Participants encoded a mixed list of enacted and read words, then completed Test 1, and a later Test 2. Encoding and Test 2 were always completed under full attention (FA). Critically though, Test 1 was completed either under FA, or under divided attention (DA) with a distracting task requiring semantic and phonological processing. We predicted a larger enactment effect following DA relative to FA, indicating greater preservation of enacted words from dual-task interference. In Experiment 1, we demonstrated that the enactment effect was indeed larger following DA than FA, indicating greater preservation of enacted words after dual-task interference. In Experiment 2, we showed that this effect was even more potent over longer time scales, which served as a conceptual replication. In Experiment 3, we showed that enactment provides little to no protection when the distracting task requires motor processing, and in Experiment 4, we returned to the phonological distracting task and showed that in contrast with enactment, generation at encoding does not afford the same protection to memory. Taken together, these finding suggest that enactment renders words relatively immune to the detrimental effects of dual-tasking during testing, through the addition of a different kind, rather than a greater degree, of processing to the memory trace at encoding. Copyright © 2017 Elsevier B.V. All rights reserved.

Low-dose energetic protons induce adaptive and bystander effects that protect human cells against DNA damage caused by a subsequent exposure to energetic iron ions

PubMed Central

Buonanno, Manuela; De Toledo, Sonia M.; Howell, Roger W.; Azzam, Edouard I.

2015-01-01

During interplanetary missions, astronauts are exposed to mixed types of ionizing radiation. The low ‘flux’ of the high atomic number and high energy (HZE) radiations relative to the higher ‘flux’ of low linear energy transfer (LET) protons makes it highly probable that for any given cell in the body, proton events will precede any HZE event. Whereas progress has been made in our understanding of the biological effects of low-LET protons and high-LET HZE particles, the interplay between the biochemical processes modulated by these radiations is unclear. Here we show that exposure of normal human fibroblasts to a low mean absorbed dose of 20 cGy of 0.05 or 1-GeV protons (LET ∼ 1.25 or 0.2 keV/μm, respectively) protects the irradiated cells (P < 0.0001) against chromosomal damage induced by a subsequent exposure to a mean absorbed dose of 50 cGy from 1 GeV/u iron ions (LET ∼ 151 keV/μm). Surprisingly, unirradiated (i.e. bystander) cells with which the proton-irradiated cells were co-cultured were also significantly protected from the DNA-damaging effects of the challenge dose. The mitigating effect persisted for at least 24 h. These results highlight the interactions of biological effects due to direct cellular traversal by radiation with those due to bystander effects in cell populations exposed to mixed radiation fields. They show that protective adaptive responses can spread from cells targeted by low-LET space radiation to bystander cells in their vicinity. The findings are relevant to understanding the health hazards of space travel. PMID:25805407

Effects of different cytokines on immune responses of rainbow trout in a virus DNA vaccination model

PubMed Central

Cao, Yongsheng; Zhang, Qiya; Xu, Liming; Li, Shaowu; Wang, Di; Zhao, Jingzhuang; Liu, Hongbai; Feng, Jian; Lu, Tongyan

2017-01-01

Seven rainbow trout cytokine genes (interleukin (IL)-2, IL-8, IL-15, IL-17, IL-1β, intracellular interferon (iIFN) 1a, and IFN-γ2) were evaluated for their adjuvant effects on a DNA vaccine, called pG, containing the glycoprotein gene of infectious hematopoietic necrosis virus (IHNV). Distinct DNA constructs in expression plasmid pcDNA3.1 encoding a cytokine gene were generated. Immunofluorescence assays in rainbow trout gonadal cells demonstrated successful protein expression from all these constructs. Subsequently, fish were immunized with pG alone or together with a cytokine expression plasmid. Results showed that each cytokine plasmids at an appropriate dose showed notable effects on immune gene expression. IL-17 and IFN-γ2 can enhance early specific IgM response. All cytokines, except IL-8, can benefit initial neutralizing antibody (NAb) titers. At 35 days post immunization (dpi), NAb titers of fish immunized with pG and IL-2, iIFN1a, or IFN-γ2 plasmids remained at high levels (1:160). NAb titers of fish immunized with pG alone decreased to 1:40. IL-8 or IL-1β can enhance antigen-specific proliferative T-cell responses at 14 dpi. At 28 dpi, coinjection of pG with IL-2, IL-8, IL-15, or IL-17 plasmids induced considerably stronger lymphocyte proliferation than that with injection of pG alone. All cytokine plasmids delivered with pG plasmid enhanced protection of trout against IHNV-mediated mortality. These results indicate that the type and dose of trout cytokine genes injected into fish affect quality of immune response to DNA vaccination. PMID:29348820

Aqueous extract of Crataegus azarolus protects against DNA damage in human lymphoblast Cell K562 and enhances antioxidant activity.

PubMed

Mustapha, Nadia; Bouhlel, Inès; Chaabane, Fadwa; Bzéouich, Imèn Mokdad; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

2014-02-01

The present study was carried out to characterize the cellular antioxidant effect of the aqueous extract of Crataegus azarolus and its antigenotoxic potential using human myelogenous cells, K562. The antioxidant capacity of this extract was evaluated by determining its cellular antioxidant activity (CAA) in K562 cells. Also, preceding antigenotoxicity assessment, its eventual genotoxicity property was investigated by evaluating its capacity to induce the DNA degradation of treated cell nuclei. As no genotoxicity was detected at different exposure times, its ability to protect cell DNA against H2O2 oxidative effect was investigated, using the "comet assay." It appears that 800 μg/mL of extract inhibited the genotoxicity induced by H2O2 with a rate of 41.30 %, after 4 h of incubation. In addition, this extract revealed a significant cellular antioxidant capacity against the reactive oxygen species in K562 cells.

Effect of 2',3'-dideoxythymidine-5'-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase alpha is the only polymerase required for cellular DNA replication.

PubMed Central

Waqar, M A; Evans, M J; Huberman, J A

1978-01-01

We have studied the effects of the nucleotide analogue, 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100-500 micrometer when the dTTP concentration is maintained at 10 micrometer. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases beta or gamma is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase alpha is very resistant. These observations suggest that DNA polymerase alpha may be the only DNA polymerase required for all aspects of cellular DNA synthesis. PMID:673840

Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice

PubMed Central

Safdar, Adeel; Bourgeois, Jacqueline M.; Ogborn, Daniel I.; Little, Jonathan P.; Hettinga, Bart P.; Akhtar, Mahmood; Thompson, James E.; Melov, Simon; Mocellin, Nicholas J.; Kujoth, Gregory C.; Prolla, Tomas A.; Tarnopolsky, Mark A.

2011-01-01

A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities. PMID:21368114

Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes

PubMed Central

Rosada, Rogério S; Torre, Lucimara Gaziola de la; Frantz, Fabiani G; Trombone, Ana PF; Zárate-Bladés, Carlos R; Fonseca, Denise M; Souza, Patrícia RM; Brandão, Izaíra T; Masson, Ana P; Soares, Édson G; Ramos, Simone G; Faccioli, Lúcia H; Silva, Célio L; Santana, Maria HA; Coelho-Castelo, Arlete AM

2008-01-01

Background The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally. Results We developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65) or complexed (COMP-hsp65), and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 μg of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-γ and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 μg). Conclusion Our objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease. PMID

Inactivation of NADPH oxidases NOX4 and NOX5 protects human primary fibroblasts from ionizing radiation-induced DNA damage.

PubMed

Weyemi, Urbain; Redon, Christophe E; Aziz, Towqir; Choudhuri, Rohini; Maeda, Daisuke; Parekh, Palak R; Bonner, Michael Y; Arbiser, Jack L; Bonner, William M

2015-03-01

Human exposure to ionizing radiation from medical procedures has increased sharply in the last three decades. Recent epidemiological studies suggest a direct relationship between exposure to ionizing radiation and health problems, including cancer incidence. Therefore, minimizing the impact of radiation exposure in patients has become a priority in the development of future clinical practices. Crucial players in radiation-induced DNA damage include reactive oxygen species (ROS), but the sources of these have remained elusive. To the best of our knowledge, we show here for the first time that two members of the ROS-generating NADPH oxidase family (NOXs), NOX4 and NOX5, are involved in radiation-induced DNA damage. Depleting these two NOXs in human primary fibroblasts resulted in reduced levels of DNA damage as measured by levels of radiation-induced foci, a marker of DNA double-strand breaks (DSBs) and the comet assay coupled with increased cell survival. NOX involvement was substantiated with fulvene-5, a NOXs-specific inhibitor. Moreover, fulvene-5 mitigated radiation-induced DNA damage in human peripheral blood mononuclear cells ex vivo. Our results provide evidence that the inactivation of NOXs protects cells from radiation-induced DNA damage and cell death. These findings suggest that NOXs inhibition may be considered as a future pharmacological target to help minimize the negative effects of radiation exposure for millions of patients each year.

Inactivation of NADPH Oxidases NOX4 and NOX5 Protects Human Primary Fibroblasts from Ionizing Radiation-Induced DNA Damage

PubMed Central

Weyemi, Urbain; Redon, Christophe E.; Aziz, Towqir; Choudhuri, Rohini; Maeda, Daisuke; Parekh, Palak R.; Bonner, Michael Y.; Arbiser, Jack L.; Bonner, William M.

2015-01-01

Human exposure to ionizing radiation from medical procedures has increased sharply in the last three decades. Recent epidemiological studies suggest a direct relationship between exposure to ionizing radiation and health problems, including cancer incidence. Therefore, minimizing the impact of radiation exposure in patients has become a priority in the development of future clinical practices. Crucial players in radiation-induced DNA damage include reactive oxygen species (ROS), but the sources of these have remained elusive. To the best of our knowledge, we show here for the first time that two members of the ROS-generating NADPH oxidase family (NOXs), NOX4 and NOX5, are involved in radiation-induced DNA damage. Depleting these two NOXs in human primary fibroblasts resulted in reduced levels of DNA damage as measured by levels of radiation-induced foci, a marker of DNA double-strand breaks (DSBs) and the comet assay coupled with increased cell survival. NOX involvement was substantiated with fulvene-5, a NOXs-specific inhibitor. Moreover, fulvene-5 mitigated radiation-induced DNA damage in human peripheral blood mononuclear cells ex vivo. Our results provide evidence that the inactivation of NOXs protects cells from radiation-induced DNA damage and cell death. These findings suggest that NOXs inhibition may be considered as a future pharmacological target to help minimize the negative effects of radiation exposure for millions of patients each year. PMID:25706776

DNA Vaccines - A Modern Gimmick or a Boon to Vaccinology?

PubMed

Manickan, Elanchezhiyan; Karem, Kevin L; Rouse, Barry T

2017-01-01

The reports in 1993 that naked DNA encoding viral genes conferred protective immunity came as a surprise to most vaccinologists. This review analyses the expanding number of examples where plasmid DNA induces immune responses. Issues such as the type of immunity induced, mechanisms of immune protection, and how DNA vaccines compare with other approaches are emphasized. Additional issues discussed include the likely means by which DNA vaccines induce CTL, how the potency and type of immunity induced can be modified, and whether DNA vaccines represent a practical means of manipulating unwanted immune response occurring during immunoinflammatory diseases. It seems doubtful if DNA vaccines will replace currently effective vaccines, but they may prove useful for prophylactic use against some agents that at present lack an effective vaccine. DNA vaccines promise to be valuable to manipulate the immune response in situations where responses to agents are inappropriate or ineffective.

Fungal beta glucan protects radiation induced DNA damage in human lymphocytes.

PubMed

Pillai, Thulasi G; Maurya, Dharmendra K; Salvi, Veena P; Janardhanan, Krishnankutty K; Nair, Cherupally K K

2014-02-01

Ganoderma lucidum (Ling Zhi), a basidiomycete white rot macrofungus has been used extensively for therapeutic use in China, Japan, Korea and other Asian countries for 2,000 years. The present study is an attempt to investigate its DNA protecting property in human lymphocytes. Beta glucan (BG) was isolated by standard procedure and the structure and composition were studied by infrared radiation (IR) and nuclear magnetic resonance (NMR) spectroscopy, gel filtration chromatography and paper chromatography. The radioprotective properties of BG isolated from the macro fungi Ganoderma lucidum was assessed by single cell gel electrophoresis (comet assay). Human lymphocytes were exposed to 0, 1, 2 and 4 Gy gamma radiation in the presence and absence of BG. The comet parameters were reduced by BG. The results indicate that the BG of G. lucidum possessed significant radioprotective activity with DNA repairing ability and antioxidant activity as the suggestive mechanism. The findings suggest the potential use of this mushroom for the prevention of radiation induced cellular damages.

DNA-based watermarks using the DNA-Crypt algorithm.

PubMed

Heider, Dominik; Barnekow, Angelika

2007-05-29

The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms.

DNA-based watermarks using the DNA-Crypt algorithm

PubMed Central

Heider, Dominik; Barnekow, Angelika

2007-01-01

Background The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. Results The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. Conclusion The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms. PMID:17535434

Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhatt, Shweta; Gupta, Manoj K.; Khamaisi, Mogher

The mechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D (disease duration ≥ 50 years) with severe (Medalist +C) or absent to mild complications (Medalist -C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist -C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated inmore » Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage. Lastly, we propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.« less

Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes

DOE PAGES

Bhatt, Shweta; Gupta, Manoj K.; Khamaisi, Mogher; ...

2015-08-04

The mechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D (disease duration ≥ 50 years) with severe (Medalist +C) or absent to mild complications (Medalist -C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist -C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated inmore » Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage. Lastly, we propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.« less

Mitochondrial-targeted DNA repair enzyme 8-oxoguanine DNA glycosylase 1 protects against ventilator-induced lung injury in intact mice.

PubMed

Hashizume, Masahiro; Mouner, Marc; Chouteau, Joshua M; Gorodnya, Olena M; Ruchko, Mykhaylo V; Potter, Barry J; Wilson, Glenn L; Gillespie, Mark N; Parker, James C

2013-02-15

This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage triggered ventilator-induced lung injury (VILI). Control mice and mice infused with a fusion protein targeting the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1) to mitochondria were mechanically ventilated with a range of peak inflation pressures (PIP) for specified durations. In minimal VILI (1 h at 40 cmH(2)O PIP), lung total extravascular albumin space increased 2.8-fold even though neither lung wet/dry (W/D) weight ratios nor bronchoalveolar lavage (BAL) macrophage inflammatory protein (MIP)-2 or IL-6 failed to differ from nonventilated or low PIP controls. This increase in albumin space was attenuated by OGG1. Moderately severe VILI (2 h at 40 cmH(2)O PIP) produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio and marked increases in BAL MIP-2 and IL-6, accompanied by oxidative mitochondrial DNA damage, as well as decreases in the total tissue glutathione (GSH) and GSH/GSSH ratio compared with nonventilated lungs. All of these injury indices were attenuated in OGG1-treated mice. At the highest level of VILI (2 h at 50 cmH(2)O PIP), OGG1 failed to protect against massive lung edema and BAL cytokines or against depletion of the tissue GSH pool. Interestingly, whereas untreated mice died before completing the 2-h protocol, OGG1-treated mice lived for the duration of observation. Thus mitochondrially targeted OGG1 prevented VILI over a range of ventilation times and pressures and enhanced survival in the most severely injured group. These findings support the concept that oxidative mtDNA damage caused by high PIP triggers induction of acute lung inflammation and injury.

Evaluation of protective effect of multiantigenic DNA vaccine encoding MIC3 and ROP18 antigen segments of Toxoplasma gondii in mice.

PubMed

Qu, Daofeng; Han, Jianzhong; Du, Aifang

2013-07-01

The high incidence and severe damage caused by Toxoplasma gondii infection clearly indicates the need for the development of a vaccine. In this study, we evaluated the immune responses and protection against toxoplasmosis by immunizing ICR mice with a multiantigenic DNA vaccine. To develop the multiantigenic vaccine, two T. gondii antigens, MIC3 and ROP18, selected on the basis of previous studies were chosen. ICR mice were immunized subcutaneously with PBS, empty pcDNA3.1 vector, pMIC3, pROP18, and pROP18-MIC3, respectively. The results of lymphocyte proliferation assay, cytokine, and antibody determinations showed that mice immunized with pROP18-MIC3 elicited stronger humoral and Th1-type cellular immune responses than those immunized with single-gene plasmids, empty plasmid, or phosphate-buffered saline. After a lethal challenge with the highly virulent T. gondii RH strain, a prolonged survival time in pROP18-MIC3-immunized mice was observed in comparison to control groups. Our study indicates that the introduction of multiantigenic DNA vaccine is more powerful and efficient than single-gene vaccine, and deserves further evaluation and development.

Gingerol Synergizes the Cytotoxic Effects of Doxorubicin against Liver Cancer Cells and Protects from Its Vascular Toxicity.

PubMed

Al-Abbasi, Fahad A; Alghamdi, Eman A; Baghdadi, Mohammed A; Alamoudi, Abdulmohsin J; El-Halawany, Ali M; El-Bassossy, Hany M; Aseeri, Ali H; Al-Abd, Ahmed M

2016-07-08

Hydroxyphenylalkanes and diarylheptanoids possess potential therapeutic value in different pathophysiological conditions, such as malignancy. In the current study, naturally isolated hydroxyphenylalkane and diarylheptanoid compounds were investigated for potential chemo-modulatory effects in addition to potential vascular protective roles with doxorubicin. Diarylheptanoids showed stronger antioxidant effects, in comparison to hydroxyphenylalkanes, as demonstrated by DPPH assay and amelioration of CCl₄-induced disturbed intracellular GSH/GSSG balance. Shogaol and 4'-methoxygingerol showed considerable cytotoxic effects against HCT116, HeLa, HepG2 and MCF7 cells, with IC50 values ranging from 3.1 to 19.4 µM. Gingerol significantly enhanced the cytotoxic profile of doxorubicin against HepG₂ and Huh7, cells decreasing its IC50s by 10- and 4-fold, respectively. Cell cycle distribution was studied using DNA cytometry. Doxorubicin alone induced cell accumulation at S-phase and G₂/M-phase, while in combination with gingerol it significantly induced cell cycle arrest at the G₂/M-phase. Additionally, the vascular protective effect of gingerol against doxorubicin (10 µM) was examined on isolated aortic rings. Co-incubation with 6-gingerol (30 µM) completely blocked the exaggerated vasoconstriction and impaired vascular relaxation induced by doxorubicin. In conclusion, despite its relatively weak antioxidant properties, gingerol protected from DOX-induced vascular damage, apparently not through a ROS scavenging mechanism. Besides, gingerol synergized the cytotoxic effects of DOX against liver cancer cells without influencing the cellular pharmacokinetics.

Slowing DNA Translocation in a Nanofluidic Field-Effect Transistor.

PubMed

Liu, Yifan; Yobas, Levent

2016-04-26

Here, we present an experimental demonstration of slowing DNA translocation across a nanochannel by modulating the channel surface charge through an externally applied gate bias. The experiments were performed on a nanofluidic field-effect transistor, which is a monolithic integrated platform featuring a 50 nm-diameter in-plane alumina nanocapillary whose entire length is surrounded by a gate electrode. The field-effect transistor behavior was validated on the gating of ionic conductance and protein transport. The gating of DNA translocation was subsequently studied by measuring discrete current dips associated with single λ-DNA translocation events under a source-to-drain bias of 1 V. The translocation speeds under various gate bias conditions were extracted by fitting event histograms of the measured translocation time to the first passage time distributions obtained from a simple 1D biased diffusion model. A positive gate bias was observed to slow the translocation of single λ-DNA chains markedly; the translocation speed was reduced by an order of magnitude from 18.4 mm/s obtained under a floating gate down to 1.33 mm/s under a positive gate bias of 9 V. Therefore, a dynamic and flexible regulation of the DNA translocation speed, which is vital for single-molecule sequencing, can be achieved on this device by simply tuning the gate bias. The device is realized in a conventional semiconductor microfabrication process without the requirement of advanced lithography, and can be potentially further developed into a compact electronic single-molecule sequencer.

Comparative analysis of the relative potential of silver, Zinc-oxide and titanium-dioxide nanoparticles against UVB-induced DNA damage for the prevention of skin carcinogenesis.

PubMed

Tyagi, Nikhil; Srivastava, Sanjeev K; Arora, Sumit; Omar, Yousef; Ijaz, Zohaib Mohammad; Al-Ghadhban, Ahmed; Deshmukh, Sachin K; Carter, James E; Singh, Ajay P; Singh, Seema

2016-12-01

Sunscreen formulations containing UVB filters, such as Zinc-oxide (ZnO) and titanium-dioxide (TiO 2 ) nanoparticles (NPs) have been developed to limit the exposure of human skin to UV-radiations. Unfortunately, these UVB protective agents have failed in controlling the skin cancer incidence. We recently demonstrated that silver nanoparticles (Ag-NPs) could serve as novel protective agents against UVB-radiations. Here our goal was to perform comparative analysis of direct and indirect UVB-protection efficacy of ZnO-, TiO 2 - and Ag-NPs. Sun-protection-factor calculated based on their UVB-reflective/absorption abilities was the highest for TiO 2 -NPs followed by Ag- and ZnO-NPs. This was further confirmed by studying indirect protection of UVB radiation-induced death of HaCaT cells. However, only Ag-NPs were active in protecting HaCaT cells against direct UVB-induced DNA-damage by repairing bulky-DNA lesions through nucleotide-excision-repair mechanism. Moreover, Ag-NPs were also effective in protecting HaCaT cells from UVB-induced oxidative DNA damage by enhancing SOD/CAT/GPx activity. In contrast, ZnO- and TiO 2 -NPs not only failed in providing any direct protection from DNA-damage, but rather enhanced oxidative DNA-damage by increasing ROS production. Together, these findings raise concerns about safety of ZnO- and TiO 2 -NPs and establish superior protective efficacy of Ag-NPs. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Comparative analysis of the relative potential of silver, zinc-oxide and titanium-dioxide nanoparticles against UVB-induced DNA damage for the prevention of skin carcinogenesis

PubMed Central

Arora, Sumit; Omar, Yousef; Ijaz, Zohaib Mohammad; AL-Ghadhban, Ahmed; Deshmukh, Sachin K.; Carter, James E.; Singh, Ajay P.; Singh, Seema

2016-01-01

Sunscreen formulations containing UVB filters, such as Zinc-oxide (ZnO) and titanium-dioxide (TiO2) nanoparticles (NPs) have been developed to limit the exposure of human skin to UV-radiations. Unfortunately, these UVB protective agents have failed in controlling the skin cancer incidence. We recently demonstrated that silver nanoparticles (Ag-NPs) could serve as novel protective agents against UVB-radiations. Here our goal was to perform comparative analysis of direct and indirect UVB-protection efficacy of ZnO-, TiO2- and Ag-NPs. Sun-protection-factor calculated based on their UVB-reflective/absorption abilities was the highest for TiO2-NPs followed by Ag- and ZnO-NPs. This was further confirmed by studying indirect protection of UVB radiation-induced death of HaCaT cells. However, only Ag-NPs were active in protecting HaCaT cells against direct UVB-induced DNA-damage by repairing bulky-DNA lesions through nucleotide-excision-repair mechanism. Moreover, Ag-NPs were also effective in protecting HaCaT cells from UVB-induced oxidative DNA damage by enhancing SOD/CAT/GPx activity. In contrast, ZnO- and TiO2-NPs not only failed in providing any direct protection from DNA-damage, but rather enhanced oxidative DNA-damage by increasing ROS production. Together, these findings raise concerns about safety of ZnO- and TiO2-NPs and establish superior protective efficacy of Ag-NPs. PMID:27693632

Effects of DNA Methylation and Chromatin State on Rates of Molecular Evolution in Insects.

PubMed

Glastad, Karl M; Goodisman, Michael A D; Yi, Soojin V; Hunt, Brendan G

2015-12-04

Epigenetic information is widely appreciated for its role in gene regulation in eukaryotic organisms. However, epigenetic information can also influence genome evolution. Here, we investigate the effects of epigenetic information on gene sequence evolution in two disparate insects: the fly Drosophila melanogaster, which lacks substantial DNA methylation, and the ant Camponotus floridanus, which possesses a functional DNA methylation system. We found that DNA methylation was positively correlated with the synonymous substitution rate in C. floridanus, suggesting a key effect of DNA methylation on patterns of gene evolution. However, our data suggest the link between DNA methylation and elevated rates of synonymous substitution was explained, in large part, by the targeting of DNA methylation to genes with signatures of transcriptionally active chromatin, rather than the mutational effect of DNA methylation itself. This phenomenon may be explained by an elevated mutation rate for genes residing in transcriptionally active chromatin, or by increased structural constraints on genes in inactive chromatin. This result highlights the importance of chromatin structure as the primary epigenetic driver of genome evolution in insects. Overall, our study demonstrates how different epigenetic systems contribute to variation in the rates of coding sequence evolution. Copyright © 2016 Glastad et al.

Immune response at birth, long-term immune memory and 2 years follow-up after in-utero anti-HBV DNA immunization.

PubMed

Fazio, V M; Ria, F; Franco, E; Rosati, P; Cannelli, G; Signori, E; Parrella, P; Zaratti, L; Iannace, E; Monego, G; Blogna, S; Fioretti, D; Iurescia, S; Filippetti, R; Rinaldi, M

2004-03-01

Infections occurring at the end of pregnancy, during birth or by breastfeeding are responsible for the high toll of death among first-week infants. In-utero DNA immunization has demonstrated the effectiveness in inducing specific immunity in newborns. A major contribution to infant immunization would be achieved if a vaccine proved able to be protective as early as at the birth, preventing the typical 'first-week infections'. To establish its potential for use in humans, in-utero DNA vaccination efficiency has to be evaluated for short- and long-term safety, protection at delivery, efficacy of boosts in adults and effective window/s for modulation of immune response during pregnancy, in an animal model suitable with human development. Here we show that a single intramuscular in-utero anti-HBV DNA immunization at two-thirds of pig gestation produces, at birth, antibody titers considered protective in humans. The boost of antibody titers in every animal following recall at 4 and 10 months demonstrates the establishment of immune memory. The safety of in-utero fetus manipulation is guaranteed by short-term (no fetus loss, lack of local alterations, at-term spontaneous delivery, breastfeeding) and long-term (2 years) monitoring. Treatment of fetuses closer to delivery results in immune ignorance without induction of tolerance. This result highlights the repercussion of selecting the appropriate time point when this approach is used to deliver therapeutic genes. All these findings illustrate the relevance of naked DNA-based vaccination technology in therapeutic efforts aimed to prevent the high toll of death among first-week infants.

Organic honey supplementation reverses pesticide-induced genotoxicity by modulating DNA damage response.

PubMed

Alleva, Renata; Manzella, Nicola; Gaetani, Simona; Ciarapica, Veronica; Bracci, Massimo; Caboni, Maria Fiorenza; Pasini, Federica; Monaco, Federica; Amati, Monica; Borghi, Battista; Tomasetti, Marco

2016-10-01

Glyphosate (GLY) and organophosphorus insecticides such as chlorpyrifos (CPF) may cause DNA damage and cancer in exposed individuals through mitochondrial dysfunction. Polyphenols ubiquitously present in fruits and vegetables, have been viewed as antioxidant molecules, but also influence mitochondrial homeostasis. Here, honey containing polyphenol compounds was evaluated for its potential protective effect on pesticide-induced genotoxicity. Honey extracts from four floral organic sources were evaluated for their polyphenol content, antioxidant activity, and potential protective effects on pesticide-related mitochondrial destabilization, reactive oxygen and nitrogen species formation, and DNA damage response in human bronchial epithelial and neuronal cells. The protective effect of honey was, then evaluated in a residential population chronically exposed to pesticides. The four honey types showed a different polyphenol profile associated with a different antioxidant power. The pesticide-induced mitochondrial dysfunction parallels ROS formation from mitochondria (mtROS) and consequent DNA damage. Honey extracts efficiently inhibited pesticide-induced mtROS formation, and reduced DNA damage by upregulation of DNA repair through NFR2. Honey supplementation enhanced DNA repair activity in a residential population chronically exposed to pesticides, which resulted in a marked reduction of pesticide-induced DNA lesions. These results provide new insight regarding the effect of honey containing polyphenols on pesticide-induced DNA damage response. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Experimental and theoretical studies on radiation protective effect of a lighter non-lead protective apron].

PubMed

Takano, Yoshihisa; Okazaki, Keiichiro; Ono, Koji; Kai, Michiaki

2005-07-20

Non-lead aprons using composite materials are often used for radiation protective aprons instead of heavy lead aprons. However, the protective effect of the lighter, non-lead aprons has not been well evaluated, and it is not yet clear how they compare with lead aprons. Therefore, we investigated the protective performance of non-lead aprons theoretically and experimentally by comparing them with lead aprons under clinical conditions. We measured the energy spectra for direct and scattered X-rays passing through protective aprons or not, and measured doses with glass dosimeters for validation of theoretical calculations based on the energy spectra. We found that the protective effect of non-lead aprons was higher than that of lead aprons at X-ray of tube voltages of 70-100 kV, which are often used for radiography and fluorography. This demonstrated that the non-lead aprons are more useful in many situations than heavy lead aprons.

Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidence in rodent bioassays.

PubMed

Paini, Alicia; Scholz, Gabriele; Marin-Kuan, Maricel; Schilter, Benoît; O'Brien, John; van Bladeren, Peter J; Rietjens, Ivonne M C M

2011-09-01

This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate whether a correlation can be obtained, using a benchmark dose (BMD) approach. Dose-response data on both carcinogenicity and in vivo DNA adduct formation were available for six compounds, i.e. 2-acetylaminofluorene, aflatoxin B1, methyleugenol, safrole, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and tamoxifen. BMD(10) values for liver carcinogenicity were calculated using the US Environmental Protection Agency BMD software. DNA adduct levels at this dose were extrapolated assuming linearity of the DNA adduct dose response. In addition, the levels of DNA adducts at the BMD(10) were compared to available data on endogenous background DNA damage in the target organ. Although for an individual carcinogen the tumour response increases when adduct levels increase, our results demonstrate that when comparing different carcinogens, no quantitative correlation exists between the level of DNA adduct formation and carcinogenicity. These data confirm that the quantity of DNA adducts formed by a DNA-reactive compound is not a carcinogenicity predictor but that other factors such as type of adduct and mutagenic potential may be equally relevant. Moreover, comparison to background DNA damage supports the notion that the mere occurrence of DNA adducts above or below the level of endogenous DNA damage is neither correlated to development of cancer. These data strongly emphasise the need to apply the mode of action framework to understand the contribution of other biological effect markers playing a role in carcinogenicity.

High Glutathione and Glutathione Peroxidase-2 Levels Mediate Cell-Type-Specific DNA Damage Protection in Human Induced Pluripotent Stem Cells

PubMed Central

Dannenmann, Benjamin; Lehle, Simon; Hildebrand, Dominic G.; Kübler, Ayline; Grondona, Paula; Schmid, Vera; Holzer, Katharina; Fröschl, Mirjam; Essmann, Frank; Rothfuss, Oliver; Schulze-Osthoff, Klaus

2015-01-01

Summary Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs), we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic insults. Intriguingly, despite the elevated apoptosis sensitivity, both mitochondrial and nuclear DNA lesions induced by genotoxins were less frequent in iPSCs compared to fibroblasts. Gene profiling identified that mRNA expression of several antioxidant proteins was considerably upregulated in iPSCs. Knockdown of glutathione peroxidase-2 and depletion of glutathione impaired protection against DNA lesions. Thus, iPSCs ensure genomic integrity through enhanced apoptosis induction and increased antioxidant defense, contributing to protection against DNA damage. PMID:25937369

Protective Effect of Unacylated Ghrelin on Compression-Induced Skeletal Muscle Injury Mediated by SIRT1-Signaling

PubMed Central

Ugwu, Felix N.; Yu, Angus P.; Sin, Thomas K.; Tam, Bjorn T.; Lai, Christopher W.; Wong, S. C.; Siu, Parco M.

2017-01-01

Unacylated ghrelin, the predominant form of circulating ghrelin, protects myotubes from cell death, which is a known attribute of pressure ulcers. In this study, we investigated whether unacylated ghrelin protects skeletal muscle from pressure-induced deep tissue injury by abolishing necroptosis and apoptosis signaling and whether these effects were mediated by SIRT1 pathway. Fifteen adult Sprague Dawley rats were assigned to receive saline or unacylated ghrelin with or without EX527 (a SIRT1 inhibitor). Animals underwent two 6-h compression cycles with 100 mmHg static pressure applied over the mid-tibialis region of the right limb whereas the left uncompressed limb served as the intra-animal control. Muscle tissues underneath the compression region, and at the similar region of the opposite uncompressed limb, were collected for analysis. Unacylated ghrelin attenuated the compression-induced muscle pathohistological alterations including rounding contour of myofibers, extensive nucleus accumulation in the interstitial space, and increased interstitial space. Unacylated ghrelin abolished the increase in necroptosis proteins including RIP1 and RIP3 and attenuated the elevation of apoptotic proteins including p53, Bax, and AIF in the compressed muscle. Furthermore, unacylated ghrelin opposed the compression-induced phosphorylation and acetylation of p65 subunit of NF-kB. The anti-apoptotic effect of unacylated ghrelin was shown by a decrease in apoptotic DNA fragmentation and terminal dUTP nick-end labeling index in the compressed muscle. The protective effects of unacylated ghrelin vanished when co-treated with EX527. Our findings demonstrated that unacylated ghrelin protected skeletal muscle from compression-induced injury. The myoprotective effects of unacylated ghrelin on pressure-induced tissue injury were associated with SIRT1 signaling. PMID:29225581

Potential Protective Effects of Ursolic Acid against Gamma Irradiation-Induced Damage Are Mediated through the Modulation of Diverse Inflammatory Mediators

PubMed Central

Wang, Hong; Sim, Meng-Kwoon; Loke, Weng Keong; Chinnathambi, Arunachalam; Alharbi, Sulaiman Ali; Tang, Feng Ru; Sethi, Gautam

2017-01-01

This study was aimed to evaluate the possible protective effects of ursolic acid (UA) against gamma radiation induced damage both in vitro as well as in vivo. It was observed that the exposure to gamma radiation dose- and time-dependently caused a significant decrease in the cell viability, while the treatment of UA attenuated this cytotoxicity. The production of free radicals including reactive oxygen species (ROS) and NO increased significantly post-irradiation and further induced lipid peroxidation and oxidative DNA damage in cells. These deleterious effects could also be effectively blocked by UA treatment. In addition, UA also reversed gamma irradiation induced inflammatory responses, as indicated by the decreased production of TNF-α, IL-6, and IL-1β. NF-κB signaling pathway has been reported to be a key mediator involved in gamma radiation-induced cellular damage. Our results further demonstrated that gamma radiation dose- and time-dependently enhanced NF-κB DNA binding activity, which was significantly attenuated upon UA treatment. The post-irradiation increase in the expression of both phospho-p65, and phospho-IκBα was also blocked by UA. Moreover, the treatment of UA was found to significantly prolong overall survival in mice exposed to whole body gamma irradiation, and reduce the excessive inflammatory responses. Given its radioprotective efficacy as described here, UA as an antioxidant and NF-κB pathway blocker, may function as an important pharmacological agent in protecting against gamma irradiation-induced injury. PMID:28670276

Early DNA vaccination of puppies against canine distemper in the presence of maternally derived immunity.

PubMed

Griot, Christian; Moser, Christian; Cherpillod, Pascal; Bruckner, Lukas; Wittek, Riccardo; Zurbriggen, Andreas; Zurbriggen, Rinaldo

2004-01-26

Canine distemper (CD) is a disease in carnivores caused by CD virus (CDV), a member of the morbillivirus genus. It still is a threat to the carnivore and ferret population. The currently used modified attenuated live vaccines have several drawbacks of which lack of appropriate protection from severe infection is the most outstanding one. In addition, puppies up to the age of 6-8 weeks cannot be immunized efficiently due to the presence of maternal antibodies. In this study, a DNA prime modified live vaccine boost strategy was investigated in puppies in order to determine if vaccinated neonatal dogs induce a neutralizing immune response which is supposed to protect animals from a CDV challenge. Furthermore, a single DNA vaccination of puppies, 14 days after birth and in the presence of high titers of CDV neutralizing maternal antibodies, induced a clear and significant priming effect observed as early as 3 days after the subsequent booster with a conventional CDV vaccine. It was shown that the priming effect develops faster and to higher titers in puppies preimmunized with DNA 14 days after birth than in those vaccinated 28 days after birth. Our results demonstrate that despite the presence of maternal antibodies puppies can be vaccinated using the CDV DNA vaccine, and that this vaccination has a clear priming effect leading to a solid immune response after a booster with a conventional CDV vaccine.

Low-dose energetic protons induce adaptive and bystander effects that protect human cells against DNA damage caused by a subsequent exposure to energetic iron ions.

PubMed

Buonanno, Manuela; De Toledo, Sonia M; Howell, Roger W; Azzam, Edouard I

2015-05-01

During interplanetary missions, astronauts are exposed to mixed types of ionizing radiation. The low 'flux' of the high atomic number and high energy (HZE) radiations relative to the higher 'flux' of low linear energy transfer (LET) protons makes it highly probable that for any given cell in the body, proton events will precede any HZE event. Whereas progress has been made in our understanding of the biological effects of low-LET protons and high-LET HZE particles, the interplay between the biochemical processes modulated by these radiations is unclear. Here we show that exposure of normal human fibroblasts to a low mean absorbed dose of 20 cGy of 0.05 or 1-GeV protons (LET ∼ 1.25 or 0.2 keV/μm, respectively) protects the irradiated cells (P < 0.0001) against chromosomal damage induced by a subsequent exposure to a mean absorbed dose of 50 cGy from 1 GeV/u iron ions (LET ∼ 151 keV/μm). Surprisingly, unirradiated (i.e. bystander) cells with which the proton-irradiated cells were co-cultured were also significantly protected from the DNA-damaging effects of the challenge dose. The mitigating effect persisted for at least 24 h. These results highlight the interactions of biological effects due to direct cellular traversal by radiation with those due to bystander effects in cell populations exposed to mixed radiation fields. They show that protective adaptive responses can spread from cells targeted by low-LET space radiation to bystander cells in their vicinity. The findings are relevant to understanding the health hazards of space travel. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Investigating CSI: portrayals of DNA testing on a forensic crime show and their potential effects.

PubMed

Ley, Barbara L; Jankowski, Natalie; Brewer, Paul R

2012-01-01

The popularity of forensic crime shows such as CSI has fueled debate about their potential social impact. This study considers CSI's potential effects on public understandings regarding DNA testing in the context of judicial processes, the policy debates surrounding crime laboratory procedures, and the forensic science profession, as well as an effect not discussed in previous accounts: namely, the show's potential impact on public understandings of DNA and genetics more generally. To develop a theoretical foundation for research on the "CSI effect," it draws on cultivation theory, social cognitive theory, and audience reception studies. It then uses content analysis and textual analysis to illuminate how the show depicts DNA testing. The results demonstrate that CSI tends to depict DNA testing as routine, swift, useful, and reliable and that it echoes broader discourses about genetics. At times, however, the show suggests more complex ways of thinking about DNA testing and genetics.

Dual Targeting Biomimetic Liposomes for Paclitaxel/DNA Combination Cancer Treatment

PubMed Central

Liu, Guo-Xia; Fang, Gui-Qing; Xu, Wei

2014-01-01

Combinations of chemotherapeutic drugs with nucleic acid has shown great promise in cancer therapy. In the present study, paclitaxel (PTX) and DNA were co-loaded in the hyaluronic acid (HA) and folate (FA)-modified liposomes (HA/FA/PPD), to obtain the dual targeting biomimetic nanovector. The prepared HA/FA/PPD exhibited nanosized structure and narrow size distributions (247.4 ± 4.2 nm) with appropriate negative charge of −25.40 ± 2.7 mV. HA/FA/PD (PTX free HA/FA/PPD) showed almost no toxicity on murine malignant melanoma cell line (B16) and human hepatocellular carcinoma cell line (HepG2) (higher than 80% cell viability), demonstrating the safety of the blank nanovector. In comparison with the FA-modified PTX/DNA co-loaded liposomes (FA/PPD), HA/FA/PPD showed significant superiority in protecting the nanoparticles from aggregation in the presence of plasma and degradation by DNase I. Moreover, HA/FA/PPD could also significantly improve the transfection efficiency and cellular internalization rates on B16 cells comparing to that of FA/PPD (p < 0.05) and PPD (p < 0.01), demonstrating the great advantages of dual targeting properties. Furthermore, fluorescence microscope and flow cytometry results showed that PTX and DNA could be effectively co-delivered into the same tumor cell via HA/FA/PPD, contributing to PTX/DNA combination cancer treatment. In conclusion, the obtained HA/FA/PPD in the study could effectively target tumor cells, enhance transfection efficiency and subsequently achieve the co-delivery of PTX and DNA, displaying great potential for optimal combination therapy. PMID:25177862

CD40L-adjuvanted DNA/modified vaccinia virus Ankara simian immunodeficiency virus SIV239 vaccine enhances SIV-specific humoral and cellular immunity and improves protection against a heterologous SIVE660 mucosal challenge.

PubMed

Kwa, Suefen; Lai, Lilin; Gangadhara, Sailaja; Siddiqui, Mariam; Pillai, Vinod B; Labranche, Celia; Yu, Tianwei; Moss, Bernard; Montefiori, David C; Robinson, Harriet L; Kozlowski, Pamela A; Amara, Rama Rao

2014-09-01

It remains a challenge to develop a successful human immunodeficiency virus (HIV) vaccine that is capable of preventing infection. Here, we utilized the benefits of CD40L, a costimulatory molecule that can stimulate both dendritic cells (DCs) and B cells, as an adjuvant for our simian immunodeficiency virus (SIV) DNA vaccine in rhesus macaques. We coexpressed the CD40L with our DNA/SIV vaccine such that the CD40L is anchored on the membrane of SIV virus-like particle (VLP). These CD40L containing SIV VLPs showed enhanced activation of DCs in vitro. We then tested the potential of DNA/SIV-CD40L vaccine to adjuvant the DNA prime of a DNA/modified vaccinia virus Ankara (MVA) vaccine in rhesus macaques. Our results demonstrated that the CD40L adjuvant enhanced the functional quality of anti-Env antibody response and breadth of anti-SIV CD8 and CD4 T cell responses, significantly delayed the acquisition of heterologous mucosal SIV infection, and improved viral control. Notably, the CD40L adjuvant enhanced the control of viral replication in the gut at the site of challenge that was associated with lower mucosal CD8 immune activation, one of the strong predictors of disease progression. Collectively, our results highlight the benefits of CD40L adjuvant for enhancing antiviral humoral and cellular immunity, leading to enhanced protection against a pathogenic SIV. A single adjuvant that enhances both humoral and cellular immunity is rare and thus underlines the importance and practicality of CD40L as an adjuvant for vaccines against infectious diseases, including HIV-1. Despite many advances in the field of AIDS research, an effective AIDS vaccine that can prevent infection remains elusive. CD40L is a key stimulator of dendritic cells and B cells and can therefore enhance T cell and antibody responses, but its overly potent nature can lead to adverse effects unless used in small doses. In order to modulate local expression of CD40L at relatively lower levels, we expressed

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.

Protective effect of Pterostilbene against free radical mediated oxidative damage

PubMed Central

2013-01-01

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

Protective effects of transduced Tat-DJ-1 protein against oxidative stress and ischemic brain injury.

PubMed

Jeong, Hoon Jae; Kim, Dae Won; Kim, Mi Jin; Woo, Su Jung; Kim, Hye Ri; Kim, So Mi; Jo, Hyo Sang; Hwang, Hyun Sook; Kim, Duk Soo; Cho, Sung Woo; Won, Moo Ho; Han, Kyu Hyung; Park, Jin Seu; Eum, Won Sik; Choi, Soo Young

2012-10-31

Reactive oxygen species (ROS) contribute to the development of a number of neuronal diseases including ischemia. DJ-1, also known to PARK7, plays an important role in transcriptional regulation, acting as molecular chaperone and antioxidant. In the present study, we investigated whether DJ-1 protein shows a protective effect against oxidative stress-induced neuronal cell death in vitro and in ischemic animal models in vivo. To explore DJ-1 protein's potential role in protecting against ischemic cell death, we constructed cell permeable Tat-DJ-1 fusion proteins. Tat-DJ-1 protein efficiently transduced into neuronal cells in a doseand time-dependent manner. Transduced Tat-DJ-1 protein increased cell survival against hydrogen peroxide (H2O2) toxicity and also reduced intracellular ROS. In addition, Tat-DJ-1 protein inhibited DNA fragmentation induced by H2O2. Furthermore, in animal models, immunohistochemical analysis revealed that Tat-DJ-1 protein prevented neuronal cell death induced by transient forebrain ischemia in the CA1 region of the hippocampus. These results demonstrate that transduced Tat-DJ-1 protein protects against cell death in vitro and in vivo, suggesting that the transduction of Tat-DJ-1 may be useful as a therapeutic agent for ischemic injuries related to oxidative stress.

Effect of Drying Methods on Protein and DNA Conformation Changes in Lactobacillus rhamnosus GG Cells by Fourier Transform Infrared Spectroscopy.

PubMed

Hlaing, Mya M; Wood, Bayden R; McNaughton, Don; Ying, DanYang; Dumsday, Geoff; Augustin, Mary Ann

2017-03-01

Microencapsulation protects cells against environmental stress encountered during the production of probiotics, which are used as live microbial food ingredients. Freeze-drying and spray-drying are used in the preparation of powdered microencapsulated probiotics. This study examines the ability of Fourier transform infrared (FTIR) spectroscopy to detect differences in cells exposed to freeze-drying and spray-drying of encapsulated Lactobacillus rhamnosus GG cells. The FTIR analysis clearly demonstrated there were more significant molecular changes in lipid, fatty acid content, protein, and DNA conformation of nonencapsulated compared to encapsulated bacterial cells. The technique was also able to differentiate between spray-dried and freeze-dried cells. The results also revealed the extent of protection from a protein-carbohydrate-based encapsulant matrix on the cells depending on the type drying process. The extent of this protection to the dehydration stress was shown to be less in spray-dried cells than in freeze-dried cells. This suggests that FTIR could be used as a rapid, noninvasive, and real-time measurement technique to detect detrimental drying effects on cells.

Genotoxicity and Cytotoxicity Evaluation of the Neolignan Analogue 2-(4-Nitrophenoxy)-1Phenylethanone and its Protective Effect Against DNA Damage

PubMed Central

Hanusch, Alex Lucas; de Oliveira, Guilherme Roberto; de Sabóia-Morais, Simone Maria Teixeira; Machado, Rafael Cosme; dos Anjos, Murilo Machado; Chen Chen, Lee

2015-01-01

Neolignans are secondary metabolites found in various groups of Angiosperms. They belong to a class of natural compounds with great diversity of chemical structures and pharmacological activities. These compounds are formed by linking two phenylpropanoid units. Several compounds that have ability to prevent genetic damage have been isolated from plants, and can be used to prevent or delay the development of tumor cells. Genetic toxicology evaluation is widely used in risk assessment of new drugs in preclinical screening tests. In this study, we evaluated the genotoxicity and cytotoxicity of the neolignan analogue 2-(4-nitrophenoxy)-1-phenylethanone (4NF) and its protective effect against DNA damage using the mouse bone marrow micronucleus test and the comet assay in mouse peripheral blood. Our results showed that this neolignan analogue had no genotoxic activity and was able to reduce induced damage both in mouse bone marrow and peripheral blood. Although the neolignan analogue 4NF was cytotoxic, it reduced cyclophosphamide-induced cytotoxicity. In conclusion, it showed no genotoxic action, but exhibited cytotoxic, antigenotoxic, and anticytotoxic activities. PMID:26554835

Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin

PubMed Central

Bandaria, Jigar N.; Qin, Peiwu; Berk, Veysel; Chu, Steven; Yildiz, Ahmet

2016-01-01

SUMMARY Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the DNA damage response (DDR) by the shelterin complex. To understand how shelterin protects telomere ends, we investigated the structural organization of telomeric chromatin in human cells using super-resolution microscopy. We found that telomeres form compact globular structures through a complex network of interactions between shelterin subunits and telomeric DNA, and not by DNA methylation, histone deacetylation or histone trimethylation at telomeres and subtelomeric regions. Mutations that abrogate shelterin assembly or removal of individual subunits from telomeres cause up to a 10-fold increase in telomere volume. Decompacted telomeres become more accessible to telomere-associated proteins and accumulate DDR signals. Recompaction of telomeric chromatin using an orthogonal method displaces DDR signals from telomeres. These results reveal the chromatin remodeling activity of shelterin and demonstrate that shelterin-mediated compaction of telomeric chromatin provides robust protection of chromosome ends against the DDR machinery. PMID:26871633

Turning self-destructing Salmonella into a universal DNA vaccine delivery platform.

PubMed

Kong, Wei; Brovold, Matthew; Koeneman, Brian A; Clark-Curtiss, Josephine; Curtiss, Roy

2012-11-20

We previously developed a biological containment system using recombinant Salmonella Typhimurium strains that are attenuated yet capable of synthesizing protective antigens. The regulated delayed attenuation and programmed self-destructing features designed into these S. Typhimurium strains enable them to efficiently colonize host tissues and allow release of the bacterial cell contents after lysis. To turn such a recombinant attenuated Salmonella vaccine (RASV) strain into a universal DNA vaccine-delivery vehicle, our approach was to genetically modify RASV strains to display a hyperinvasive phenotype to maximize Salmonella host entry and host cell internalization, to enable Salmonella endosomal escape to release a DNA vaccine into the cytosol, and to decrease Salmonella-induced pyroptosis/apoptosis that allows the DNA vaccine time to traffic to the nucleus for efficient synthesis of encoded protective antigens. A DNA vaccine vector that encodes a domain that contributes to the arabinose-regulated lysis phenotype but has a eukaryotic promoter was constructed. The vector was then improved by insertion of multiple DNA nuclear-targeting sequences for efficient nuclear trafficking and gene expression, and by increasing nuclease resistance to protect the plasmid from host degradation. A DNA vaccine encoding influenza WSN virus HA antigen delivered by the RASV strain with the best genetic attributes induced complete protection to mice against a lethal influenza virus challenge. Adoption of these technological improvements will revolutionize means for effective delivery of DNA vaccines to stimulate mucosal, systemic, and cellular protective immunities, and lead to a paradigm shift in cost-effective control and prevention of a diversity of diseases.

Turning self-destructing Salmonella into a universal DNA vaccine delivery platform

PubMed Central

Kong, Wei; Brovold, Matthew; Koeneman, Brian A.; Clark-Curtiss, Josephine; Curtiss, Roy

2012-01-01

We previously developed a biological containment system using recombinant Salmonella Typhimurium strains that are attenuated yet capable of synthesizing protective antigens. The regulated delayed attenuation and programmed self-destructing features designed into these S. Typhimurium strains enable them to efficiently colonize host tissues and allow release of the bacterial cell contents after lysis. To turn such a recombinant attenuated Salmonella vaccine (RASV) strain into a universal DNA vaccine-delivery vehicle, our approach was to genetically modify RASV strains to display a hyperinvasive phenotype to maximize Salmonella host entry and host cell internalization, to enable Salmonella endosomal escape to release a DNA vaccine into the cytosol, and to decrease Salmonella-induced pyroptosis/apoptosis that allows the DNA vaccine time to traffic to the nucleus for efficient synthesis of encoded protective antigens. A DNA vaccine vector that encodes a domain that contributes to the arabinose-regulated lysis phenotype but has a eukaryotic promoter was constructed. The vector was then improved by insertion of multiple DNA nuclear-targeting sequences for efficient nuclear trafficking and gene expression, and by increasing nuclease resistance to protect the plasmid from host degradation. A DNA vaccine encoding influenza WSN virus HA antigen delivered by the RASV strain with the best genetic attributes induced complete protection to mice against a lethal influenza virus challenge. Adoption of these technological improvements will revolutionize means for effective delivery of DNA vaccines to stimulate mucosal, systemic, and cellular protective immunities, and lead to a paradigm shift in cost-effective control and prevention of a diversity of diseases. PMID:23129620

DNA Prime/Adenovirus Boost Malaria Vaccine Encoding P. falciparum CSP and AMA1 Induces Sterile Protection Associated with Cell-Mediated Immunity

PubMed Central

Chuang, Ilin; Sedegah, Martha; Cicatelli, Susan; Spring, Michele; Polhemus, Mark; Tamminga, Cindy; Patterson, Noelle; Guerrero, Melanie; Bennett, Jason W.; McGrath, Shannon; Ganeshan, Harini; Belmonte, Maria; Farooq, Fouzia; Abot, Esteban; Banania, Jo Glenna; Huang, Jun; Newcomer, Rhonda; Rein, Lisa; Litilit, Dianne; Richie, Nancy O.; Wood, Chloe; Murphy, Jittawadee; Sauerwein, Robert; Hermsen, Cornelus C.; McCoy, Andrea J.; Kamau, Edwin; Cummings, James; Komisar, Jack; Sutamihardja, Awalludin; Shi, Meng; Epstein, Judith E.; Maiolatesi, Santina; Tosh, Donna; Limbach, Keith; Angov, Evelina; Bergmann-Leitner, Elke; Bruder, Joseph T.; Doolan, Denise L.; King, C. Richter; Carucci, Daniel; Dutta, Sheetij; Soisson, Lorraine; Diggs, Carter; Hollingdale, Michael R.; Ockenhouse, Christian F.; Richie, Thomas L.

2013-01-01

Background Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection. Methodology/Principal Findings The vaccine regimen was three monthly doses of two DNA plasmids (DNA) followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad). The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea), possibly related to immunization, was severe (Grade 3), preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27%) were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44–817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5–102) and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13–408; AMA1 348, range 88–1270) and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019). Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant. Significance The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%). Protection was

Fungal beta glucan protects radiation induced DNA damage in human lymphocytes

PubMed Central

Maurya, Dharmendra K.; Salvi, Veena P.; Janardhanan, Krishnankutty K; Nair, Cherupally K. K.

2014-01-01

Background Ganoderma lucidum (Ling Zhi), a basidiomycete white rot macrofungus has been used extensively for therapeutic use in China, Japan, Korea and other Asian countries for 2,000 years. The present study is an attempt to investigate its DNA protecting property in human lymphocytes. Materials and methods Beta glucan (BG) was isolated by standard procedure and the structure and composition were studied by infrared radiation (IR) and nuclear magnetic resonance (NMR) spectroscopy, gel filtration chromatography and paper chromatography. The radioprotective properties of BG isolated from the macro fungi Ganoderma lucidum was assessed by single cell gel electrophoresis (comet assay). Human lymphocytes were exposed to 0, 1, 2 and 4 Gy gamma radiation in the presence and absence of BG. Results The comet parameters were reduced by BG. The results indicate that the BG of G. lucidum possessed significant radioprotective activity with DNA repairing ability and antioxidant activity as the suggestive mechanism. Conclusions The findings suggest the potential use of this mushroom for the prevention of radiation induced cellular damages. PMID:25332989

Melanin distribution in human epidermis affords localized protection against DNA photodamage and concurs with skin cancer incidence difference in extreme phototypes.

PubMed

Fajuyigbe, Damilola; Lwin, Su M; Diffey, Brian L; Baker, Richard; Tobin, Desmond J; Sarkany, Robert P E; Young, Antony R

2018-02-02

Epidermal DNA damage, especially to the basal layer, is an established cause of keratinocyte cancers (KCs). Large differences in KC incidence (20- to 60-fold) between white and black populations are largely attributable to epidermal melanin photoprotection in the latter. The cyclobutane pyrimidine dimer (CPD) is the most mutagenic DNA photolesion; however, most studies suggest that melanin photoprotection against CPD is modest and cannot explain the considerable skin color-based differences in KC incidence. Along with melanin quantity, solar-simulated radiation-induced CPD assessed immediately postexposure in the overall epidermis and within 3 epidermal zones was compared in black West Africans and fair Europeans. Melanin in black skin protected against CPD by 8.0-fold in the overall epidermis and by 59.0-, 16.5-, and 5.0-fold in the basal, middle, and upper epidermis, respectively. Protection was related to the distribution of melanin, which was most concentrated in the basal layer of black skin. These results may explain, at least in part, the considerable skin color differences in KC incidence. These data suggest that a DNA protection factor of at least 60 is necessary in sunscreens to reduce white skin KC incidence to a level that is comparable with that of black skin.-Fajuyigbe, D., Lwin, S. M., Diffey, B. L., Baker, R., Tobin, D. J., Sarkany, R. P. E., Young, A. R. Melanin distribution in human epidermis affords localized protection against DNA photodamage and concurs with skin cancer incidence difference in extreme phototypes.

Novel Antigen Identification Method for Discovery of Protective Malaria Antigens by Rapid Testing of DNA Vaccines Encoding Exons from the Parasite Genome

PubMed Central

Haddad, Diana; Bilcikova, Erika; Witney, Adam A.; Carlton, Jane M.; White, Charles E.; Blair, Peter L.; Chattopadhyay, Rana; Russell, Joshua; Abot, Esteban; Charoenvit, Yupin; Aguiar, Joao C.; Carucci, Daniel J.; Weiss, Walter R.

2004-01-01

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens. PMID:14977966

A multiagent filovirus DNA vaccine delivered by intramuscular electroporation completely protects mice from ebola and Marburg virus challenge.

PubMed

Grant-Klein, Rebecca J; Van Deusen, Nicole M; Badger, Catherine V; Hannaman, Drew; Dupuy, Lesley C; Schmaljohn, Connie S

2012-11-01

We evaluated the immunogenicity and protective efficacy of DNA vaccines expressing the codon-optimized envelope glycoprotein genes of Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus (Musoke and Ravn). Intramuscular or intradermal delivery of the vaccines in BALB/c mice was performed using the TriGrid™ electroporation device. Mice that received DNA vaccines against the individual viruses developed robust glycoprotein-specific antibody titers as determined by ELISA and survived lethal viral challenge with no display of clinical signs of infection. Survival curve analysis revealed there was a statistically significant increase in survival compared to the control groups for both the Ebola and Ravn virus challenges. These data suggest that further analysis of the immune responses generated in the mice and additional protection studies in nonhuman primates are warranted.

Unexpected effect of dry olive leaf extract on the level of DNA damage in lymphocytes of lead intoxicated workers, before and after CaNa2EDTA chelation therapy.

PubMed