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Sample records for protect cellular dna

  1. Adenovirus terminal protein protects single stranded DNA from digestion by a cellular exonuclease.

    PubMed Central

    Dunsworth-Browne, M; Schell, R E; Berk, A J

    1980-01-01

    Adenovirus 5 DNA-protein complex is isolated from virions as a duplex DNA molecule covalently attached by the 5' termini of each strand to virion protein of unknown function. The DNA-protein complex can be digested with E. coli exonuclease III to generate molecules analogous to DNA replication intermediates in that they contain long single stranded regions ending in 5' termini bound to terminal protein. The infectivity of pronase digested Adenovirus 5 DNA is greatly diminished by exonuclease III digestion. However, the infectivity of the DNA-protein complex is not significantly altered when up to at least 2400 nucleotides are removed from the 3' ends of each strand. This indicates that the terminal protein protects 5' terminated single stranded regions from digestion by a cellular exonuclease. DNA-protein complex prepared from a host range mutant with a mutation mapping in the left 4% of the genome was digested with exonuclease III, hybridized to a wild type restriction fragment comprising the left 8% of the genome, and transfected into HeLa cells. Virus with wild type phenotype was recovered at high frequency. Images PMID:6255444

  2. XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

    PubMed Central

    Horton, Julie K; Watson, Mary; Stefanick, Donna F; Shaughnessy, Daniel T; Taylor, Jack A; Wilson, Samuel H

    2008-01-01

    Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5′Œ-phosphate and 3′Œ-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1−/− mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β (pol β) is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMStreated XRCC1−/−, and to a lesser extent in pol β−/−cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and pol β −/−cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1−/− cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage. PMID:18166976

  3. Protective Effect of Carvacrol on Oxidative Stress and Cellular DNA Damage Induced by UVB Irradiation in Human Peripheral Lymphocytes.

    PubMed

    Aristatile, Balakrishnan; Al-Numair, Khalid S; Al-Assaf, Abdullah H; Veeramani, Chinnadurai; Pugalendi, Kodukkur Viswanathan

    2015-11-01

    Exposure to ultraviolet B (UVB; 280-320 nm) radiation induces the formation of reactive oxygen species (ROS) in the biological system. In this study, we examined the protective effect of carvacrol on UVB-induced lipid peroxidation and oxidative DNA damage with reference to alterations in cellular an-tioxidant status in human lymphocytes. A series of in vitro assays (hydroxyl radical, superoxide, nitric oxide, DPPH (2,2-Diphenyl-1-picryl hydrazyl), and ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assays) demonstrate antioxidant property of carvacrol in our study. UVB exposure significantly increased thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LHPs), % tail DNA and tail moment; decreased % cell viability and antioxidant status in UVB-irradiated lymphocytes. Treatment with carvacrol 30 min prior to UVB-exposure resulted in a significant decline of TBARS, LHP, % tail DNA, and tail moment and increased % cell viability as carvacrol concentration increased. UVB irradiated lymphocytes with carvacrol alone (at 10 μg/mL) gave no significant change in cell viability, TBARS, LHP, % tail DNA, and tail moment when compared with normal lymphocytes. On the basis of our results, we conclude that carvacrol, a dietary antioxidant, mediates its protective effect through modulation of UVB-induced ROS.

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

    PubMed Central

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

    2015-01-01

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

  5. 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.

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

    SciTech Connect

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

    2008-06-01

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

  7. Canine distemper virus DNA vaccination induces humoral and cellular immunity and protects against a lethal intracerebral challenge.

    PubMed

    Sixt, N; Cardoso, A; Vallier, A; Fayolle, J; Buckland, R; Wild, T F

    1998-11-01

    We have studied the immune responses to the two glycoproteins of the Morbillivirus canine distemper virus (CDV) after DNA vaccination of BALB/c mice. The plasmids coding for both CDV hemagglutinin (H) and fusion protein (F) induce high levels of antibodies which persist for more than 6 months. Intramuscular inoculation of the CDV DNA induces a predominantly immunoglobulin G2a (IgG2a) response (Th1 response), whereas gene gun immunization with CDV H evokes exclusively an IgG1 response (Th2 response). In contrast, the CDV F gene elicited a mixed, IgG1 and IgG2a response. Mice vaccinated (by gene gun) with either the CDV H or F DNA showed a class I-restricted cytotoxic lymphocyte response. Immunized mice challenged intracerebrally with a lethal dose of a neurovirulent strain of CDV were protected. However, approximately 30% of the mice vaccinated with the CDV F DNA became obese in the first 2 months following the challenge. This was not correlated with the serum antibody levels.

  8. Cellular responses to environmental DNA damage

    SciTech Connect

    Not Available

    1994-08-01

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

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

    PubMed

    Gapeyev, A B; Lukyanova, N A

    2015-01-01

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

  10. STING Recognition of Cytoplasmic DNA Instigates Cellular Defense

    PubMed Central

    Abe, Takayuki; Harashima, Ai; Xia, Tianli; Konno, Hiroyasu; Konno, Keiko; Morales, Alejo; Ahn, Jeonghyun; Gutman, Delia; Barber, Glen N.

    2013-01-01

    Summary How the cell recognizes cytosolic DNA including DNA based microbes to trigger host defense related gene activation remains to be fully resolved. Here, we demonstrate that STING (for Stimulator of Interferon Genes), an endoplasmic reticulum (ER) translocon associated transmembrane protein, acts to detect cytoplasmic DNA species. STING homodimers were able to complex with self (apoptotic, necrotic) or pathogen related ssDNA and dsDNA and were indispensible for HSV-1-mediated transcriptional activation of a wide array of innate immune and pro-inflammatory genes in addition to type I IFN. Our data indicates that STING instigates cytoplasmic DNA-mediated cellular defense gene transcription and facilitates adoptive responses that are required for protection of the host. In contrast, chronic STING activation may manifest inflammatory responses and possibly autoimmune disease triggered by self-DNA. PMID:23478444

  11. Modification of cellular DNA by synthetic aziridinomitosenes

    PubMed Central

    Mallory, Chris M.; Carfi, Ryan P.; Moon, SangPhil; Cornell, Kenneth A.; Warner, Don L.

    2015-01-01

    Two synthetic aziridinomitosenes (AZMs), Me-AZM and H-AZM, structurally related to mitomycin C (MC) were evaluated for their anticancer activity against six cancer cell lines (HeLa, Jurkat, T47D, HepG2, HL-60, and HuT-78) and tested for their DNA-modifying abilities in Jurkat cells. Cytotoxicity assays showed that Me-AZM is up to 72-fold and 520-fold more potent than MC and H-AZM, respectively. Me-AZM also demonstrated increased DNA modification over MC and H-AZM in alkaline COMET and Hoechst fluorescence assays that measured crosslinks in cellular DNA. Me-AZM and H-AZM treatment of Jurkat cells was found to sponsor significant DNA-protein crosslinks using a K-SDS assay. The results clearly indicate that the AZM C6/C7 substitution pattern plays an important role in drug activity and supports both DNA-DNA and DNA-protein adduct formation as mechanisms for inducing cytotoxic effects. PMID:26541587

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

    PubMed

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

    2011-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1995-02-01

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

  14. [Experimental study on impact of salmon milt DNA on T-cellular immunity].

    PubMed

    Fedianina, L N; Zaĭtseva, E A; Epshteĭn, L M; Besednova, N N; Somov, G P

    2005-01-01

    The impact of a biologically active food supplement containing salmon milt DNA on cellular immunity was experimentally studied. It was shown that the DNA had a dose-dependent stimulating effect on lymphocyte transformation, stimulated the DTH response and protected from experimental listeriosis infection.

  15. Protective cellular antigen of Clostridium chauvoei.

    PubMed

    Stevenson, J R; Stonger, K A

    1980-04-01

    Cellular antigens of Clostridium chauvoei, strain IRP-128, were demonstrated to be important in induction of immunity against this bacterium in guinea pigs. At least one major component of the cellular antigen complex was heat-labile. Acid extraction of the bacterial cells, followed by selective purification for flagella, led to the preparation of an acid extract antigen that possessed a high degree of immunogenicity. The acid extract antigen contained flagellar components and was resolved into two major and approximately five minor protein components by polyacrylamide-gel electrophoresis.

  16. DNA origami: Nanorobots grab cellular control

    NASA Astrophysics Data System (ADS)

    Elbaz, Johann; Willner, Itamar

    2012-04-01

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

  17. Preparing Cellular DNA from Nuclei or Whole Cells.

    PubMed

    Nilsen, Timothy W

    2015-09-01

    It is often desirable to have cellular DNA on hand. DNA is stable and can be maintained intact for many years. This protocol describes the preparation of DNA from nuclei after the cytoplasmic extract has been removed. The resulting DNA is suitable for polymerase chain reactions and Southern blots to determine copy number and sites of integration of plasmids in stable cell lines. Quantitation of DNA may not be exact because RNA is not completely removed. The method can also be used on whole cells, but there will be more RNA contamination.

  18. Cellular effects of UVA: DNA damages

    SciTech Connect

    Peak, M.J.; Peak, J.G.

    1988-01-01

    Ultraviolet radiation between 320 nm and visible light (UVA) is a major component of both solar radiation and suntan lamps, which are being increasingly used in tanning booths. UVA has generally been considered innocuous, partially because DNA does not absorb appreciably in this region, but UVB radiation (290-320 nm) has widely been considered to be the major etiological factor in human skin carcinogenesis caused by solar uv radiation, largely because DNA absorbs photons of UVB, which is known to produce thymine photoproducts (cyclobutane dimers and adducts). It is true that patients with xeroderma pigmentosum are particularly prone to solar-uv-induced skin cancer, and cells derived from these people have been shown to lack ability to repair pyrimidine photoproducts by excision, evidence that pyrimidine photoproducts might play a role in carcinogenesis in certain specialized situations. Normal cells have the ability to repair these UVB-induced lesions. However, UVA is considerably more penetrating and more abundant than UVB, and others have performed a spectral analysis that claimed that 20-60% (depending upon the solar zenith angle) of the toxic biological effects of solar radiation can be attributed to UVA. The fact that UVA radiations are mutagenic provides motivation for studying DNA changes that might be effected by this region of the electromagnetic spectrum.

  19. Analysis of cellular and extracellular DNA in fingerprints

    SciTech Connect

    Button, Julie M.

    2014-09-09

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

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

    PubMed

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

    2011-12-01

    All the self-reproducing cellular organisms so far examined have DNA as the genome. However, a DNA-less organism carrying an RNA genome is suggested by the fact that many RNA viruses exist and the widespread view that an RNA world existed before the present DNA world. Such a possibility is most plausible in the microbial world where biological diversity is enormous and most organisms have not been identified. We have developed experimental methodology to search DNA-less microorganisms, which is based on cultivation with drugs that inhibit replication or expression of DNA, detection of DNA in colonies with a fluorescent dye and double staining for DNA and RNA at a cellular level. These methods have been applied for about 100 microbial samples from various waters including hot springs, soils including deep sea sediments, and organisms. We found many colonies and cells which apparently looked DNA-less and examined them further. So far, all such colonies that reformed colonies on isolation were identified to be DNA-positive. However, considering the difficulty in cultivation, we think it possible for DNA-less microorganisms to live around us. We believe that our ideas and results will be of interest and useful to discover one in the future. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  1. 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.

  2. Mechanisms for Initiating Cellular DNA Replication

    PubMed Central

    Costa, Alessandro; Hood, Iris V.; Berger, James M.

    2015-01-01

    The initiation of DNA replication represents a committing step to cell proliferation. Appropriate replication onset depends on multiprotein complexes that help properly distinguish origin regions, generate nascent replication bubbles, and promote replisome formation. This review describes initiation systems employed by bacteria, archaea, and eukaryotes, with a focus on comparing and contrasting molecular mechanisms among organisms. Although commonalities can be found in the functional domains and strategies used to carry out and regulate initiation, many key participants have markedly different activities and appear to have evolved convergently. Despite significant advances in the field, major questions still persist in understanding how initiation programs are executed at the molecular level. PMID:23746253

  3. Protection from lethal and sub-lethal whole body exposures of mice to γ-radiation by Acorus calamus L.: studies on tissue antioxidant status and cellular DNA damage.

    PubMed

    Sandeep, Divyasree; Nair, Cherupally Krishnan Krishnan

    2012-01-01

    The radioprotecting activity of Acorus calamus extract after whole body exposure of mice to lethal and sub-lethal doses of γ-irradiation in terms of radiation induced mortality and damages to cellular DNA and tissue antioxidant levels were studied. A. calamus extract (250 mg/kg body weight) was orally administered to mice 1 h prior to whole body γ-radiation exposure. The antioxidant levels in the tissue homogenates of brain, liver and kidney of the irradiated mice were determined and cellular DNA damage was monitored by comet assay. Effect of administration of the extract on survival of the animals exposed to acute lethal dose of 10 Gy whole body γ-radiations was also monitored. Administration of the extract significantly increased the activities of major enzymes of the antioxidant defense system specially SOD, catalase and GPx and levels of GSH in 2, 6 and 10 Gy irradiated mice and decreased the formation MDA. The extract also decreased DNA strand breaks. The survival rate was found to be increased up to 5%. These studies highlight the role of A. calamus extract as good source of natural radioprotecting agent and its therapeutic implications for radiation-induced injuries.

  4. Borrowing Nuclear DNA Helicases to Protect Mitochondrial DNA

    PubMed Central

    Ding, Lin; Liu, Yilun

    2015-01-01

    In normal cells, mitochondria are the primary organelles that generate energy, which is critical for cellular metabolism. Mitochondrial dysfunction, caused by mitochondrial DNA (mtDNA) mutations or an abnormal mtDNA copy number, is linked to a range of human diseases, including Alzheimer’s disease, premature aging‎ and cancer. mtDNA resides in the mitochondrial lumen, and its duplication requires the mtDNA replicative helicase, Twinkle. In addition to Twinkle, many DNA helicases, which are encoded by the nuclear genome and are crucial for nuclear genome integrity, are transported into the mitochondrion to also function in mtDNA replication and repair. To date, these helicases include RecQ-like helicase 4 (RECQ4), petite integration frequency 1 (PIF1), DNA replication helicase/nuclease 2 (DNA2) and suppressor of var1 3-like protein 1 (SUV3). Although the nuclear functions of some of these DNA helicases have been extensively studied, the regulation of their mitochondrial transport and the mechanisms by which they contribute to mtDNA synthesis and maintenance remain largely unknown. In this review, we attempt to summarize recent research progress on the role of mammalian DNA helicases in mitochondrial genome maintenance and the effects on mitochondria-associated diseases. PMID:25984607

  5. Borrowing nuclear DNA helicases to protect mitochondrial DNA.

    PubMed

    Ding, Lin; Liu, Yilun

    2015-05-13

    In normal cells, mitochondria are the primary organelles that generate energy, which is critical for cellular metabolism. Mitochondrial dysfunction, caused by mitochondrial DNA (mtDNA) mutations or an abnormal mtDNA copy number, is linked to a range of human diseases, including Alzheimer's disease, premature aging‎ and cancer. mtDNA resides in the mitochondrial lumen, and its duplication requires the mtDNA replicative helicase, Twinkle. In addition to Twinkle, many DNA helicases, which are encoded by the nuclear genome and are crucial for nuclear genome integrity, are transported into the mitochondrion to also function in mtDNA replication and repair. To date, these helicases include RecQ-like helicase 4 (RECQ4), petite integration frequency 1 (PIF1), DNA replication helicase/nuclease 2 (DNA2) and suppressor of var1 3-like protein 1 (SUV3). Although the nuclear functions of some of these DNA helicases have been extensively studied, the regulation of their mitochondrial transport and the mechanisms by which they contribute to mtDNA synthesis and maintenance remain largely unknown. In this review, we attempt to summarize recent research progress on the role of mammalian DNA helicases in mitochondrial genome maintenance and the effects on mitochondria-associated diseases.

  6. A fluorescence enhancement assay for cellular DNA damage. [X Radiation

    SciTech Connect

    Kanter, P.M.; Schwartz, H.S.

    1982-07-01

    A fluorescence procedure is described for quantitative measurement of DNA damage in mammalian cells. The technique is based upon the time-dependent partial alkaline unwinding of cellular DNA followed by determination of duplex:total DNA ratios with bisbenzamide, which has a differential molar fluorescence with single-stranded and duplex DNA. The method is rapid, does not require radioactive labeling of DNA, and is sufficiently sensitive to detect damage induced with 100 rads of X-irradiation. This method is standardized with respect to the alkaline unwinding unit, Mn0, and the unwinding constant, beta. Results obtained with this new technique and with hydroxylapatite chromatography for physical separation of single- and double-stranded DNA were confirmatory. The utility of the technique was demonstrated by detection of dose-related damage with X-irradiation and a variety of antineoplastic agents in unlabeled murine leukemia cells.

  7. Sequence-specific binding of simian virus 40 A protein to nonorigin and cellular DNA.

    PubMed Central

    Wright, P J; DeLucia, A L; Tegtmeyer, P

    1984-01-01

    The simian virus 40 A protein (T antigen) recognized and bound to the consensus sequence 5'-GAGGC-3' in DNA from many sources. Sequence-specific binding to single pentanucleotides in randomly chosen DNA predominated over binding to nonspecific sequences. The asymmetric orientation of protein bound to nonorigin recognition sequences also resembled that of protein bound to the origin region of simian virus 40 DNA. Sequence variations in the DNA adjacent to single pentanucleotides influenced binding affinities even though methylation interference and protection studies did not reveal specific interactions outside of pentanucleotides. Thus, potential locations of A protein bound to any DNA can be predicted although the determinants of binding affinity are not yet understood. Sequence-specific binding of A protein to cellular DNA would provide a mechanism for specific alterations of host gene expression that facilitate viral function. Images PMID:6570189

  8. APOBEC3A damages the cellular genome during DNA replication.

    PubMed

    Green, Abby M; Landry, Sébastien; Budagyan, Konstantin; Avgousti, Daphne C; Shalhout, Sophia; Bhagwat, Ashok S; Weitzman, Matthew D

    2016-01-01

    The human APOBEC3 family of DNA-cytosine deaminases comprises 7 members (A3A-A3H) that act on single-stranded DNA (ssDNA). The APOBEC3 proteins function within the innate immune system by mutating DNA of viral genomes and retroelements to restrict infection and retrotransposition. Recent evidence suggests that APOBEC3 enzymes can also cause damage to the cellular genome. Mutational patterns consistent with APOBEC3 activity have been identified by bioinformatic analysis of tumor genome sequences. These mutational signatures include clusters of base substitutions that are proposed to occur due to APOBEC3 deamination. It has been suggested that transiently exposed ssDNA segments provide substrate for APOBEC3 deamination leading to mutation signatures within the genome. However, the mechanisms that produce single-stranded substrates for APOBEC3 deamination in mammalian cells have not been demonstrated. We investigated ssDNA at replication forks as a substrate for APOBEC3 deamination. We found that APOBEC3A (A3A) expression leads to DNA damage in replicating cells but this is reduced in quiescent cells. Upon A3A expression, cycling cells activate the DNA replication checkpoint and undergo cell cycle arrest. Additionally, we find that replication stress leaves cells vulnerable to A3A-induced DNA damage. We propose a model to explain A3A-induced damage to the cellular genome in which cytosine deamination at replication forks and other ssDNA substrates results in mutations and DNA breaks. This model highlights the risk of mutagenesis by A3A expression in replicating progenitor cells, and supports the emerging hypothesis that APOBEC3 enzymes contribute to genome instability in human tumors.

  9. APOBEC3A damages the cellular genome during DNA replication

    PubMed Central

    Green, Abby M.; Landry, Sébastien; Budagyan, Konstantin; Avgousti, Daphne C.; Shalhout, Sophia; Bhagwat, Ashok S.; Weitzman, Matthew D.

    2016-01-01

    ABSTRACT The human APOBEC3 family of DNA-cytosine deaminases comprises 7 members (A3A-A3H) that act on single-stranded DNA (ssDNA). The APOBEC3 proteins function within the innate immune system by mutating DNA of viral genomes and retroelements to restrict infection and retrotransposition. Recent evidence suggests that APOBEC3 enzymes can also cause damage to the cellular genome. Mutational patterns consistent with APOBEC3 activity have been identified by bioinformatic analysis of tumor genome sequences. These mutational signatures include clusters of base substitutions that are proposed to occur due to APOBEC3 deamination. It has been suggested that transiently exposed ssDNA segments provide substrate for APOBEC3 deamination leading to mutation signatures within the genome. However, the mechanisms that produce single-stranded substrates for APOBEC3 deamination in mammalian cells have not been demonstrated. We investigated ssDNA at replication forks as a substrate for APOBEC3 deamination. We found that APOBEC3A (A3A) expression leads to DNA damage in replicating cells but this is reduced in quiescent cells. Upon A3A expression, cycling cells activate the DNA replication checkpoint and undergo cell cycle arrest. Additionally, we find that replication stress leaves cells vulnerable to A3A-induced DNA damage. We propose a model to explain A3A-induced damage to the cellular genome in which cytosine deamination at replication forks and other ssDNA substrates results in mutations and DNA breaks. This model highlights the risk of mutagenesis by A3A expression in replicating progenitor cells, and supports the emerging hypothesis that APOBEC3 enzymes contribute to genome instability in human tumors. PMID:26918916

  10. HSV-I and the cellular DNA damage response.

    PubMed

    Smith, Samantha; Weller, Sandra K

    2015-04-01

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

  11. The sub-cellular localization of Sulfolobus DNA replication.

    PubMed

    Gristwood, Tamzin; Duggin, Iain G; Wagner, Michaela; Albers, Sonja V; Bell, Stephen D

    2012-07-01

    Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.

  12. Cellular Uptake of Tile-Assembled DNA Nanotubes

    PubMed Central

    Kocabey, Samet; Meinl, Hanna; MacPherson, Iain S.; Cassinelli, Valentina; Manetto, Antonio; Rothenfusser, Simon; Liedl, Tim; Lichtenegger, Felix S.

    2014-01-01

    DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP-expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.

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

    PubMed

    Stone, H R; Morris, J R

    2014-02-13

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

  14. DNA double-strand break repair in a cellular context.

    PubMed

    Shibata, A; Jeggo, P A

    2014-05-01

    Substantial insight into the mechanisms responding to DNA double-strand breaks has been gained from molecular, biochemical and structural approaches. Attention is now focusing on understanding the interplay between the pathways, how they interface through the cell cycle and the communication with other DNA transactions, such as replication and transcription. Understanding these aspects will facilitate an assessment of how cancer cells have modified these processes to achieve unlimited proliferative capacity and adaptability, and pave the way to identify targets suitable for therapy. Here, we briefly overview the processes responding to double-strand breaks and discuss our current understanding of their interplay in a cellular context.

  15. DNA-Protected Silver Clusters for Nanophotonics

    PubMed Central

    Gwinn, Elisabeth; Schultz, Danielle; Copp, Stacy M.; Swasey, Steven

    2015-01-01

    DNA-protected silver clusters (AgN-DNA) possess unique fluorescence properties that depend on the specific DNA template that stabilizes the cluster. They exhibit peak emission wavelengths that range across the visible and near-IR spectrum. This wide color palette, combined with low toxicity, high fluorescence quantum yields of some clusters, low synthesis costs, small cluster sizes and compatibility with DNA are enabling many applications that employ AgN-DNA. Here we review what is known about the underlying composition and structure of AgN-DNA, and how these relate to the optical properties of these fascinating, hybrid biomolecule-metal cluster nanomaterials. We place AgN-DNA in the general context of ligand-stabilized metal clusters and compare their properties to those of other noble metal clusters stabilized by small molecule ligands. The methods used to isolate pure AgN-DNA for analysis of composition and for studies of solution and single-emitter optical properties are discussed. We give a brief overview of structurally sensitive chiroptical studies, both theoretical and experimental, and review experiments on bringing silver clusters of distinct size and color into nanoscale DNA assemblies. Progress towards using DNA scaffolds to assemble multi-cluster arrays is also reviewed.

  16. Rational design of human DNA ligase inhibitors that target cellular DNA replication and repair.

    PubMed

    Chen, Xi; Zhong, Shijun; Zhu, Xiao; Dziegielewska, Barbara; Ellenberger, Tom; Wilson, Gerald M; MacKerell, Alexander D; Tomkinson, Alan E

    2008-05-01

    Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents.

  17. Alkene-tetrazine ligation for imaging cellular DNA.

    PubMed

    Rieder, Ulrike; Luedtke, Nathan W

    2014-08-25

    5-Vinyl-2'-deoxyuridine (VdU) is the first reported metabolic probe for cellular DNA synthesis that can be visualized by using an inverse electron demand Diels-Alder reaction with a fluorescent tetrazine. VdU is incorporated by endogenous enzymes into the genomes of replicating cells, where it exhibits reduced genotoxicity compared to 5-ethynyl-2'-deoxyuridine (EdU). The VdU-tetrazine ligation reaction is rapid (k≈0.02 M(-1) s(-1)) and chemically orthogonal to the alkyne-azide "click" reaction of EdU-modified DNA. Alkene-tetrazine ligation reactions provide the first alternative to azide-alkyne click reactions for the bioorthogonal chemical labeling of nucleic acids in cells and facilitate time-resolved, multicolor labeling of DNA synthesis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Measurement of oxidatively generated base damage in cellular DNA.

    PubMed

    Cadet, Jean; Douki, Thierry; Ravanat, Jean-Luc

    2011-06-03

    This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and (32)P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.

  19. A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria.

    PubMed

    Rainczuk, A; Scorza, T; Spithill, T W; Smooker, P M

    2004-10-01

    The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in production and secretion of both AMA-1 and MSP4/5 in vitro. Vaccination of BALB/c mice via intraepidermal gene gun and intramuscular routes against AMA-1 and MSP4/5 resulted in antibody production and significant in vitro proliferation of splenocytes stimulated by both AMA-1 and MSP4/5. Survival of BALB/c mice vaccinated with bicistronic constructs after lethal Plasmodium chabaudi adami DS erythrocytic-stage challenge was variable, although significant increases in survival and reductions in peak parasitemia were observed in several challenge trials when the vaccine was delivered by the intramuscular route. This study using a murine model demonstrates that the delivery of malarial antigens via bicistronic vectors is feasible. Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against malaria.

  20. Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps.

    PubMed

    Martinez, A; Kolter, R

    1997-08-01

    Reactive oxygen species can damage most cellular components, but DNA appears to be the most sensitive target of these agents. Here we present the first evidence of DNA protection against the toxic and mutagenic effects of oxidative damage in metabolically active cells: direct protection of DNA by Dps, an inducible nonspecific DNA-binding protein from Escherichia coli. We demonstrate that in a recA-deficient strain, expression of Dps from an inducible promoter prior to hydrogen peroxide challenge increases survival and reduces the number of chromosomal single-strand breaks. dps mutants exhibit increased levels of the G x C-->T x A mutations characteristic of oxidative damage after treatment with hydrogen peroxide. In addition, expression of Dps from the inducible plasmid reduces the frequency of spontaneous G x C-->T x A and A x T-->T x A mutations and can partially suppress the mutator phenotype of mutM (fpg) and mutY alleles. In a purified in vitro system, Dps reduces the number of DNA single-strand breaks and Fpg-sensitive sites introduced by hydrogen peroxide treatment, indicating that the protection observed in vivo is a direct effect of DNA binding by Dps. The widespread conservation of Dps homologs among prokaryotes suggests that this may be a general strategy for coping with oxidative stress.

  1. Circadian Clock Control of the Cellular Response to DNA Damage

    PubMed Central

    Sancar, Aziz; Lindsey-Boltz, Laura A.; Kang, Tae-Hong; Reardon, Joyce T.; Lee, Jin Hyup; Ozturk, Nuri

    2010-01-01

    Mammalian cells possess a cell-autonomous molecular clock which controls the timing of many biochemical reactions and hence the cellular response to environmental stimuli including genotoxic stress. The clock consists of an autoregulatory transcription-translation feedback loop made up of four genes/proteins, BMal1, Clock, Cryptochrome, and Period. The circadian clock has an intrinsic period of about 24 hours, and it dictates the rates of many biochemical reactions as a function of the time of the day. Recently, it has become apparent that the circadian clock plays an important role in determining the strengths of cellular responses to DNA damage including repair, checkpoints, and apoptosis. These new insights are expected to guide development of novel mechanism-based chemotherapeutic regimens. PMID:20227409

  2. Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

    PubMed

    Kwant, M M; van der Vliet, P C

    1980-09-11

    There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.

  3. Cellular protection with proanthocyanidins derived from grape seeds.

    PubMed

    Bagchi, Debasis; Bagchi, Manashi; Stohs, Sidney j; Ray, Sidhatha D; Sen, Chandan K; Preuss, Harry G

    2002-05-01

    Grape seed proanthocyanidins have been reported to possess a broad spectrum of pharmacological and medicinal properties against oxidative stress. We have demonstrated that IH636 proanthocyanidin extract (GSPE) provides excellent protection against free radicals in both in vitro and in vivo models. GSPE had significantly better free radical scavenging ability than vitamins C, E and beta-carotene and demonstrated significant cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal cells. GSPE protected against tobacco-induced apoptotic cell death in human oral keratinocytes and provided protection against cancer chemotherapeutic drug-induced cytotoxicity in human liver cells by modulating cell cycle/apoptosis regulatory genes such as bcl2, p53 and c-myc. Recently, the bioavailability and mechanistic pathways of cytoprotection by GSPE were examined on acetaminophen-induced hepatotoxicity and nephrotoxicity, amiodarone-induced pulmonary toxicity, doxorubicin-induced cardiotoxicity, DMN-induced immunotoxicity and MOCAP-induced neurotoxicity in mice. Serum chemistry changes, integrity of genomic DNA and histopathology were assessed. GSPE pre-exposure provided near complete protection in terms of serum chemistry changes and DNA damage, as well as abolished apoptotic and necrotic cell death in all tissues. Histopathological examination reconfirmed these findings. GSPE demonstrated concentration-/dose-dependent inhibitory effects on the drug metabolizing enzyme cytochrome P450 2E1, and this may be a major pathway for the anti-toxic potential exerted by GSPE. Furthermore, GSPE treatment significantly decreased TNFalpha-induced adherence of T-cells to HUVEC by inhibiting VCAM-1 expression. These results demonstrate that GSPE is highly bioavailable and may serve as a potential therapeutic tool in protecting multiple target organs from structurally diverse drug- and chemical-induced toxicity.

  4. The ATP requirements of adenovirus type 5 DNA replication and cellular DNA replication.

    PubMed

    De Jong, P J; Kwant, M M; van Driel, W; Jansz, H S; van der Vliet, P C

    1983-01-15

    Several in vitro DNA replication systems were employed to characterize the ATP dependency of adenovirus type 5 (Ad5) DNA replication. Ad5 DNA synthesis in isolated nuclei, representing the elongation of nascent DNA chains, was slightly ATP dependent. Reduction of the ATP concentration from the optimum (8 mM) to the endogenous value (0.16 microM) reduced Ad5 DNA replication only to 70%. No change in the pattern of replication was observed as indicated by the analysis of replicative intermediates using agarose gel electrophoresis. ATP could be replaced by dATP, but not by GTP or other nucleoside triphosphates. By contrast, cellular DNA replication in isolated nuclei from HeLa cells was reduced to 12% by the omission of ATP. These differences could not be explained by different ATP pools or by effects of ATP on dNTP pools. Cellular DNA replication in contrast to viral DNA replication was sensitive to low concentrations of adenosine 5'-O-(3-thiotriphosphate). Inhibition by this ATP analog was competitive with ATP (Ki = 0.4 mM). Adenovirus DNA replication by DNA-free nuclear extracts, representing initiation plus elongation (Challberg and Kelly, Proc. Nat. Acad. Sci. USA 76, 655-659, 1979), exhibited a nearly absolute requirement for ATP. ATP could be substituted not only by dATP, but also by GTP and dGTP and to a lesser extent by pyrimidine triphosphates. Similar results were found when the formation of a covalent complex between dCTP and the precursor terminal protein was studied. This reaction is essential for the initiation of Ad5 DNA replication. The results indicate that different ATP-requiring functions are employed during the initiation and elongation stages of adenovirus DNA replication.

  5. The DNA damage response in viral-induced cellular transformation.

    PubMed

    Nikitin, P A; Luftig, M A

    2012-01-31

    The DNA damage response (DDR) has emerged as a critical tumour suppressor pathway responding to cellular DNA replicative stress downstream of aberrant oncogene over-expression. Recent studies have now implicated the DDR as a sensor of oncogenic virus infection. In this review, we discuss the mechanisms by which tumour viruses activate and also suppress the host DDR. The mechanism of tumour virus induction of the DDR is intrinsically linked to the need for these viruses to promote an S-phase environment to replicate their nucleic acid during infection. However, inappropriate expression of viral oncoproteins can also activate the DDR through various mechanisms including replicative stress, direct interaction with DDR components and induction of reactive oxygen species. Given the growth-suppressive consequences of activating the DDR, tumour viruses have also evolved mechanisms to attenuate these pathways. Aberrant expression of viral oncoproteins may therefore promote tumourigenesis through increased somatic mutation and aneuploidy due to DDR inactivation. This review will focus on the interplay between oncogenic viruses and the DDR with respect to cellular checkpoint control and transformation.

  6. Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair.

    PubMed

    German, Peter; Szaniszlo, Peter; Hajas, Gyorgy; Radak, Zsolt; Bacsi, Attila; Hazra, Tapas K; Hegde, Muralidhar L; Ba, Xueqing; Boldogh, Istvan

    2013-10-01

    Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.

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

    NASA Technical Reports Server (NTRS)

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

    2015-01-01

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

  8. Salt shield: intracellular salts provide cellular protection against ionizing radiation in the halophilic archaeon, Halobacterium salinarum NRC-1.

    PubMed

    Kish, A; Kirkali, G; Robinson, C; Rosenblatt, R; Jaruga, P; Dizdaroglu, M; DiRuggiero, J

    2009-05-01

    The halophilic archaeon Halobacterium salinarum NRC-1 was used as a model system to investigate cellular damage induced by exposure to high doses of ionizing radiation (IR). Oxidative damages are the main lesions from IR and result from free radicals production via radiolysis of water. This is the first study to quantify DNA base modification in a prokaryote, revealing a direct relationship between yield of DNA lesions and IR dose. Most importantly, our data demonstrate the significance of DNA radiation damage other than strand breaks on cell survival. We also report the first in vivo evidence of reactive oxygen species scavenging by intracellular halides in H. salinarum NRC-1, resulting in increased protection against nucleotide modification and carbonylation of protein residues. Bromide ions, which are highly reactive with hydroxyl radicals, provided the greatest protection to cellular macromolecules. Modified DNA bases were repaired in 2 h post irradiation, indicating effective DNA repair systems. In addition, measurements of H. salinarum NRC-1 cell interior revealed a high Mn/Fe ratio similar to that of Deinococcus radiodurans and other radiation-resistant microorganisms, which has been shown to provide a measure of protection for proteins against oxidative damage. The work presented here supports previous studies showing that radiation resistance is the product of mechanisms for cellular protection and detoxification, as well as for the repair of oxidative damage to cellular macromolecules. The finding that not only Mn/Fe but also the presence of halides can decrease the oxidative damage to DNA and proteins emphasizes the significance of the intracellular milieu in determining microbial radiation resistance.

  9. Sulforaphane mobilizes cellular defenses that protect skin against damage by UV radiation

    PubMed Central

    Talalay, Paul; Fahey, Jed W.; Healy, Zachary R.; Wehage, Scott L.; Benedict, Andrea L.; Min, Christine; Dinkova-Kostova, Albena T.

    2007-01-01

    UV radiation (UVR) is a complete carcinogen that elicits a constellation of pathological events, including direct DNA damage, generation of reactive oxidants that peroxidize lipids and damage other cellular components, initiation of inflammation, and suppression of the immune response. Recent dramatic increases in the incidence of nonmelanoma skin cancers are largely attributable to higher exposure of an aging population to UVR. Therefore, the development of cellular strategies for intrinsic protection of the skin against the deleterious effects of UVR is imperative. Here we show that erythema resulting from UVR is a comprehensive and noninvasive biomarker for assessing UVR damage and can be precisely and easily quantified in human skin. Topical application of sulforaphane-rich extracts of 3-day-old broccoli sprouts up-regulated phase 2 enzymes in the mouse and human skin, protected against UVR-induced inflammation and edema in mice, and reduced susceptibility to erythema arising from narrow-band 311-nm UVR in humans. In six human subjects (three males and three females, 28–53 years of age), the mean reduction in erythema across six doses of UVR (300–800 mJ/cm2 in 100 mJ/cm2 increments) was 37.7% (range 8.37–78.1%; P = 0.025). This protection against a carcinogen in humans is catalytic and long lasting. PMID:17956979

  10. Antioxidant Activity of Lawsonia inermis Extracts Inhibits Chromium(VI)-Induced Cellular and DNA Toxicity

    PubMed Central

    Guha, Gunjan; Rajkumar, V.; Kumar, R. Ashok; Mathew, Lazar

    2011-01-01

    Hexavalent chromium Cr(VI) is a very strong oxidant which consequently causes high cytotoxicity through oxidative stress. Prevention of Cr(VI)-induced cellular damage has been sought in this study in aqueous and methanolic extracts of Lawsonia inermis Linn. (Lythraceae), commonly known as Henna. The extracts showed significant (P < .05) potential in scavenging free radicals (DPPH• and ABTS•+) and Fe3+, and in inhibiting lipid peroxidation. DNA damage caused by exposure of pBR322 to Cr(VI)-UV is markedly inhibited by both extracts in varying degrees. A distinct decline in Cr(VI)-induced cytotoxicity was noticed in MDA-MB-435S (human breast carcinoma) cells with an increase in dosage of both extracts individually. Furthermore, both extracts proved to contain a high content of phenolic compounds which were found to have a strong and significant (P < .05) positive correlation to the radical scavenging potential, lipid peroxidation inhibition capacity and cyto-protective efficiency against Cr(VI)-induced oxidative cellular damage. HPLC analysis identified some of the major phenolic compounds in both extracts, which might be responsible for the antioxidant potential and the properties of DNA and cyto-protection. This study contributes to the search for natural resources that might yield potent therapeutic drugs against Cr(VI)-induced oxidative cell damage. PMID:20008460

  11. Radiation-induced damage to cellular DNA: measurement and biological role

    NASA Astrophysics Data System (ADS)

    Cadet, Jean; Douki, Thierry; Gasparutto, Didier; Ravanat, Jean-Luc

    2005-02-01

    Emphasis is placed in this short review on recent developments concerning several aspects of the chemical and biochemical effects of ionizing radiation on both isolated and cellular DNA. This includes the mechanism of formation of single and tandem DNA lesions upon one-electron oxidation and one hydroxyl radical hit only. Information is also provided on the specificity of DNA repair enzymes and the measurement of radiation-induced damage in cellular DNA.

  12. Delivery methods to increase cellular uptake and immunogenicity of DNA vaccines.

    PubMed

    Jorritsma, S H T; Gowans, E J; Grubor-Bauk, B; Wijesundara, D K

    2016-11-04

    DNA vaccines are ideal candidates for global vaccination purposes because they are inexpensive and easy to manufacture on a large scale such that even people living in low-income countries can benefit from vaccination. However, the potential of DNA vaccines has not been realized owing mainly to the poor cellular uptake of DNA in vivo resulting in the poor immunogenicity of DNA vaccines. In this review, we discuss the benefits and shortcomings of several promising and innovative non-biological methods of DNA delivery that can be used to increase cellular delivery and efficacy of DNA vaccines.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  16. Dichotomy of protective cellular immune responses to human visceral leishmaniasis.

    PubMed

    Khalil, E A G; Ayed, N B; Musa, A M; Ibrahim, M E; Mukhtar, M M; Zijlstra, E E; Elhassan, I M; Smith, P G; Kieny, P M; Ghalib, H W; Zicker, F; Modabber, F; Elhassan, A M

    2005-05-01

    Healing/protective responses in human visceral leishmaniasis (VL) are associated with stimulation/production of Th1 cytokines, such as interferon IFN-gamma, and conversion in the leishmanin skin test (LST). Such responses were studied for 90 days in 44 adult healthy volunteers from VL non-endemic areas, with no past history of VL/cutaneous leishmaniasis (CL) and LST non-reactivity following injection with one of four doses of Alum-precipitated autoclaved Leishmania major (Alum/ALM) +/- bacille Calmette-Guerin (BCG), a VL candidate vaccine. The vaccine was well tolerated with minimal localized side-effects and without an increase in antileishmanial antibodies or interleukin (IL)-5. Five volunteers (5/44; 11.4%) had significant IFN-gamma production by peripheral blood mononuclear cells (PBMCs) in response to Leishmania antigens in their prevaccination samples (P = 0.001) but were LST non-reactive. On day 45, more than half the volunteers (26/44; 59.0%) had significantly high LST indurations (mean 9.2 +/- 2.7 mm) and high IFN-gamma levels (mean 1008 +/- 395; median 1247 pg/ml). Five volunteers had significant L. donovani antigen-induced IFN-gamma production (mean 873 +/- 290; median 902; P = 0.001), but were non-reactive in LST. An additional five volunteers (5/44; 11.4%) had low IFN-gamma levels (mean 110 +/- 124 pg/ml; median 80) and were non-reactive in LST (induration = 00 mm). The remaining eight volunteers had low IFN-gamma levels, but significant LST induration (mean 10 +/- 2.9 mm; median 11). By day 90 the majority of volunteers (27/44; 61.4%) had significant LST induration (mean 10.8 +/- 9.9 mm; P < 0.001), but low levels of L. donovani antigen-induced IFN-gamma (mean 66.0 +/- 62 pg/ml; P > 0.05). Eleven volunteers (11/44; 25%) had significantly high levels of IFN-gamma and LST induration, while five volunteers had low levels of IFN-gamma (<100 pg/ml) and no LST reactivity (00 mm). One volunteer was lost to follow-up. In conclusion, it is hypothesized that

  17. Threonines 2638/2647 in DNA-PK are essential for cellular resistance to ionizing radiation.

    PubMed

    Soubeyrand, Sébastien; Pope, Louise; Pakuts, Benjamin; Haché, Robert J G

    2003-03-15

    DNA-dependent protein kinase (DNA-PK) is required for the repair of double-stranded DNA breaks through the nonhomologous DNA end joining pathway. DNA-PK activity is required for DNA repair, but kinase activity also appears to be attenuated through an autoregulatory feedback loop. We show that autophosphorylation of DNA-PK catalytic subunit occurs in trans at least three sites NH(2) terminal to the catalytic domain and that two sites, threonine 2638 and 2647, determine DNA-PK autophosphorylation in vitro. Thr2638/2647ala substitution in DNA-PK catalytic subunit compromised cellular resistance to ionizing radiation without affecting DNA end joining, suggesting a requirement for DNA-PK inactivation for cell survival at a step after the rejoining of double-stranded DNA breaks.

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  19. The arginine metabolite agmatine protects mitochondrial function and confers resistance to cellular apoptosis.

    PubMed

    Arndt, Mary Ann; Battaglia, Valentina; Parisi, Eva; Lortie, Mark J; Isome, Masato; Baskerville, Christopher; Pizzo, Donald P; Ientile, Riccardo; Colombatto, Sebastiano; Toninello, Antonio; Satriano, Joseph

    2009-06-01

    Agmatine, an endogenous metabolite of arginine, selectively suppresses growth in cells with high proliferative kinetics, such as transformed cells, through depletion of intracellular polyamine levels. In the present study, we depleted intracellular polyamine content with agmatine to determine if attrition by cell death contributes to the growth-suppressive effects. We did not observe an increase in necrosis, DNA fragmentation, or chromatin condensation in Ha-Ras-transformed NIH-3T3 cells administered agmatine. In response to Ca(2+)-induced oxidative stress in kidney mitochondrial preparations, agmatine demonstrated attributes of a free radical scavenger by protecting against the oxidation of sulfhydryl groups and decreasing hydrogen peroxide content. The functional outcome was a protective effect against Ca(2+)-induced mitochondrial swelling and mitochondrial membrane potential collapse. We also observed decreased expression of proapoptotic Bcl-2 family members and of execution caspase-3, implying antiapoptotic potential. Indeed, we found that apoptosis induced by camptothecin or 5-fluorourocil was attenuated in cells administered agmatine. Agmatine may offer an alternative to the ornithine decarboxylase inhibitor difluoromethyl ornithine for depletion of intracellular polyamine content while avoiding the complications of increasing polyamine import and reducing the intracellular free radical scavenger capacity of polyamines. Depletion of intracellular polyamine content with agmatine suppressed cell growth, yet its antioxidant capacity afforded protection from mitochondrial insult and resistance to cellular apoptosis. These results could explain the beneficial outcomes observed with agmatine in models of injury and disease.

  20. 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

  1. Global DNA hypomethylation coupled to cellular transformation and metastatic ability.

    PubMed

    Funaki, Soichiro; Nakamura, Toshinobu; Nakatani, Tsunetoshi; Umehara, Hiroki; Nakashima, Hiroyuki; Okumura, Meinoshin; Oboki, Keisuke; Matsumoto, Kenji; Saito, Hirohisa; Nakano, Toru

    2015-12-21

    Global DNA hypomethylation and DNA hypermethylation of promoter regions are frequently detected in human cancers. Although many studies have suggested a contribution to carcinogenesis, it is still unclear whether the aberrant DNA hypomethylation observed in tumors is a consequence or a cause of cancer. Here, we show that the enforced expression of Stella (also known as PGC7 and Dppa3) induced not only global DNA demethylation but also transformation of NIH3T3 cells. Furthermore, overexpression of Stella enhanced the metastatic ability of B16 melanoma cells, presumably through the induction of metastasis-related genes. These results provide new insights into the function of global DNA hypomethylation in carcinogenesis.

  2. 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

  3. Mitochondrial transcription factor A (TFAM): roles in maintenance of mtDNA and cellular functions.

    PubMed

    Kang, Dongchon; Kim, Sang Ho; Hamasaki, Naotaka

    2007-01-01

    A growing body of evidence suggests that mammalian mitochondrial DNA takes on higher structure called nucleoid or mitochromosome corresponding to that of nuclear DNA. Mitochondrial transcription factor A (TFAM), which was cloned as a transcription factor for mitochondrial DNA, has known to be essential for the maintenance of mitochondrial DNA. Human TFAM has an ability to bind to DNA in a sequence-independent manner and is abundant enough to cover whole region of mitochondrial DNA, owing to which TFAM stabilizes mitochondrial DNA through formation of nucleoid and regulates (or titrates) the amount of mitochondrial DNA. Overexpression of human TFAM in mice increases the amount of mitochondrial DNA and dramatically ameliorates the cardiac dysfunctions caused by myocardial infarction. The maintenance of integrity of mitochondrial DNA is important for keeping proper cellular functions both under physiological and pathological conditions. TFAM may play a crucial role in maintaining mitochondrial DNA as a main component of the nucleoid.

  4. Reticuloendotheliosis Virus Nucleic Acid Sequences in Cellular DNA

    PubMed Central

    Kang, Chil-Yong; Temin, Howard M.

    1974-01-01

    Reticuloendotheliosis virus 60S RNA labeled with 125I, or reticuloendotheliosis virus complementary DNA labeled with 3H, were hybridized to DNAs from infected chicken and pheasant cells. Most of the sequences of the viral RNA were found in the infected cell DNAs. The reticuloendotheliosis viruses, therefore, replicate through a DNA intermediate. The same labeled nucleic acids were hybridized to DNA of uninfected chicken, pheasant, quail, turkey, and duck. About 10% of the sequences of reticuloendotheliosis virus RNA were present in the DNA of uninfected chicken, pheasant, quail, and turkey. None were detected in DNA of duck. The specificity of the hybridization was shown by competition between unlabeled and 125I-labeled viral RNAs and by determination of melting temperatures. In contrast, 125I-labeled RNA of Rous-associated virus-O, an avian leukosis-sarcoma virus, hybridized 55% to DNA of uninfected chicken, 20% to DNA of uninfected pheasant, 15% to DNA of uninfected quail, 10% to DNA of uninfected turkey, and less than 1% to DNA of uninfected duck. PMID:4372393

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

    PubMed Central

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

    2015-01-01

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

  6. Protective Cellular Immunity Against Influenza Virus Induced by Plasmid Inoculation of Newborn Mice

    PubMed Central

    Bot, Adrian; Bot, Simona; García-Sastre, Adolfo

    1998-01-01

    Neonate organisms display an intrinsic disability to mount effective immune responses to infectious agents or conventional vaccines. Whereas low. doses of antigens trigger a suboptimal response, higher doses are frequently associated with tolerance induction. We investigated the ability of a plasmid-expressing nucleoprotein of influenza virus to prime a specific cellular immune response when administered to newborn mice. We found that persistent exposure to antigen following plasmid inoculation of neonates leads to a vigorous priming of specific CTLs rather than tolerance induction. The CTLs were cross-reactive against multiple strains of type A influenza viruses and produced IFNγ but no IL-4. The immunity triggered by plasmid inoculation of neonates was protective in terms of pulmonary virus clearance as well as survival rate following lethal challenge with influenza virus. Whereas the persistence of the plasmid at the site of injection was readily demonstrable in adult mice at 3 months after inoculation, mice immunized as newborns displayed no plasmid at 3 months and very little at 1 month after injection. Thus, DNA-based immunization of neonates may prove an effective and safe vaccination strategy for induction of cellular immunity against microbes that cause serious infectious diseases in the early period of life. PMID:9851359

  7. DNA Vaccination in the Skin Using Microneedles Improves Protection Against Influenza

    PubMed Central

    Song, Jae-Min; Kim, Yeu-Chun; O, Eunju; Compans, Richard W; Prausnitz, Mark R; Kang, Sang-Moo

    2012-01-01

    In this study, we tested the hypothesis that DNA vaccination in the skin using microneedles improves protective immunity compared to conventional intramuscular (IM) injection of a plasmid DNA vaccine encoding the influenza hemagglutinin (HA). In vivo fluorescence imaging demonstrated the expression of a reporter gene delivered to the skin using a solid microneedle patch coated with plasmid DNA. Vaccination at a low dose (3 µg HA DNA) using microneedles generated significantly stronger humoral immune responses and better protective responses post-challenge compared to IM vaccination at either low or high (10 µg HA DNA) dose. Vaccination using microneedles at a high (10 µg) dose further generated improved post-challenge protection, as measured by survival, recall antibody-secreting cell responses in spleen and bone marrow, and interferon (IFN)-γ cytokine T-cell responses. This study demonstrates that DNA vaccination in the skin using microneedles induces higher humoral and cellular immune responses as well as improves protective immunity compared to conventional IM injection of HA DNA vaccine. PMID:22508490

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

    PubMed

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

    2008-06-01

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

  9. Methods of introducing nucleic acids into cellular DNA

    DOEpatents

    Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

    2017-06-27

    A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

  10. DNA damage induction and/or repair as mammalian cell biomarker for the prediction of cellular radiation response

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, C.

    DNA damage and its repair processes are key factors in cancer induction and also in the treatment of malignancies. Cancer prevention during extended space missions becomes a topic of great importance for space radiobiology. The knowledge of individual responsiveness would allow the protection strategy to be tailored optimally in each case. Radiobiological analysis of cultured cells derived from tissue explants from individuals has shown that measurement of the surviving fraction after 2 Gy (SF2) may be used to predict the individual responsiveness. However, clonogenic assays are timeconsuming, thus alternative assays for the determination of radiore-sponse are being sought. For that reason CHO cell strains having different repair capacities were used for examining whether DNA strand break repair is a suitable experimental design to allow predictive statements. Cellular survival (CFA assay) and DNA strand breaks (total DNA strand breaks: FADU technique; DSBs: non-denaturing elution) were determined in parallel immediately after irradiation as well as after a 24 hour recovery period according to dose. There were no correlations between the dose-response curves of the initial level of DNA strand breaks and parameters that describe clonogenic survival curves (SF2). A good correlation exists between intrinsic cellular radioresistance and the extent of residual DNA strand breaks.

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

    PubMed Central

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

    2016-01-01

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

  12. Distinct Mechanisms Underlying Resveratrol-Mediated Protection from Types of Cellular Stress in C6 Glioma Cells.

    PubMed

    Means, John C; Gerdes, Bryan C; Koulen, Peter

    2017-07-14

    The polyphenolic phytostilbene, trans-resveratrol, is found in high amounts in several types and tissues of plants, including grapes, and has been proposed to have beneficial effects in the central nervous system due to its activity as an antioxidant. The objective of the present study was to identify the mechanisms underlying the protective effects of resveratrol under conditions of oxidative stress or DNA damage, induced by the extracellularly applied oxidant, tert-butyl hydrogen peroxide, or UV-irradiation, respectively. In C6 glioma cells, a model system for glial cell biology and pharmacology, resveratrol was protective against both types of insult. Prevention of tau protein cleavage and of the formation of neurofibrillary tangles were identified as mechanisms of action of resveratrol-mediated protection in both paradigms of cellular damage. However, depending on the type of insult, resveratrol exerted its protective activity differentially: under conditions of chemically induced oxidative stress, inhibition of caspase activity, while with DNA damage, resveratrol regulated tau phosphorylation at Ser(422). Results advance our understanding of resveratrol's complex impact on cellular signaling pathway and contribute to the notion of resveratrol's role as a pleiotropic therapeutic agent.

  13. Radiation protection by disulfiram: protection of membrane and DNA in vitro and in vivo against gamma-radiation.

    PubMed

    Gandhi, Nitin Motilal; Gopalaswamy, Usulumarty Venu; Nair, Cherupally rishnan K

    2003-09-01

    Disufiram (a drug used for the treatment of alcoholism) protected microsomal membranes and plasmid DNA against damages induced by gamma-radiation. The peroxidation of membrane lipids increased linearly with the radiation dose up to 600 Gy, and the presence of disulfiram inhibited membrane lipid peroxidation as assayed by the presence of thiobarbituric acid reacting substances. The reduction of the quantity of the supercoiled (ccc) form of plasmid pBR322 DNA is directly related to the radiation-induced damage, particularly to DNA strand breaks. There was a complete protection of plasmid DNA when exposed to gamma-radiation in the presence of disufiram (0.1 mM) at 300 Gy. This drug also protected deoxyribose against damages caused by hydroxyl radicals produced by the Fenton reaction. The administration of DSF to mice prior to whole-body radiation exposure (4 Gy) resulted in a reduction of peroxidation of membrane lipids in mice liver as well as a decrease in radiation-induced damage to cellular DNA, as assayed by single-cell gel electrophoresis (comet assay). The results thus suggest the possible use of DSF as a radioprotector.

  14. 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.

  15. [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.

  16. Intra- and extra-cellular DNA damage by harmine and 9-methyl-harmine.

    PubMed

    Vignoni, Mariana; Erra-Balsells, Rosa; Epe, Bernd; Cabrerizo, Franco M

    2014-03-05

    It is known that β-carbolines are able to produce photosensitized damage in cell-free DNA, but there is little information on their effects on cellular DNA. Therefore, we have analyzed the DNA damage produced by harmine and 9-methyl-harmine under UVA irradiation in V79 cells, together with the associated generation of micronuclei and photocytotoxicity. The results indicate that the most frequent photoproducts generated in the cellular DNA are modified purines such as 8-oxo-7,8-dihydroguanine. Only relatively few single-strand breaks were observed. CPDs were absent, although they were generated in cell-free DNA irradiated under the same conditions. The overall extent of DNA damage in the cells was considerably smaller than the one observed in cell free DNA. The generation of cellular DNA damage was associated with a significant generation of micronuclei and decreased cell proliferation. The data indicate that β-carbolines act as photosensitizers in mammalian cells. The spectrum of DNA modification, and therefore the mechanism of DNA damage generation, differs considerably from that observed with cell-free DNA.

  17. Evolution of viruses by acquisition of cellular RNA or DNA nucleotide sequences and genes: an introduction.

    PubMed

    Becker, Y

    2000-01-01

    The origins of virus evolution may be traced to Archeabacteria since Inouye and Inouye (6) discovered a retroelement with a gene for reverse transcriptase in the bacterial genome and in the satellite, multiple copy single stranded DNA (msDNA) in the soil bacterium Myxococcus xanthus. It was possible (8) to define the evolution of retroelements in eukaryotic cells of plants, insects (gypsy retrovirus) and vertebrates. The replication of RNA viruses in eukaryotic cells allowed for the viral RNA genome to integrate a cellular ubiquitin mRNA, as reported for BVDV (24). Another example is the integration of 28S ribosomal RNA into the hemagglutinin gene of an influenza virus. This change in the hemagglutinin gene led to an increased pathogenicity of the influenza virus (25). In contrast to RNA viruses, DNA viruses had evolved by inserting cDNA molecules derived from mRNA transcripts of cellular genes or foreign viral RNA. It is of interest that the virus acquired cellular genes in the genomes of DNA viruses represent genes that code for proteins that inhibit cellular molecular processes related to HLA class I and II molecules. The other acquired genes are cellular genes that code for cytokines that are capable of inhibiting antigen presentation to T cells by antigen presenting cells (APC) by dendritic Langerhans cells. The acquisition of cellular genes by DNA viruses enhances their pathogenicity by inhibiting the hosts' defense systems.

  18. Protective Effect of Juglans regia L. Walnut Extract Against Oxidative DNA Damage.

    PubMed

    Calcabrini, Cinzia; De Bellis, Roberta; Mancini, Umberto; Cucchiarini, Luigi; Stocchi, Vilberto; Potenza, Lucia

    2017-06-01

    Walnuts (Juglans regia L.) are relevant components of the Mediterranean diet providing important macronutrients, micronutrients and other bioactive constituents including unsaturated fatty acids, proteins, fiber, vitamins, minerals, phytosterols and polyphenols. Although the walnut beneficial effects in human health are widely recognized by a lot of epidemiologic studies very little is known regarding its effect on damaged DNA. The aim of the present study was to investigate the effect of Juglans regia L. ethanolic extract from kernel on the induction of DNA strand breaks by thiol/Fe(3+)/O2 mixed function oxidase, tert-butyl hydroperoxide or UVC radiations in acellular and cellular models. Plasmid DNA cleavage and fast Halo assay were used to monitor oxidative damage to DNA. Both approaches showed protection of oxidatively injured DNA. These results agree with a lot of scientific proofs which recommend walnut as dietary adjunct in health promotion and prevention as well as in treatment of lifestyle-related oxidative diseases.

  19. Methylene Blue Protects Astrocytes against Glucose Oxygen Deprivation by Improving Cellular Respiration

    PubMed Central

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

    2015-01-01

    Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration. PMID:25848957

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2011-01-01

    This study aims at the development of a safe and effective formulation to counter the effects of lethal irradiation. The sub-fraction (G-001M), prepared from Podophyllum hexandrum has rendered high degree of survival (>90%) at a dose of 6 mg kg−1 body weight (intramuscular) in lethally irradiated mice. Therapeutic dose of G-001M, at about 20 times lower concentration than its LD100, has revealed a DRF of 1.62. Comet assay studies in peripheral blood leukocytes have reflected that, treatment of G-001M before irradiation has significantly reduced DNA tail length (P < .001) and DNA damage score (P < .001), as compared to radiation-only group. Spleen cell counts in irradiated animals had declined drastically at the very first day of exposure, and the fall continued till the 5th day (P < .001). In the treated irradiated groups, there was a steep reduction in the counts initially, but this phase did not prolong. More than 60% decline in thymocytes of irradiated group animals was registered at 5 h of irradiation when compared with controls, and the fall progressed further downwards with the similar pace till 5th day of exposure (P < .001). At later intervals, thymus was found fully regressed. In G-001M pre-treated irradiated groups also, thymocytes decreased till the 5th day but thereafter rejuvenated and within 30 days of treatment the values were close to normal. Current studies have explicitly indicated that, G-001M in very small doses has not only rendered high survivability in lethally irradiated mice, but also protected their cellular DNA, besides supporting fast replenishment of the immune system. PMID:19553386

  4. Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus.

    PubMed Central

    Silver, J; Keerikatte, V

    1989-01-01

    We describe a modification of the polymerase chain reaction technique which allows amplification of cellular DNA adjacent to an integrated provirus given sequence information for the provirus only. The modified technique should be generally useful for studies of insertional mutagenesis and other situations in which one wishes to isolate DNA adjacent to a region of known sequence. Images PMID:2704070

  5. Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells

    PubMed Central

    Kong, Xiangduo; Mohanty, Samarendra K.; Stephens, Jared; Heale, Jason T.; Gomez-Godinez, Veronica; Shi, Linda Z.; Kim, Jong-Soo; Yokomori, Kyoko; Berns, Michael W.

    2009-01-01

    Proper recognition and repair of DNA damage is critical for the cell to protect its genomic integrity. Laser microirradiation ranging in wavelength from ultraviolet A (UVA) to near-infrared (NIR) can be used to induce damage in a defined region in the cell nucleus, representing an innovative technology to effectively analyze the in vivo DNA double-strand break (DSB) damage recognition process in mammalian cells. However, the damage-inducing characteristics of the different laser systems have not been fully investigated. Here we compare the nanosecond nitrogen 337 nm UVA laser with and without bromodeoxyuridine (BrdU), the nanosecond and picosecond 532 nm green second-harmonic Nd:YAG, and the femtosecond NIR 800 nm Ti:sapphire laser with regard to the type(s) of damage and corresponding cellular responses. Crosslinking damage (without significant nucleotide excision repair factor recruitment) and single-strand breaks (with corresponding repair factor recruitment) were common among all three wavelengths. Interestingly, UVA without BrdU uniquely produced base damage and aberrant DSB responses. Furthermore, the total energy required for the threshold H2AX phosphorylation induction was found to vary between the individual laser systems. The results indicate the involvement of different damage mechanisms dictated by wavelength and pulse duration. The advantages and disadvantages of each system are discussed. PMID:19357094

  6. Smallpox DNA Vaccine Protects Nonhuman Primates Against Lethal Monkeypox

    DTIC Science & Technology

    2004-05-01

    PCR. Monkeys were challenged with MPOV on day 0. †, fatality . VOL. 78, 2004 SMALLPOX DNA VACCINE AGAINST LETHAL MONKEYPOX 4441 FIG. 5. Antibody...01 MAY 2004 2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Smallpox DNA vaccine protects nonhuman primates against lethal ...vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals

  7. Fluorescent cyanine probe for DNA detection and cellular imaging

    NASA Astrophysics Data System (ADS)

    Zheng, Yong-Chao; Zheng, Mei-Ling; Zhao, Zhen-Sheng; Duan, Xuan-Ming

    2014-03-01

    In our study, two carbazole-based cyanines, 3,6-bis[2-(1-methylpyridinium)vinyl]-9-methyl carbazole diiodide (A) and 6,6'-bis[2-(1-methylpyridinium)vinyl]-bis(9-methyl-carbazol-3yl)methane diiodide (B) were synthesized and employed as light-up probes for DNA and cell imaging. Both of the cyanine probes possess a symmetric structure and bis-cationic center. The obvious induced circular dichroism signals in circular dichroism spectra reveal that the molecules can specifically interact with DNA. Strong fluorescence enhancement is observed when these two cyanines are bound to DNA. These cyanine probes show high binding affinity to oligonucleotides but different binding preferences to various secondary structures. Confocal microscopy images of fixed cell stained by the probes exhibit strong brightness and high contrast in nucleus with a very low cytoplasmic background.

  8. Nucleosome remodelling, DNA repair and transcriptional regulation build negative feedback loops in cancer and cellular ageing.

    PubMed

    Watanabe, Reiko; Kanno, Shin-Ichiro; Mohammadi Roushandeh, Amaneh; Ui, Ayako; Yasui, Akira

    2017-10-05

    Nucleosome remodelling (NR) regulates transcription in an ATP-dependent manner, and influences gene expression required for development and cellular functions, including those involved in anti-cancer and anti-ageing processes. ATP-utilizing chromatin assembly and remodelling factor (ACF) and Brahma-associated factor (BAF) complexes, belonging to the ISWI and SWI/SNF families, respectively, are involved in various types of DNA repair. Suppression of several BAF factors makes U2OS cells significantly sensitive to X-rays, UV and especially to cisplatin, and these BAF factors contribute to the accumulation of repair proteins at various types of DNA damage and to DNA repair. Recent cancer genome sequencing and expression analysis has shown that BAF factors are frequently mutated or, more frequently, silenced in various types of cancer cells. Thus, those cancer cells are potentially X-ray- and especially cisplatin-sensitive, suggesting a way of optimizing current cancer therapy. Recent single-stem cell analysis suggests that mutations and epigenetic changes influence stem cell functionality leading to cellular ageing. Genetic and epigenetic changes in the BAF factors diminish DNA repair as well as transcriptional regulation activities, and DNA repair defects in turn negatively influence NR and transcriptional regulation. Thus, they build negative feedback loops, which accelerate both cellular senescence and transformation as common and rare cellular events, respectively, causing cellular ageing.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

  9. Baseline Cellular HIV DNA Load Predicts HIV DNA Decline and Residual HIV Plasma Levels during Effective Antiretroviral Therapy

    PubMed Central

    Andreis, Samantha; Mengoli, Carlo; Scaggiante, Renzo; Ferretto, Roberto; Manfrin, Vinicio; Cruciani, Mario; Giobbia, Mario; Boldrin, Caterina; Basso, Monica; Andreoni, Massimo; Palù, Giorgio; Sarmati, Loredana

    2012-01-01

    Cellular human immunodeficiency virus type 1 (HIV-1) DNA may be considered a marker of disease progression with significant predictive power, but published data on its correlation with plasma HIV RNA levels and CD4 counts in acute and chronic patients are not conclusive. We evaluated a cohort of 180 patients naïve for antiretroviral therapy before the beginning of treatment and after a virological response in order to define the indicators correlated with HIV DNA load decrease until undetectability. The following variables were evaluated as continuous variables: age, CD4 cell count and log10 HIV DNA level at baseline and follow-up, and baseline log10 HIV RNA level. Primary HIV infection at the start of therapy, an HIV RNA level at follow-up of <2.5 copies/ml, origin, gender, and transmission risk were evaluated as binary variables. The decline of HIV DNA values during effective therapy was directly related to baseline HIV DNA and HIV RNA values, to an increase in the number of CD4 cells, and to the achievement of an HIV RNA load of <2.5 copies/ml. An undetectable cellular HIV DNA load was achieved by 21.6% of patients at the follow-up time point and correlated significantly with lower baseline cellular HIV DNA values and with being in the primary stage of infection when therapy started. In conclusion, early treatment facilitated the achievement of undetectable levels of plasma viremia and cellular HIV DNA and a better recovery of CD4 lymphocytes. HIV DNA levels before and during highly active antiretroviral therapy may be used as a new tool for monitoring treatment efficacy. PMID:22135262

  10. Radiation protection by diethyldithiocarbamate: protection of membrane and DNA in vitro and in vivo against gamma-radiation.

    PubMed

    Gandhi, Nitin Motilal; Nair, Cherupally Krishnan Krishnan

    2004-06-01

    Diethyldithiocarbamate (DDTC) is studied for its antioxidant and radioprotective abilities. DDTC at a concentration of 0.5 mM reduced DPPH radical. DDTC reduced the damage to deoxyribose resulting from hydroxyl radicals generated by Fenton reaction, indicating that the radioprotective abilities of this compound could be due to the free radical scavenging. DDTC protected rat liver microsomal membranes in vitro from peroxidative damage in lipids (measured as TBARS) resulting from 50 Gy gamma-radiation. It also protected plasmid pBR322 DNA from radiation-induced strand breaks. An oral administration of DDTC to mice before whole body gamma-radiation exposure (4 Gy) resulted in a reduction of radiation-induced lipid peroxides in the liver homogenates. An administration of DDTC to mice before gamma-radiation reduced the radiation-induced DNA damage as studied by single cell gel-electrophoresis (comet assay). The comet parameters such as tail length, tail moment, and percent of DNA in tail were found to increase in the blood leukocytes of mice exposed to 4 Gy gamma-radiation. When DDTC was administered to mice before the radiation exposure, the increase in the comet parameters as a result of radiation was prevented, indicating a protection of cellular DNA. The present study has implication for the potential use of DDTC as a radioprotector.

  11. Noncatalytic, N-terminal Domains of DNA Polymerase Lambda Affect Its Cellular Localization and DNA Damage Response.

    PubMed

    Stephenson, Anthony A; Taggart, David J; Suo, Zucai

    2017-04-13

    Specialized DNA polymerases, such as DNA polymerase lambda (Polλ), are important players in DNA damage tolerance and repair pathways. Knowing how DNA polymerases are regulated and recruited to sites of DNA damage is imperative to understanding these pathways. Recent work has suggested that Polλ plays a role in several distinct DNA damage tolerance and repair pathways. In this paper, we report previously unknown roles of the N-terminal domains of human Polλ for modulating its involvement in DNA damage tolerance and repair. By using Western blot analysis, fluorescence microscopy, and cell survival assays, we found that the BRCA1 C-terminal (BRCT) and proline/serine-rich (PSR) domains of Polλ affect its cellular localization and DNA damage responses. The nuclear localization signal (NLS) of Polλ was necessary to overcome the impediment of its nuclear localization caused by its BRCT and PSR domains. Induction of DNA damage resulted in recruitment of Polλ to chromatin, which was controlled by its BRCT and PSR domains. In addition, the presence of both domains was required for Polλ-mediated tolerance of oxidative DNA damage but not DNA methylation damage. These findings suggest that the N-terminal domains of Polλ are important for regulating its responses to DNA damage.

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

    PubMed

    Syldona, Maria

    2007-01-01

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

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

    SciTech Connect

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

    2007-09-01

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

  14. Cellular delivery of enzyme-loaded DNA origami.

    PubMed

    Ora, Ari; Järvihaavisto, Erika; Zhang, Hongbo; Auvinen, Henni; Santos, Hélder A; Kostiainen, Mauri A; Linko, Veikko

    2016-12-01

    In this communication, we show that active enzymes can be delivered into HEK293 cells in vitro when they are attached to tubular DNA origami nanostructures. We use bioluminescent enzymes as a cargo and monitor their activity from a cell lysate. The results show that the enzymes stay intact and retain their activity in the transfection process. The method is highly modular, which makes it a compelling candidate for a great variety of delivery applications.

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

    PubMed

    Taylor, Duncan

    2016-09-01

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

  16. Insertion of Foreign DNA into an Established Mammalian Genome Can Alter the Methylation of Cellular DNA Sequences†

    PubMed Central

    Remus, Ralph; Kämmer, Christina; Heller, Hilde; Schmitz, Birgit; Schell, Gudrun; Doerfler, Walter

    1999-01-01

    The insertion of adenovirus type 12 (Ad12) DNA into the hamster genome and the transformation of these cells by Ad12 can lead to marked alterations in the levels of DNA methylation in several cellular genes and DNA segments. Since such alterations in DNA methylation patterns are likely to affect the transcription patterns of cellular genes, it is conceivable that these changes have played a role in the generation or the maintenance of the Ad12-transformed phenotype. We have now isolated clonal BHK21 hamster cell lines that carry in their genomes bacteriophage λ and plasmid pSV2neo DNAs in an integrated state. Most of these cell lines contain one or multiple copies of integrated λ DNA, which often colocalize with the pSV2neo DNA, usually in a single chromosomal site as determined by the fluorescent in situ hybridization technique. In different cell lines, the loci of foreign DNA insertion are different. The inserted bacteriophage λ DNA frequently becomes de novo methylated. In some of the thus-generated hamster cell lines, the levels of DNA methylation in the retrotransposon genomes of the endogenous intracisternal A particles (IAP) are increased in comparison to those in the non-λ-DNA-transgenic BHK21 cell lines. These changes in the methylation patterns of the IAP subclone I (IAPI) segment have been documented by restriction analyses with methylation-sensitive restriction endonucleases followed by Southern transfer hybridization and phosphorimager quantitation. The results of genomic sequencing experiments using the bisulfite protocol yielded additional evidence for alterations in the patterns of DNA methylation in selected segments of the IAPI sequences. In these experiments, the nucleotide sequences in >330 PCR-generated cloned DNA molecules were determined. Upon prolonged cultivation of cell lines with altered cellular methylation patterns, these differences became less apparent, perhaps due to counterselection of the transgenic cells. The possibility

  17. Cellular prion protein protects from inflammatory and neuropathic pain.

    PubMed

    Gadotti, Vinicius M; Zamponi, Gerald W

    2011-08-16

    Cellular prion protein (PrPC) inhibits N-Methyl-D-Aspartate (NMDA) receptors. Since NMDA receptors play an important role in the transmission of pain signals in the dorsal horn of spinal cord, we thus wanted to determine if PrPC null mice show a reduced threshold for various pain behaviours.We compared nociceptive thresholds between wild type and PrPC null mice in models of inflammatory and neuropathic pain, in the presence and the absence of a NMDA receptor antagonist. 2-3 months old male PrPC null mice exhibited an MK-801 sensitive decrease in the paw withdrawal threshold in response both mechanical and thermal stimuli. PrPC null mice also exhibited significantly longer licking/biting time during both the first and second phases of formalin-induced inflammation of the paw, which was again prevented by treatment of the mice with MK-801, and responded more strongly to glutamate injection into the paw. Compared to wild type animals, PrPC null mice also exhibited a significantly greater nociceptive response (licking/biting) after intrathecal injection of NMDA. Sciatic nerve ligation resulted in MK-801 sensitive neuropathic pain in wild-type mice, but did not further augment the basal increase in pain behaviour observed in the null mice, suggesting that mice lacking PrPC may already be in a state of tonic central sensitization. Altogether, our data indicate that PrPC exerts a critical role in modulating nociceptive transmission at the spinal cord level, and fit with the concept of NMDA receptor hyperfunction in the absence of PrPC.

  18. Analysis of Cellular DNA Content by Flow and Laser Scanning Cytometry

    PubMed Central

    Darzynkiewicz, Zbigniew; Halicka, H. Dorota; Zhao, Hong

    2010-01-01

    This chapter covers several aspects of methodology of DNA content analysis in individual cells that is most commonly used for assessment of DNA ploidy and for enumeration of cells in particular phases of the cell cycle. Briefly presented are general principles of instrumentation and cell analysis by flow- and laser scanning- cytometry. Described are major methods designed to stain DNA with fluorochromes in live cells, in detergent-permeabilized cells, in cells fixed prior to DNA staining as well as in nuclei of cells isolated from paraffin-embedded tissues. Briefly addressed are approaches to estimate cellular DNA content in conjunction with cellular immunophenotype. Discussed are factors that affect accuracy of DNA content measurement such as: (i) differences in chromatin structure of the analyzed cells that restrict DNA accessibility to fluorochromes, (ii) stoichiometry of interaction between fluorochromes and DNA in chromatin and (iii) chemical mass action law defining dependency of fluorochrome binding to DNA in relation to fluorochrome concentration and number of potential binding sites in a sample. Described also are controls used to ensure accuracy of DNA ploidy determination, the principles in ploidy assessment and possible pitfalls in analysis. PMID:20687474

  19. Oral vaccination with a liposome-encapsulated influenza DNA vaccine protects mice against respiratory challenge infection.

    PubMed

    Liu, Jing; Wu, Jianqi; Wang, Bing; Zeng, Sheng; Qi, Feifei; Lu, Changlong; Kimura, Yoshinobu; Liu, Beixing

    2014-05-01

    It is well accepted that vaccination by oral administration has many advantages over injected parenteral immunization. The present study focuses on whether oral vaccination with a DNA vaccine could induce protective immunity against respiratory challenge infection. The M1 gene of influenza A virus was used to construct DNA vaccine using pcDNA 3.1(+) plasmid, a eukaryotic expression vector. The cationic liposomes were used to deliver the constructed DNA vaccine. In vitro and in vivo expression of M1 gene was observed in the cell line and in the intestine of orally vaccinated C57BL/6 mice, respectively. It became clear that this type of oral DNA vaccination was capable of inducing both humoral and cellular immune responses, together with an augmentation of IFN-γ production. In addition, oral vaccination with liposome-encapsulated DNA vaccine could protect the mice against respiratory challenge infection. These results suggest that gastrointestinal tract, a constituent member of the common mucosal immune system, is a potent candidate applicable as a DNA vaccine route against virus respiratory diseases.

  20. Parvovirus B19 Nonstructural Protein-Induced Damage of Cellular DNA and Resultant Apoptosis

    PubMed Central

    Poole, Brian D.; Kivovich, Violetta; Gilbert, Leona; Naides, Stanley J.

    2011-01-01

    Parvovirus B19 is a widespread virus with diverse clinical presentations. The viral nonstructural protein, NS1, binds to and cleaves the viral genome, and induces apoptosis when transfected into nonpermissive cells, such as hepatocytes. We hypothesized that the cytotoxicity of NS1 in such cells results from chromosomal DNA damage caused by the DNA-nicking and DNA-attaching activities of NS1. Upon testing this hypothesis, we found that NS1 covalently binds to cellular DNA and is modified by PARP, an enzyme involved in repairing single-stranded DNA nicks. We furthermore discovered that the DNA nick repair pathway initiated by poly(ADPribose)polymerase and the DNA repair pathways initiated by ATM/ATR are necessary for efficient apoptosis resulting from NS1 expression. PMID:21278893

  1. Rearrangements of DNA-protein interactions in animal cells coupled with cellular growth-quiescence transitions.

    PubMed Central

    Lichtenstein, A V; Sjakste, N I; Zaboykin, M M; Shapot, V S

    1982-01-01

    Overall DNA-protein interactions in animal cells undergo drastic changes coupled with cellular transitions from quiescence to growth and reversely as revealed by nucleoprotein-Celite chromatography. DNA of chromatin was found to exist in one of the two sharply distinct alternative forms, namely, either tightly or weakly bound to protein moiety. These forms are specific for cycling and quiescent cells, respectively. The tight DNA-protein interactions characterize all cycling cells independent of the cell cycle phase. Transition of DNA of cycling cells from one form to another was observed as a result of treatment of isolated nuclei with DNase I. PMID:7063419

  2. An Overview of Chemical Processes That Damage Cellular DNA: Spontaneous Hydrolysis, Alkylation, and Reactions with Radicals

    PubMed Central

    Gates, Kent S.

    2009-01-01

    The sequence of heterocyclic bases on the interior of the DNA double helix constitutes the genetic code that drives the operation of all living organisms. With this said, it is not surprising that chemical modification of cellular DNA can have profound biological consequences. Therefore, the organic chemistry of DNA damage is fundamentally important to diverse fields including medicinal chemistry, toxicology, and biotechnology. This review is designed to provide a brief overview of the common types of chemical reactions that lead to DNA damage under physiological conditions. PMID:19757819

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

    PubMed Central

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

    2014-01-01

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

  4. Evaluating the immunogenicity and protective efficacy of a DNA vaccine encoding Lassa virus nucleoprotein.

    PubMed

    Rodriguez-Carreno, Maria P; Nelson, Michael S; Botten, Jason; Smith-Nixon, Kim; Buchmeier, Michael J; Whitton, J Lindsay

    2005-04-25

    Several viruses in the Arenavirus genus of the family Arenaviridae cause severe, often fatal, hemorrhagic fever. One such virus, Lassa virus (LV), is a frequent cause of disease in Africa, and survivors often are left with substantial neurological impairment. The feasibility of protective immunization against LV infection, and the associated disease, has been demonstrated in animal models, using recombinant vaccinia viruses to deliver Lassa proteins. Circumstantial evidence implicates cellular immunity in this Lassa-induced protection, but this has not been confirmed. Here, we describe DNA vaccines that encode LV proteins. A single inoculation of a plasmid encoding full-length Lassa nucleoprotein (LNP) can induce CD8(+) T cell responses in mice and can protect against challenge with two arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV). A DNA minigene vaccine encoding a 9 amino acid sequence from LNP also induces CD8(+) T cells and protects against arenavirus challenge, thus confirming prior speculation that protective cellular immunity is induced by LV proteins.

  5. Unreserved application of epigenetic methods to define differences of DNA methylation between urinary cellular and cell-free DNA.

    PubMed

    Ghanjati, Foued; Beermann, Agnes; Hermanns, Thomas; Poyet, Cedric; Araúzo-Bravo, Marcos J; Seifert, Hans-Helge; Schmidtpeter, Martin; Goering, Wolfgang; Sorg, Rüdiger; Wernet, Peter; Santourlidis, Simeon

    2014-01-01

    Urinary DNA is increasingly gaining importance in diagnosis of urological malignancies. Especially cell-free DNA originating from apoptotic and necrotic cells of the early tumor could become a key target for early stage tumor diagnosis. Aberrant DNA methylation forms tumor cell characteristic epigenetic profiles which are covalently established before any tumor related aberration at transcriptional or protein level has occurred. In addition, these epigenetic signatures are alterably adapted to and accompanying the individual stages of multistep, progressive tumorigenesis. Hence, they seem very promising for diagnosis as well as for monitoring the patient's follow-up care and even for decisions regarding personalized therapeutic options. The essential prerequisite at this approach will be a reliable methodological handling of the biological material of interest. In this study we present detailed analyses of LINE-1 DNA methylation profiles and demonstrate the sensitive detection of LINE-1 DNA methylation differences as well as between cancer patients and healthy individuals, between urinary cellular and cell-free DNA. In addition, we show methylome differences between both DNA fractions from a healthy individual and bladder cancer patients. In conclusion, we demonstrate here the unrestricted amenability of urinary cell-free DNA for both, a detailed characterization of a distinct DNA methylation alteration and its sensitive detection and a comprehensive global, array-based screening for DNA methylation differences.

  6. Mechanisms of cellular penetration and nuclear localization of an anti-double strand DNA autoantibody.

    PubMed

    Zack, D J; Stempniak, M; Wong, A L; Taylor, C; Weisbart, R H

    1996-09-01

    An anti-dsDNA Ab, mAb 3E10, was identified that bound membranes of fixed human renal tubular cells, penetrated live murine renal tubular cells in vivo, and localized in the cell nucleus. mAb 3E10 binds both dsDNA and an extracellular matrix protein, HP8/HEVIN, expressed in high endothelial venules. Previous studies showed both shared and distinct binding determinants of mAb 3E10 VH for DNA and HP8/HEVIN. To independently assess the requirement of DNA and HP8/HEVIN in cellular penetration, site-directed mutants of mAb 3E10 VH and V kappa were studied for penetrating kidney cell lines. The results showed that residues required for binding DNA, but not HP8/HEVIN, were necessary for Ab penetration, indicating that cellular penetration required the presence of DNA or binding of Ab to a membrane determinant precisely resembling DNA. Ab Fab penetrated cells, indicating that neither the Fc nor multivalent Ab binding is necessary for cellular penetration. Ab synthesized in the cytoplasm as a result of deleting heavy and light chain signal peptides was not translocated to the nucleus, indicating a mechanism distinct from the usual protein nuclear localization signals and suggesting the need for a membrane-mediated pathway or for post-translational modification of the Ab.

  7. Homozygous mutation of MTPAP causes cellular radiosensitivity and persistent DNA double-strand breaks

    PubMed Central

    Martin, N T; Nakamura, K; Paila, U; Woo, J; Brown, C; Wright, J A; Teraoka, S N; Haghayegh, S; McCurdy, D; Schneider, M; Hu, H; Quinlan, A R; Gatti, R A; Concannon, P

    2014-01-01

    The study of rare human syndromes characterized by radiosensitivity has been instrumental in identifying novel proteins and pathways involved in DNA damage responses to ionizing radiation. In the present study, a mutation in mitochondrial poly-A-polymerase (MTPAP), not previously recognized for its role in the DNA damage response, was identified by exome sequencing and subsequently associated with cellular radiosensitivity. Cell lines derived from two patients with the homozygous MTPAP missense mutation were radiosensitive, and this radiosensitivity could be abrogated by transfection of wild-type mtPAP cDNA into mtPAP-deficient cell lines. Further analysis of the cellular phenotype revealed delayed DNA repair, increased levels of DNA double-strand breaks, increased reactive oxygen species (ROS), and increased cell death after irradiation (IR). Pre-IR treatment of cells with the potent anti-oxidants, α-lipoic acid and n-acetylcysteine, was sufficient to abrogate the DNA repair and clonogenic survival defects. Our results firmly establish that mutation of the MTPAP gene results in a cellular phenotype of increased DNA damage, reduced repair kinetics, increased cell death by apoptosis, and reduced clonogenic survival after exposure to ionizing radiation, suggesting a pathogenesis that involves the disruption of ROS homeostasis. PMID:24651433

  8. DNA nanoflowers for multiplexed cellular imaging and traceable targeted drug delivery.

    PubMed

    Hu, Rong; Zhang, Xiaobing; Zhao, Zilong; Zhu, Guizhi; Chen, Tao; Fu, Ting; Tan, Weihong

    2014-06-02

    We present a facile approach to make aptamer-conjugated FRET (fluorescent resonance energy transfer) nanoflowers (NFs) through rolling circle replication for multiplexed cellular imaging and traceable targeted drug delivery. The NFs can exhibit multi-fluorescence emissions by a single-wavelength excitation as a result of the DNA matrix covalently incorporated with three dye molecules able to perform FRET. Compared with the conventional DNA nanostructure assembly, NF assembly is independent of template sequences, avoiding the otherwise complicated design of DNA building blocks assembled into nanostructures by base-pairing. The NFs were uniform and exhibited high fluorescence intensity and excellent photostability. Combined with the ability of traceable targeted drug delivery, these colorful DNA NFs provide a novel system for applications in multiplex fluorescent cellular imaging, effective screening of drugs, and therapeutic protocol development. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed Central

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

    2013-01-01

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

  10. Cellular interactions of doxorubicin-loaded DNA-modified halloysite nanotubes

    NASA Astrophysics Data System (ADS)

    Lee, Yeonju; Jung, Goo-Eun; Cho, Sang Joon; Geckeler, Kurt E.; Fuchs, Harald

    2013-08-01

    Halloysite nanotube (HNT)-based supramolecular complexes are synthesized and evaluated with respect to their cytotoxicity and effects on cellular structures. As HNTs are water-insoluble, DNA is applied for wrapping the surface of HNTs to enhance their water-dispersibility. To investigate the potential of DNA-wrapped HNTs (HD) as a promising drug delivery carrier, doxorubicin (DOX) is introduced as a model anticancer agent and loaded onto HD. The DOX-loaded, DNA-wrapped HNTs (HDD) show sustained DOX release over two weeks without initial burst of DOX indicating delayed DOX release inside cells. In addition, effects of DNA-wrapped HNTs (HD) or HDD on the cytoskeleton organization of A549 cells are studied by visualizing the distribution of F-actin filaments using confocal laser scanning microscopy, and cellular morphological changes are observed by scanning electron microscopy and scanning ion conductance microscopy.Halloysite nanotube (HNT)-based supramolecular complexes are synthesized and evaluated with respect to their cytotoxicity and effects on cellular structures. As HNTs are water-insoluble, DNA is applied for wrapping the surface of HNTs to enhance their water-dispersibility. To investigate the potential of DNA-wrapped HNTs (HD) as a promising drug delivery carrier, doxorubicin (DOX) is introduced as a model anticancer agent and loaded onto HD. The DOX-loaded, DNA-wrapped HNTs (HDD) show sustained DOX release over two weeks without initial burst of DOX indicating delayed DOX release inside cells. In addition, effects of DNA-wrapped HNTs (HD) or HDD on the cytoskeleton organization of A549 cells are studied by visualizing the distribution of F-actin filaments using confocal laser scanning microscopy, and cellular morphological changes are observed by scanning electron microscopy and scanning ion conductance microscopy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr02665e

  11. Irreparable telomeric DNA damage and persistent DDR signalling as a shared causative mechanism of cellular senescence and ageing.

    PubMed

    Rossiello, Francesca; Herbig, Utz; Longhese, Maria Pia; Fumagalli, Marzia; d'Adda di Fagagna, Fabrizio

    2014-06-01

    The DNA damage response (DDR) orchestrates DNA repair and halts cell cycle. If damage is not resolved, cells can enter into an irreversible state of proliferative arrest called cellular senescence. Organismal ageing in mammals is associated with accumulation of markers of cellular senescence and DDR persistence at telomeres. Since the vast majority of the cells in mammals are non-proliferating, how do they age? Are telomeres involved? Also oncogene activation causes cellular senescence due to altered DNA replication and DDR activation in particular at the telomeres. Is there a common mechanism shared among apparently distinct types of cellular senescence? And what is the role of telomeric DNA damage?

  12. Amphiphilic Block Copolymers Enhance Cellular Uptake and Nuclear Entry of Polyplex-Delivered DNA

    PubMed Central

    Yang, Zhihui; Sahay, Gaurav; Sriadibhatla, Srikanth; Kabanov, Alexander V.

    2008-01-01

    This work for the first time demonstrates that synthetic polymers enhance uptake and nuclear import of plasmid DNA (pDNA) through the activation of cellular trafficking machinery. Nonionic block copolymers of poly(ethylene oxide) and poly(propylene oxide), Pluronics, are widely used as excipients in pharmaceutics. We previously demonstrated that Pluronics increase the phosphorylation of IκB and subsequent NFκB nuclear localization as well as upregulate numerous NFκB-related genes. In this study, we show that Pluronics enhance gene transfer by pDNA/polycation complexes (“polyplexes”) in a promoter-dependent fashion. Addition of Pluronic P123 or P85 to polyethyleneimine-based polyplexes had little effect on polyplex particle size but significantly enhanced pDNA cellular uptake, nuclear translocation and gene expression in several cell lines. When added to polyplex-transfected cells after transfection, Pluronics enhanced nuclear import of pDNA containing NFκB–binding sites, but have no effect on import of pDNA without these sites. All together, our studies suggest that Pluronics rapidly activate NFκB, which binds cytosolic pDNA that possesses promoters containing NFκB binding sites and consequently increases nuclear import of pDNA through NFκB nuclear translocation. PMID:18729495

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

    PubMed Central

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

    2012-01-01

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

  14. Inducing humoral and cellular responses to multiple sporozoite and liver-stage malaria antigens using exogenous plasmid DNA.

    PubMed

    Ferraro, B; Talbott, K T; Balakrishnan, A; Cisper, N; Morrow, M P; Hutnick, N A; Myles, D J; Shedlock, D J; Obeng-Adjei, N; Yan, J; Kayatani, A K K; Richie, N; Cabrera, W; Shiver, R; Khan, A S; Brown, A S; Yang, M; Wille-Reece, U; Birkett, A J; Sardesai, N Y; Weiner, D B

    2013-10-01

    A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4(+) and CD8(+) T cell compartments. Furthermore, hepatic CD8(+) lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8(+) granzyme B(+) T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.

  15. Depurinating acylfulvene-DNA adducts: characterizing cellular chemical reactions of a selective antitumor agent.

    PubMed

    Gong, Jiachang; Vaidyanathan, V G; Yu, Xiang; Kensler, Thomas W; Peterson, Lisa A; Sturla, Shana J

    2007-02-21

    Acylfulvenes (AFs) are a class of semisynthetic agents with high toxicity toward certain tumor cells, and for one analogue, hydroxymethylacylfulvene (HMAF), clinical trials are in progress. DNA alkylation by AFs, mediated by bioreductive activation, is believed to contribute to cytotoxicity, but the structures and chemical properties of corresponding DNA adducts are unknown. This study provides the first structural characterization of AF-specific DNA adducts. In the presence of a reductive enzyme, alkenal/one oxidoreductase (AOR), AF selectively alkylates dAdo and dGuo in reactions with a monomeric nucleoside, as well as in reactions with naked or cellular DNA, with 3-alkyl-dAdo as the apparently most abundant AF-DNA adduct. Characterization of this adduct was facilitated by independent chemical synthesis of the corresponding 3-alkyl-Ade adduct. In addition, in naked or cellular DNA, evidence was obtained for the formation of an additional type of adduct resulting from direct conjugate addition of Ade to AF followed by hydrolytic cyclopropane ring-opening, indicating the potential for a competing reaction pathway involving direct DNA alkylation. The major AF-dAdo and AF-dGuo adducts are unstable under physiologically relevant conditions and depurinate to release an alkylated nucleobase in a process that has a half-life of 8.5 h for 3-alkyladenine and less than approximately 2 h for dGuo adducts. DNA alkylation further leads to single-stranded DNA cleavage, occurring exclusively at dGuo and dAdo sites, in a nonsequence-specific manner. In AF-treated cells that were transfected with either AOR or control vectors, the DNA adducts identified match those from in vitro studies. Moreover, a positive correlation was observed between DNA adduct levels and cell sensitivity to AF. The potential contributing roles of AOR-mediated bioactivation and adduct stability to the cytotoxicity of AF are discussed.

  16. Role of Humoral versus Cellular Responses Induced by a Protective Dengue Vaccine Candidate

    PubMed Central

    Zellweger, Raphaël M.; Miller, Robyn; Eddy, William E.; White, Laura J.; Johnston, Robert E.; Shresta, Sujan

    2013-01-01

    With 2.5 billion people at risk, dengue is a major emerging disease threat and an escalating public health problem worldwide. Dengue virus causes disease ranging from a self-limiting febrile illness (dengue fever) to the potentially fatal dengue hemorrhagic fever/dengue shock syndrome. Severe dengue disease is associated with sub-protective levels of antibody, which exacerbate disease upon re-infection. A dengue vaccine should generate protective immunity without increasing severity of disease. To date, the determinants of vaccine-mediated protection against dengue remain unclear, and additional correlates of protection are urgently needed. Here, mice were immunized with viral replicon particles expressing the dengue envelope protein ectodomain to assess the relative contribution of humoral versus cellular immunity to protection. Vaccination with viral replicon particles provided robust protection against dengue challenge. Vaccine-induced humoral responses had the potential to either protect from or exacerbate dengue disease upon challenge, whereas cellular immune responses were beneficial. This study explores the immunological basis of protection induced by a dengue vaccine and suggests that a safe and efficient vaccine against dengue should trigger both arms of the immune system. PMID:24204271

  17. Isolation of Circular DNA Molecules from Whole Cellular DNA by Use of ATP-Dependent Deoxyribonuclease

    PubMed Central

    Mukai, Tsunehiro; Matsubara, Kenichi; Takagi, Yasuyuki

    1973-01-01

    A technique is described for isolation of plasmid DNA in closed and open circular double-stranded forms from bacterial cells, by use of ATP-dependent deoxyribonuclease purified from Micrococcus luteus. This DNase, acting only upon linear DNA molecules, degrades all bacterial chromosomal DNA extracted in the linear form. Circular plasmid DNAs are left intact, and are then separated by sedimentation through a sucrose gradient. Unlike previous techniques for analysis of plasmid DNA, this technique can be used to isolate not only closed circular DNA but also open circular DNA. Several plasmids, such as those from phage (λdv1 and λdv21), a colicinogenic factor (Col E2), a sex factor (F8′ gal), and “minicircles” in Escherichia coli 15, in both the open and closed circular forms, were well separated from chromosomal DNA by this technique. PMID:4355370

  18. Molecular basis for protection of ribosomal protein L4 from cellular degradation

    PubMed Central

    Huber, Ferdinand M.; Hoelz, André

    2017-01-01

    Eukaryotic ribosome biogenesis requires the nuclear import of ∼80 nascent ribosomal proteins and the elimination of excess amounts by the cellular degradation machinery. Assembly chaperones recognize nascent unassembled ribosomal proteins and transport them together with karyopherins to their nuclear destination. We report the crystal structure of ribosomal protein L4 (RpL4) bound to its dedicated assembly chaperone of L4 (Acl4), revealing extensive interactions sequestering 70 exposed residues of the extended RpL4 loop. The observed molecular recognition fundamentally differs from canonical promiscuous chaperone–substrate interactions. We demonstrate that the eukaryote-specific RpL4 extension harbours overlapping binding sites for Acl4 and the nuclear transport factor Kap104, facilitating its continuous protection from the cellular degradation machinery. Thus, Acl4 serves a dual function to facilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery. PMID:28148929

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

    PubMed Central

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

    2016-01-01

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

  20. Cysteamine protects gastric epithelial cell monolayers against drug induced damage: evidence for direct cellular protection by sulphydryl compounds.

    PubMed Central

    Romano, M; Razandi, M; Raza, A; Szabo, S; Ivey, J

    1992-01-01

    The sulphydryl containing drug cysteamine protects gastric mucosa in vivo against acute injury. It is not known whether this protection includes a direct effect on gastric cells. Using gastric epithelial cell monolayers derived from a well differentiated human cell line, we evaluated whether cysteamine protects against taurocholate or indomethacin induced damage in conditions which completely exclude the influence of vascular, hormonal, and neural factors. The effect of cysteamine on prostaglandin production by monolayer cells in vitro was also assessed. Cysteamine decreased damage brought about by sodium taurocholate and indomethacin by 40% (p less than 0.01) and 50% (p less than 0.01) respectively. The sulphydryl blocker iodoacetamide prevented the protective effect of cysteamine. Pretreatment with indomethacin, which inhibited prostaglandin E2 output by 60%, did not prevent protection by cysteamine; incubation with cysteamine decreased prostaglandin E2 production by cultured cells. We conclude that (i) cysteamine directly protected gastric epithelial cells in vitro (ii) this protection occurred with indomethacin, which interferes with cellular metabolism of prostaglandins, and taurocholate, whose damaging action at neutral pH is unrelated to interference with prostanoid metabolism, (iii) cysteamine protection in vitro is unrelated to endogenous prostaglandins and is probably mediated by endogenous sulphydryl compounds. Images Figure 1A-1B Figure 1C-1E PMID:1740273

  1. Hyperphosphorylation results in tau dysfunction in DNA folding and protection.

    PubMed

    Lu, Yang; He, Hai-Jin; Zhou, Jun; Miao, Jun-Ye; Lu, Jing; He, Ying-Ge; Pan, Rong; Wei, Yan; Liu, Ying; He, Rong-Qiao

    2013-01-01

    Hyperphosphorylation of tau occurs in preclinical and clinical stages of Alzheimer's disease (AD), and hyperphosphorylated tau is the main constituent of the paired helical filaments in the brains of mild cognitive impairment and AD patients. While most of the work described so far focused on the relationship between hyperphosphorylation of tau and microtubule disassembly as well as axonal transport impairments, both phenomena ultimately leading to cell death, little work has been done to study the correlation between tau hyperphosphorylation and DNA damage. As we showed in this study, tau hyperphosphorylation and DNA damage co-occurred under formaldehyde treatment in N2a cells, indicating that phosphorylated tau (p-Tau) induced by formaldehyde may be involved in DNA impairment. After phosphorylation, the effect of tau in preventing DNA from thermal denaturation was diminished, its ability to accelerate DNA renaturation was lost, and its function in protecting DNA from reactive oxygen species (ROS) attack was impaired. Thus, p-Tau is not only associated with the disassembly of the microtubule system, but also plays a crucial role in DNA impairment. Hyperphosphorylation-mediated dysfunction of tau protein in prevention of DNA structure from damage under the attack of ROS may provide novel insights into the mechanisms underlying tauopathies.

  2. Free radical scavenging, DNA protection, and inhibition of lipid peroxidation mediated by uric acid.

    PubMed

    Stinefelt, Beth; Leonard, Stephen S; Blemings, Kenneth P; Shi, Xianglin; Klandorf, Hillar

    2005-01-01

    Uric acid (UA) has been proposed to be the dominant antioxidant in birds. The objective of this study was to investigate the quenching effect of varying concentrations of UA, including those found in avian plasma, on specific reactive oxygen species (ROS) and to determine the ability of UA to protect DNA and cellular membranes from ROS-mediated damage. Hydroxyl (OH) and superoxide (O2-) radicals were detected by electron spin resonance (ESR) and their presence was reduced following addition of UA (p <0.05) in a concentration-dependent manner. UA inhibited hydroxyl-mediated DNA damage, indicated by the presence of more precise, dense bands of lambda Hind III DNA after agarose gel electrophoresis and ethidium bromide staining (p <0.05). Lipid peroxidation of silica-exposed RAW 264.7 cell membranes was diminished (p <0.02) after addition of UA to the cell incubation mixture. These studies demonstrate that UA scavenges hydroxyl and superoxide radicals and protects against DNA damage and lipid peroxidation. These results indicate specific antioxidant protection that UA may afford birds against ROS-mediated damage.

  3. DNA protection by ectoine from ionizing radiation: molecular mechanisms.

    PubMed

    Hahn, Marc Benjamin; Meyer, Susann; Schröter, Maria-Astrid; Kunte, Hans-Jörg; Solomun, Tihomir; Sturm, Heinz

    2017-09-27

    Ectoine, a compatible solute and osmolyte, is known to be an effective protectant of biomolecules and whole cells against heating, freezing and extreme salinity. Protection of cells (human keratinocytes) by ectoine against ultraviolet radiation has also been reported by various authors, although the underlying mechanism is not yet understood. We present the first electron irradiation of DNA in a fully aqueous environment in the presence of ectoine and at high salt concentrations. The results demonstrate effective protection of DNA by ectoine against the induction of single-strand breaks by ionizing radiation. The effect is explained by an increase in low-energy electron scattering at the enhanced free-vibrational density of states of water due to ectoine, as well as the use of ectoine as an ˙OH-radical scavenger. This was demonstrated by Raman spectroscopy and electron paramagnetic resonance (EPR).

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

    PubMed Central

    2014-01-01

    DNA damage attacks on bacterial cells have been known to activate the SOS response, a transcriptional response affecting chromosome replication, DNA recombination and repair, cell division and prophage induction. All these functions require double-stranded (ds) DNA translocation by ASCE hexameric motors. This review seeks to delineate the structural and functional characteristics of the SOS response and the SOS-regulated DNA translocases FtsK and RuvB with the phi29 bacteriophage packaging motor gp16 ATPase as a prototype to study bacterial motors. While gp16 ATPase, cellular FtsK and RuvB are similarly comprised of hexameric rings encircling dsDNA and functioning as ATP-driven DNA translocases, they utilize different mechanisms to accomplish separate functions, suggesting a convergent evolution of these motors. The gp16 ATPase and FtsK use a novel revolution mechanism, generating a power stroke between subunits through an entropy-DNA affinity switch and pushing dsDNA inward without rotation of DNA and the motor, whereas RuvB seems to employ a rotation mechanism that remains to be further characterized. While FtsK and RuvB perform essential tasks during the SOS response, their roles may be far more significant as SOS response is involved in antibiotic-inducible bacterial vesiculation and biofilm formation as well as the perspective of the bacteria-cancer evolutionary interaction. PMID:24995125

  5. Quantification of cellular uptake of DNA nanostructures by qPCR.

    PubMed

    Okholm, Anders Hauge; Nielsen, Jesper Sejrup; Vinther, Mathias; Sørensen, Rasmus Schøler; Schaffert, David; Kjems, Jørgen

    2014-05-15

    DNA nanostructures facilitating drug delivery are likely soon to be realized. In the past few decades programmed self-assembly of DNA building blocks have successfully been employed to construct sophisticated nanoscale objects. By conjugating functionalities to DNA, other molecules such as peptides, proteins and polymers can be precisely positioned on DNA nanostructures. This exceptional ability to produce modular nanoscale devices with tunable and controlled behavior has initiated an interest in employing DNA nanostructures for drug delivery. However, to obtain this the relationship between cellular interactions and structural and functional features of the DNA delivery device must be thoroughly investigated. Here, we present a rapid and robust method for the precise quantification of the component materials of DNA origami structures capable of entering cells in vitro. The quantification is performed by quantitative polymerase chain reaction, allowing a linear dynamic range of detection of five orders of magnitude. We demonstrate the use of this method for high-throughput screening, which could prove efficient to identify key features of DNA nanostructures enabling cell penetration. The method described here is suitable for quantification of in vitro uptake studies but should easily be extended to quantify DNA nanostructures in blood or tissue samples.

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

    PubMed Central

    Koonin, Eugene V

    2006-01-01

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

  7. A small molecule inhibitor of PAI-1 protects against doxorubicin-induced cellular senescence

    PubMed Central

    Ghosh, Asish K.; Rai, Rahul; Park, Kitae E.; Eren, Mesut; Miyata, Toshio; Wilsbacher, Lisa D.; Vaughan, Douglas E.

    2016-01-01

    Doxorubicin, an anthracycline antibiotic, is a commonly used anticancer drug. In spite of its widespread usage, its therapeutic effect is limited by its cardiotoxicity. On the cellular level, Doxorubicin-induced cardiotoxicity manifests as stress induced premature senescence. Previously, we demonstrated that plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of serine proteases, is an important biomarker and regulator of cellular senescence and aging. Here, we tested the hypothesis that pharmacological inhibition of cellular PAI-1 protects against stress- and aging-induced cellular senescence and delineated the molecular basis of protective action of PAI-1 inhibition. Results show that TM5441, a potent small molecule inhibitor of PAI-1, effectively prevents Doxorubicin-induced senescence in cardiomyocytes, fibroblasts and endothelial cells. TM5441 exerts its inhibitory effect on Doxorubicin-induced cellular senescence by decreasing reactive oxygen species generation, induction of antioxidants like catalase and suppression of stress-induced senescence cadre p53, p21, p16, PAI-1 and IGFBP3. Importantly, TM5441 also reduces replicative senescence of fibroblasts. Together these results for the first time demonstrate the efficacy of PAI-1 inhibitor in prevention of Doxorubicin-induced and replicative senescence in normal cells. Thus PAI-1 inhibitor may form an important adjuvant component of chemotherapy regimens, limiting not only Doxorubicin-induced cardiac senescence but also ameliorating the prothrombotic profile. PMID:27736799

  8. 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

  9. A human cellular sequence implicated in trk oncogene activation is DNA damage inducible

    SciTech Connect

    Ben-Ishai, R.; Scharf, R.; Sharon, R.; Kapten, I. )

    1990-08-01

    Xeroderma pigmentosum cells, which are deficient in the repair of UV light-induced DNA damage, have been used to clone DNA-damage-inducible transcripts in human cells. The cDNA clone designated pC-5 hybridizes on RNA gel blots to a 1-kilobase transcript, which is moderately abundant in nontreated cells and whose synthesis is enhanced in human cells following UV irradiation or treatment with several other DNA-damaging agents. UV-enhanced transcription of C-5 RNA is transient and occurs at lower fluences and to a greater extent in DNA-repair-deficient than in DNA-repair-proficient cells. Southern blot analysis indicates that the C-5 gene belongs to a multigene family. A cDNA clone containing the complete coding sequence of C-5 was isolated. Sequence analysis revealed that it is homologous to a human cellular sequence encoding the amino-terminal activating sequence of the trk-2h chimeric oncogene. The presence of DNA-damage-responsive sequences at the 5' end of a chimeric oncogene could result in enhanced expression of the oncogene in response to carcinogens.

  10. Correlation of binding efficacies of DNA to flavonoids and their induced cellular damage.

    PubMed

    Das, Asmita; Majumder, Debashis; Saha, Chabita

    2017-05-01

    Flavonoids are dietary intakes which are bestowed with several health benefits. The most studied property of flavonoids is their antioxidant efficacy. Among the chosen flavonoids Quercetin, Kaempferol and Myricetin is catagorized as flavonols whereas Apigenin and Luteolin belong to the flavone group. In the present study anti-cancer properties of flavonoids are investigated on the basis of their binding efficacy to ct-DNA and their ability to induce cytotoxicity in K562 leukaemic cells. The binding affinities of the flavonoids with calf thymus DNA (ct-DNA) are in the order Quercetin>Myricetin>Luteolin>Kaempferol>Apigenin. Quercetin with fewer OH than myricetin has higher affinity towards DNA suggesting that the number and position of OH influence the binding efficacies of flavonoids to ct-DNA. CD spectra and EtBr displacement studies evidence myricetin and apigenin to be stronger intercalators of DNA compared to quercetin. From comet assay results it is observed that quercetin and myricetin when used in combination induce higher DNA damage in K562 leukemic cells than when tested individually. Higher binding efficacy has been recorded for quercetin to DNA at lower pH, which is the micro environment of cancerous cells, and hence quercetin can act as a potential anti-cancer agent. Presence of Cu also increases cellular damage as recorded by comet assay. Copyright © 2017. Published by Elsevier B.V.

  11. Cellular factors required for multiple stages of SV40 DNA replication in vitro.

    PubMed Central

    Fairman, M P; Stillman, B

    1988-01-01

    Plasmids containing the SV40 origin replicate in the presence of SV40 T antigen and a cell free extract derived from human 293 cells. Upon fractionation of this extract, two essential replication factors have been identified. One of these is a multi-subunit DNA binding protein containing polypeptides of 70,000, 34,000 and 11,000 daltons which may function as a eukaryotic single strand DNA binding protein (SSB). The other partially purified fraction is required with T antigen for the first stage of DNA replication, the formation of a pre-synthesis complex at the replication origin. These results, and others, define multiple stages of SV40 DNA replication in vitro which are analogous to multiple stages of Escherichia coli and phage lambda replication, and may reflect similar events in the replication of cellular chromosomes. Images PMID:2841119

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

  14. Sex differences of leukocytes DNA methylation adjusted for estimated cellular proportions.

    PubMed

    Inoshita, Masatoshi; Numata, Shusuke; Tajima, Atsushi; Kinoshita, Makoto; Umehara, Hidehiro; Yamamori, Hidenaga; Hashimoto, Ryota; Imoto, Issei; Ohmori, Tetsuro

    2015-01-01

    DNA methylation, which is most frequently the transference of a methyl group to the 5-carbon position of the cytosine in a CpG dinucleotide, plays an important role in both normal development and diseases. To date, several genome-wide methylome studies have revealed sex-biased DNA methylation, yet no studies have investigated sex differences in DNA methylation by taking into account cellular heterogeneity. The aim of the present study was to investigate sex-biased DNA methylation on the autosomes in human blood by adjusting for estimated cellular proportions because cell-type proportions may vary by sex. We performed a genome-wide DNA methylation profiling of the peripheral leukocytes in two sets of samples, a discovery set (49 males and 44 females) and a replication set (14 males and 10 females) using Infinium HumanMethylation450 BeadChips for 485,764 CpG dinucleotides and then examined the effect of sex on DNA methylation with a multiple linear regression analysis after adjusting for age, the estimated 6 cell-type proportions, and the covariates identified in a surrogate variable analysis. We identified differential DNA methylation between males and females at 292 autosomal CpG site loci in the discovery set (Bonferroni-adjusted p < 0.05). Of these 292 CpG sites, significant sex differences were also observed at 98 sites in the replication set (p < 0.05). These findings provided further evidence that DNA methylation may play a role in the differentiation or maintenance of sexual dimorphisms. Our methylome mapping of the effects of sex may be useful to understanding the molecular mechanism involved in both normal development and diseases.

  15. One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA

    PubMed Central

    Cadet, Jean; Wagner, J. Richard; Shafirovich, Vladimir; Geacintov, Nicholas E.

    2014-01-01

    Purpose The aim of this survey is to critically review the available information on one-electron oxidation reactions of nucleobases in cellular DNA with emphasis on damage induced through the transient generation of purine and pyrimidine radical cations. Since the indirect effect of ionizing radiation mediated by hydroxyl radical is predominant in cells, efforts have been made to selectively ionize bases using suitable one-electron oxidants that consist among others of high intensity UVC laser pulses. Thus, the main oxidation product in cellular DNA was found to be 8-oxo-7,8-dihydroguanine as a result of direct bi-photonic ionization of guanine bases and indirect formation of guanine radical cations through hole transfer reactions from other base radical cations. The formation of 8-oxo-7,8-dihydroguanine and other purine and pyrimidine degradation products was rationalized in terms of the initial generation of related radical cations followed by either hydration or deprotonation reactions in agreement with mechanistic pathways inferred from detailed mechanistic studies. The guanine radical cation has been shown to be implicated in three other nucleophilic additions that give rise to DNA-protein and DNA-DNA cross-links in model systems. Evidence was recently provided for the occurrence of these three reactions in cellular DNA. Conclusion There is growing evidence that one-electron oxidation reactions of nucleobases whose mechanisms have been characterized in model studies involving aqueous solutions take place in a similar way in cells. It may also be pointed out that the above cross-linked lesions are only produced from the guanine radical cation and may be considered as diagnostic products of the direct effect of ionizing radiation. PMID:24369822

  16. Labeling of Cellular DNA with a Cyclosal Phosphotriester Pronucleotide Analog of 5-ethynyl-2'-deoxyuridine.

    PubMed

    Huynh, Ngoc; Dickson, Charlotte; Zencak, Dusan; Hilko, David H; Mackay-Sim, Alan; Poulsen, Sally-Ann

    2015-10-01

    DNA synthesis is a fundamental biological process central to all proliferating cells, and the design of small molecule probes that allow detection of this DNA is important for many applications. 5-Ethynyl-2'-deoxyuridine, known as EdU, has become a workhorse for metabolic labeling of DNA in mammalian cells, followed by bioconjugation to a small molecule fluorescent azide using copper-catalyzed azide-alkyne cycloaddition (CuAAC), click chemistry, to allow detection. In this study, we demonstrate that a cyclosal phosphotriester pronucleotide analog of EdU is suitable for metabolic incorporation into DNA of proliferating cells and subsequent labeling by CuAAC. This analog has two advantages over EdU; first, by delivering EdU with a preinstalled 5'-monophosphate moiety, it bypasses the need for thymidine kinase processing, and second, the increased lipophilicity compared to EdU may enable passive diffusion across the cell membrane and may circumvent the reliance on nucleoside active transport mechanisms for cellular uptake. These advantages pave the way for the development of additional novel pronucleotides to widen experimental opportunities for future bioconjugation applications involving cellular DNA.

  17. Myocyte stress 1 plays an important role in cellular hypertrophy and protection against apoptosis.

    PubMed

    Koekemoer, Andrea L; Chong, Nelson W; Goodall, Alison H; Samani, Nilesh J

    2009-09-03

    Myocyte stress 1 (MS1) is a recently described striated muscle actin-binding protein that is up-regulated in the early stages of pressure overload left ventricular hypertrophy. The aim of this study was to determine whether MS1 induces cellular hypertrophy and protects against apoptosis. Over-expressed MS1 co-localized with actin in H9c2 cells and altered expression of genes of the myocardin-related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathways and in addition the apoptosis repressor with caspase recruitment domain (Nol3) gene. The size of cells over-expressing MS1 was significantly increased by 55% and over-expression of MS1 dramatically inhibited staurosporine-induced apoptosis by 89%. These findings suggest the involvement of MS1 in cellular hypertrophy and protection against apoptosis.

  18. Synthesis of a Cationic BODIPY-Containing Conjugated Polymer for Detection of DNA and Cellular Imaging.

    PubMed

    Wang, Lingyun; Fang, Guipo; Cao, Derong

    2016-03-01

    A water-soluble cationic conjugated polyelectrolyte (P1) containing fluorene, BODIPY and diacetylene moieties was synthesized and characterized. P1 showed two main absorption bands with maxima at 360 and 574 nm as well as fluorescence maxima at 648 nm due to the incorporation of BODIPY into the polymer backbone. Addition of CT DNA can quench the emission of P1 because of the formation of a P1/CT DNA complex, which was demonstrated by UV-vis spectra and dynamic light scattering (DLS) analyses. Cellular imaging results indicated P1 could be utilized as cellular imaging of HeLa cells, where red fluorescence was observed in the partial cytoplasm. Moreover, CCK-8 assay showed P1 had a low cytotoxicity.

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  1. Dysregulated human Tyrosyl-DNA phosphodiesterase I acts as cellular toxin

    PubMed Central

    Cuya, Selma M.; Comeaux, Evan Q.; Wanzeck, Keith; Yoon, Karina J.; van Waardenburg, Robert C.A.M.

    2016-01-01

    Tyrosyl-DNA phosphodiesterase I (TDP1) hydrolyzes the drug-stabilized 3’phospho-tyrosyl bond formed between DNA topoisomerase I (TOPO1) and DNA. TDP1-mediated hydrolysis uses a nucleophilic histidine (Hisnuc) and a general acid/base histidine (Hisgab). A Tdp1Hisgab to Arg mutant identified in patients with the autosomal recessive neurodegenerative disease SCAN1 causes stabilization of the TDP1-DNA intermediate. Based on our previously reported Hisgab-substitutions inducing yeast toxicity (Gajewski et al. J. Mol. Biol. 415, 741-758, 2012), we propose that converting TDP1 into a cellular poison by stabilizing the covalent enzyme-DNA intermediate is a novel therapeutic strategy for cancer treatment. Here, we analyzed the toxic effects of two TDP1 catalytic mutants in HEK293 cells. Expression of human Tdp1HisnucAla and Tdp1HisgabAsn mutants results in stabilization of the covalent TDP1-DNA intermediate and induces cytotoxicity. Moreover, these mutants display reduced in vitro catalytic activity compared to wild type. Co-treatment of Tdp1mutant with topotecan shows more than additive cytotoxicity. Overall, these results support the hypothesis that stabilization of the TDP1-DNA covalent intermediate is a potential anti-cancer therapeutic strategy. PMID:27893431

  2. DNA Damage and Its Cellular Response in Mother and Fetus Exposed to Hyperglycemic Environment

    PubMed Central

    Moreli, Jusciele Brogin; Santos, Janine Hertzog; Rocha, Clarissa Ribeiro; Rudge, Marilza Vieira; Bevilacqua, Estela; Calderon, Iracema Mattos Paranhos

    2014-01-01

    The increased production of reactive oxygen species (ROS) plays a key role in pathogenesis of diabetic complications. ROS are generated by exogenous and endogenous factors such as during hyperglycemia. When ROS production exceeds the detoxification and scavenging capacity of the cell, oxidative stress ensues. Oxidative stress induces DNA damage and when DNA damage exceeds the cellular capacity to repair it, the accumulation of errors can overwhelm the cell resulting in cell death or fixation of genome mutations that can be transmitted to future cell generations. These mutations can lead to and/or play a role in cancer development. This review aims at (i) understanding the types and consequences of DNA damage during hyperglycemic pregnancy; (ii) identifying the biological role of DNA repair during pregnancy, and (iii) proposing clinical interventions to maintain genome integrity. While hyperglycemia can damage the maternal genetic material, the impact of hyperglycemia on fetal cells is still unclear. DNA repair mechanisms may be important to prevent the deleterious effects of hyperglycemia both in mother and in fetus DNA and, as such, prevent the development of diseases in adulthood. Hence, in clinical practice, maternal glycemic control may represent an important point of intervention to prevent the deleterious effects of maternal hyperglycemia to DNA. PMID:25197655

  3. Wogonin, a natural flavonoid, intercalates with genomic DNA and exhibits protective effects in IL-1β stimulated osteoarthritis chondrocytes.

    PubMed

    Khan, Nazir M; Ahmad, Imran; Ansari, Mohammad Y; Haqqi, Tariq M

    2017-08-25

    Wogonin has recently been shown to possess anti-inflammatory and chondroprotective properties and is of considerable interest due to its broad pharmacological activities. The present study highlights that Wogonin binds DNA and exerts chondroprotective effects in vitro. Wogonin showed strong binding with chondrocytes genomic DNA in vitro. The mode of binding of Wogonin to genomic-DNA was assessed by competing Wogonin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. EtBr fluorescence reduced significantly with increase in Wogonin concentration suggesting possible DNA intercalation of Wogonin. Further, in silico molecular docking of Wogonin on mammalian DNA also indicated possible intercalation of Wogonin with DNA. The denaturation and FRET studies revealed that Wogonin prevents denaturation of DNA strands and provide stability to genomic DNA against a variety of chemical denaturants. The cellular uptake study showed that Wogonin enters osteoarthritis chondrocytes and was mainly localized in the nucleus. Wogonin treatment to OA chondrocytes protects the fragmentation of genomic DNA in response to IL-1β as evaluated by DNA ladder and TUNEL assay. Treatment of chondrocytes with Wogonin resulted in significant suppression of IL-1β-mediated induction of ROS. Further, Wogonin exhibited protective potential through potent suppression of extrinsic and intrinsic apoptotic pathways and induction of anti-apoptotic proteins in IL-1β-stimulated osteoarthritis chondrocytes. Our data thus suggest that DNA intercalation by Wogonin may result in the stabilization of genomic DNA leading to protective activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. PGC7 suppresses TET3 for protecting DNA methylation

    PubMed Central

    Bian, Chunjing; Yu, Xiaochun

    2014-01-01

    Ten-eleven translocation (TET) family enzymes convert 5-methylcytosine to 5-hydroxylmethylcytosine. However, the molecular mechanism that regulates this biological process is not clear. Here, we show the evidence that PGC7 (also known as Dppa3 or Stella) interacts with TET2 and TET3 both in vitro and in vivo to suppress the enzymatic activity of TET2 and TET3. Moreover, lacking PGC7 induces the loss of DNA methylation at imprinting loci. Genome-wide analysis of PGC7 reveals a consensus DNA motif that is recognized by PGC7. The CpG islands surrounding the PGC7-binding motifs are hypermethylated. Taken together, our study demonstrates a molecular mechanism by which PGC7 protects DNA methylation from TET family enzyme-dependent oxidation. PMID:24322296

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

    NASA Technical Reports Server (NTRS)

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

    2015-01-01

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

  6. Gentiana asclepiadea protects human cells against oxidation DNA lesions.

    PubMed

    Hudecová, Alexandra; Hašplová, Katarína; Miadoková, Eva; Magdolenová, Zuzana; Rinna, Alessandra; Collins, Andrew R; Gálová, Eliška; Vaculčíková, Dagmar; Gregáň, Fridrich; Dušinská, Mária

    2012-03-01

    The objectives of this study were to examine whether the methanolic and aqueous extracts from the haulm and flower of Gentiana asclepiadea exhibited free radical scavenging and protective (antigenotoxic) effect against DNA oxidation induced by H(2)O(2) in human lymphocytes and human embryonic kidney cells (HEK 293). All four extracts exhibited high scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals at concentrations 2.5 and 25 mg ml(-1). The level of DNA damage was measured using the alkaline version of single-cell gel electrophoresis (comet assay). Challenge with H(2)O(2) shows that the pre-treatment of the cells with non-genotoxic doses of Gentiana extracts protected human DNA-either eliminated or significantly reduced H(2)O(2) induced DNA damage. The genotoxic activity of H(2)O(2) was most effectively decreased after 30 min of pre-incubation with 0.05 mg ml(-1) (range, 93.5%-96.3% of reduction in lymphocytes) and 0.25 mg ml(-1) (range, 59.5%-71.4% and 52.7%-66.4% of reduction in lymphocytes and HEK 293 cells, respectively) of G. asclepiadea extracts. These results suggest that the tested G. asclepiadea extracts could be considered as an effective natural antioxidant source.

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

    PubMed

    Sakasai, Ryo; Iwabuchi, Kuniyoshi

    2016-01-01

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

  8. Singlet oxygen oxidation of isolated and cellular DNA: product formation and mechanistic insights.

    PubMed

    Cadet, Jean; Ravanat, Jean-Luc; Martinez, Glaucia R; Medeiros, Marisa H G; Di Mascio, Paolo

    2006-01-01

    This survey focuses on recent aspects of the singlet oxygen oxidation of the guanine moiety of nucleosides, oligonucleotides, isolated and cellular DNA that has been shown to be the exclusive DNA target for this biologically relevant photogenerated oxidant. A large body of mechanistic data is now available from studies performed on nucleosides in both aprotic solvents and aqueous solutions. A common process to both reaction conditions is the formation of 8-oxo-7,8-dihydroguanine by reduction of 8-hydroperoxyguanine that arises from the rearrangement of initially formed endoperoxide across the 4,8-bond of the purine moiety. However, in organic solvent the hydroperoxide is converted as a major degradation pathway into a dioxirane that subsequently decomposes into a complex pattern of oxidation products. A different reaction that involved the formation of a highly reactive quinonoid intermediate consecutively to the loss of a water molecule from the 8-hydroperoxide has been shown to occur in aqueous solution. Subsequent addition of a water molecule at C5 leads to the generation of a spiroiminodihy-dantoin compound via a rearrangement that involves an acyl shift. However, in both isolated and cellular DNA the latter decomposition pathway is at the best a minor process, because only 8-oxo-7,8-dihydroguanine has been found to be generated. It is interesting to point out that singlet oxygen has been shown to contribute predominantly to the formation of 8-oxo-7,8-dihydroguanine in the DNA of bacterial and human cells upon exposure to UVA radiation. It may be added that the formation of secondary singlet-oxygen oxidation products of 8-oxo-7,8-dihydroguanine, including spiroiminodihydantoin and oxaluric acid that were characterized in nucleosides and oligonucleotide, respectively, have not yet been found in cellular DNA.

  9. 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.

  10. Induction of DNA double-strand breaks and cellular senescence by human respiratory syncytial virus

    PubMed Central

    Martínez, Isidoro; García-Carpizo, Verónica; Guijarro, Trinidad; García-Gomez, Ana; Navarro, Diego; Aranda, Ana; Zambrano, Alberto

    2016-01-01

    ABSTRACT Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and γH2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained γH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as γH2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections. PMID:26809688

  11. Induction of DNA double-strand breaks and cellular senescence by human respiratory syncytial virus.

    PubMed

    Martínez, Isidoro; García-Carpizo, Verónica; Guijarro, Trinidad; García-Gomez, Ana; Navarro, Diego; Aranda, Ana; Zambrano, Alberto

    2016-05-18

    Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and γH2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained γH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as γH2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections.

  12. Effect of sodium butyrate on induction of cellular and viral DNA syntheses in polyoma virus-infected mouse kidney cells.

    PubMed Central

    Wawra, E; Pöckl, E; Müllner, E; Wintersberger, E

    1981-01-01

    Sodium butyrate inhibited initiation of viral and cellular DNA replication in polyoma virus-infected mouse kidney cells. Ongoing viral or cellular DNA replication, however, was not affected by the presence of the substance. Butyrate had no effect on T-antigen synthesis and on the stimulation of transcription, one of the earliest reactions of the infected cells to the appearance of T-antigen, nor did it inhibit expression of late viral genes (synthesis of viral capsid proteins). In addition to blocking the onset of DNA synthesis, butyrate also inhibited stimulation of the activities of enzymes involved in DNA synthesis. When butyrate was removed, viral and cellular DNA syntheses were induced in parallel after a lag period of approximately 4 h. At the same time, the activities of enzymes involved in DNA synthesis increase. If protein synthesis was inhibited during part of the lag period, the initiation of DNA synthesis was retarded for the same time interval, suggesting that the proteins involved in the initiation of DNA replication had to be made. We have developed an in vitro system for measuring DNA synthesis in crude nuclear preparations which mimics the status of DNA replication in intact cells and may help in future experiments to study the requirements for initiation of cellular and viral DNA synthesis and the possible involvement of T-antigens in this reaction. Images PMID:6264167

  13. Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage

    PubMed Central

    Ganapathy, Vengatesh; Manyanga, Jimmy; Brame, Lacy; McGuire, Dehra; Sadhasivam, Balaji; Floyd, Evan; Rubenstein, David A.; Ramachandran, Ilangovan; Wagener, Theodore

    2017-01-01

    Background Electronic cigarette (EC) aerosols contain unique compounds in addition to toxicants and carcinogens traditionally found in tobacco smoke. Studies are warranted to understand the public health risks of ECs. Objective The aim of this study was to determine the genotoxicity and the mechanisms induced by EC aerosol extracts on human oral and lung epithelial cells. Methods Cells were exposed to EC aerosol or mainstream smoke extracts and DNA damage was measured using the primer anchored DNA damage detection assay (q-PADDA) and 8-oxo-dG ELISA assay. Cell viability, reactive oxygen species (ROS) and total antioxidant capacity (TAC) were measured using standard methods. mRNA and protein expression were evaluated by RT-PCR and western blot, respectively. Results EC aerosol extracts induced DNA damage in a dose-dependent manner, but independently of nicotine concentration. Overall, EC aerosol extracts induced significantly less DNA damage than mainstream smoke extracts, as measured by q-PADDA. However, the levels of oxidative DNA damage, as indicated by the presence of 8-oxo-dG, a highly mutagenic DNA lesion, were similar or slightly higher after exposure to EC aerosol compared to mainstream smoke extracts. Mechanistically, while exposure to EC extracts significantly increased ROS, it decreased TAC as well as the expression of 8-oxoguanine DNA glycosylase (OGG1), an enzyme essential for the removal of oxidative DNA damage. Conclusions Exposure to EC aerosol extracts suppressed the cellular antioxidant defenses and led to significant DNA damage. These findings emphasize the urgent need to investigate the potential long-term cancer risk of exposure to EC aerosol for vapers and the general public. PMID:28542301

  14. p53 pathway determines the cellular response to alcohol-induced DNA damage in MCF-7 breast cancer cells

    PubMed Central

    Zhao, Ming; Howard, Erin W.; Guo, Zhiying; Parris, Amanda B.; Yang, Xiaohe

    2017-01-01

    Alcohol consumption is associated with increased breast cancer risk; however, the underlying mechanisms that contribute to mammary tumor initiation and progression are unclear. Alcohol is known to induce oxidative stress and DNA damage; likewise, p53 is a critical modulator of the DNA repair pathway and ensures genomic integrity. p53 mutations are frequently detected in breast and other tumors. The impact of alcohol on p53 is recognized, yet the role of p53 in alcohol-induced mammary carcinogenesis remains poorly defined. In our study, we measured alcohol-mediated oxidative DNA damage in MCF-7 cells using 8-OHdG and p-H2AX foci formation assays. p53 activity and target gene expression after alcohol exposure were determined using p53 luciferase reporter assay, qPCR, and Western blotting. A mechanistic study delineating the role of p53 in DNA damage response and cell cycle arrest was based on isogenic MCF-7 cells stably transfected with control (MCF-7/Con) or p53-targeting siRNA (MCF-7/sip53), and MCF-7 cells that were pretreated with Nutlin-3 (Mdm2 inhibitor) to stabilize p53. Alcohol treatment resulted in significant DNA damage in MCF-7 cells, as indicated by increased levels of 8-OHdG and p-H2AX foci number. A p53-dependent signaling cascade was stimulated by alcohol-induced DNA damage. Moderate to high concentrations of alcohol (0.1–0.8% v/v) induced p53 activation, as indicated by increased p53 phosphorylation, reporter gene activity, and p21/Bax gene expression, which led to G0/G1 cell cycle arrest. Importantly, compared to MCF-7/Con cells, alcohol-induced DNA damage was significantly enhanced, while alcohol-induced p21/Bax expression and cell cycle arrest were attenuated in MCF-7/sip53 cells. In contrast, inhibition of p53 degradation via Nutlin-3 reinforced G0/G1 cell cycle arrest in MCF-7 control cells. Our study suggests that functional p53 plays a critical role in cellular responses to alcohol-induced DNA damage, which protects the cells from DNA damage

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

    PubMed

    Yang, Yafan; Li, Shuangshuang

    2015-01-01

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

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

    PubMed Central

    Yang, Yafan; Li, Shuangshuang

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Alizadeh, Elahe; Sanche, Léon

    2014-04-01

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

  18. Determination of cellular RNA concentrations by electron microscopy of R loop-containing DNA.

    PubMed Central

    Kaback, D B; Rosbash, M; Davidson, N

    1981-01-01

    R loop hybridizations and electron microscopy have been used to determine cellular RNA concentrations for cloned genes. In plasmid DNA sequence excess, all the complementary RNA is driven into R loop structures that can be assayed by electron microscopy. To determine the concentration of a particular poly(A)+ RNA, plasmid DNA crosslinked once every 2000-5000 base pairs with trioxsalen and UV light is hybridized in DNA sequence excess to various known amounts of total poly(A)+ RNA, and the R loops are stabilized by treatment with glyoxal. If necessary, excess nonhybridized RNA is removed by Sepharose 2B chromatography, which enables the visualization of less abundant transcripts. Reconstruction experiments demonstrated that electron microscopic determination of the fraction of plasmid DNA molecules containing specific RNA loops gives accurate values of specific RNA weight fractions or concentrations in the total poly(A)+ RNA populations. These methods were also used to determine the concentrations of five RNA species complementary to sequences on TRT3, a recombinant DNA plasmid containing yeast histone 2A and 2B genes and three other nonhistone genes. The methods described allow one to visualize the R loop structures for both abundant and nonabundant transcripts and to estimate concentrations of these RNA species simply by determining the fraction of DNA containing R loops. Images PMID:6265914

  19. Relationship between histologic grade and cytofluorometric cellular DNA and RNA content in primary bone tumors.

    PubMed

    Takeshita, H; Kusuzaki, K; Kuzuhara, A; Tsuji, Y; Ashihara, T; Gebhardt, M C; Mankin, H J; Springfield, D S; Hirasawa, Y

    2001-01-01

    The diagnosis and grading of bone tumors remains a challenging problem. We studied the relationship between histologic grade and cytofluorometric cellular DNA and RNA content in 108 primary bone tumors. The data included DNA ploidy, mean DNA content (MDC), S-phase fraction (SPF), mean RNA content (MRC) and RNA/DNA ratio (RDR; MRC/MDC) which represents the RNA content normalized for the DNA content. Benign tumors had a diploid stem line with low MDC (mean; 1.04), low SPF (0.9), high MRC (2.41) and high RDR (2.31). Giant cell tumors of bone, which are locally aggressive benign tumors, showed diploidy with relatively higher MDC (1.07, p < 0.01) and SPF (2.6, p < 0.01) and lower MRC (1.81, p < 0.01) and RDR (1.69, p < 0.01). Similar results were obtained in low-grade sarcomas. In high-grade sarcomas, the data depended on the histologic findings. Pleomorphic sarcomas such as osteosarcomas revealed aneuploidy with remarkably higher MDC (1.70 in osteosarcomas, p < 0.01) and SPF (6.5, p < 0.01), but lower RDR (1.70, p < 0.01). In contrast, small cell sarcomas, such as Ewing's sarcomas, showed diploidy with low MDC (1.11 in Ewing's sarcomas, N.S.) and SPF (2.5, p < 0.01) and extremely low RDR (1.34, p < 0.01). The RDR value was higher in well-differentiated tumors than in primitive tumors, rendering it useful in grading bone tumors with a diploid stem line. By combining the RDR value with the MDC value, 96% of diploid sarcomas could be distinguished from benign tumors. These results indicate that cellular DNA and RNA content analysis may be of value in assessing the malignant potential of diploid as well as aneuploid bone sarcomas.

  20. [The role of a lysine residue in the antioxidant and dna-protective activity of oligopeptides].

    PubMed

    Prazdnova, E V; Mazanko, M S; Zolotukhin, P V; Kharchenko, E Y; Chistyakov, V A; Arutiunov, V A; Kozina, L S

    2016-01-01

    Oligopeptides present in the living cell were found to have antioxidative activity and to be involved in the regulation of antioxidant balance by interaction with the redox-dependent cellular signaling cascades. Experiments on animal models have shown that the introduction of oligopeptides causes geroprotective and adaptogenic effects. In the present work, we investigate the biological action of a number of synthetic oligopeptides using bacterial biosensors. This approach allows us to precisely estimate the antioxidant properties of the compounds without affecting their participation in regulatory cascades typical to eukaryotic cells. It has been shown that the ability of oligopeptides to protect cells from action of physical prooxidant factors (UV irradiation) is related to the presence of a lysine residue in the molecule. For chemical pro-oxidants (dioxidine), we have observed a similar, though less strict pattern. This effect also correlates with DNA-protective activity of the investigated oligopeptides.

  1. Functional DNA-containing nanomaterials: cellular applications in biosensing, imaging, and targeted therapy.

    PubMed

    Liang, Hao; Zhang, Xiao-Bing; Lv, Yifan; Gong, Liang; Wang, Ruowen; Zhu, Xiaoyan; Yang, Ronghua; Tan, Weihong

    2014-06-17

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

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

    SciTech Connect

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

    2013-02-01

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

  3. 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.

  4. 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

  5. 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.

  6. Cellular stress induces a protective sleep-like state in C. elegans.

    PubMed

    Hill, Andrew J; Mansfield, Richard; Lopez, Jessie M N G; Raizen, David M; Van Buskirk, Cheryl

    2014-10-20

    Sleep is recognized to be ancient in origin, with vertebrates and invertebrates experiencing behaviorally quiescent states that are regulated by conserved genetic mechanisms. Despite its conservation throughout phylogeny, the function of sleep remains debated. Hypotheses for the purpose of sleep include nervous-system-specific functions such as modulation of synaptic strength and clearance of metabolites from the brain, as well as more generalized cellular functions such as energy conservation and macromolecule biosynthesis. These models are supported by the identification of synaptic and metabolic processes that are perturbed during prolonged wakefulness. It remains to be seen whether perturbations of cellular homeostasis in turn drive sleep. Here we show that under conditions of cellular stress, including noxious heat, cold, hypertonicity, and tissue damage, the nematode Caenorhabditis elegans engages a behavioral quiescence program. The stress-induced quiescent state displays properties of sleep and is dependent on the ALA neuron, which mediates the conserved soporific effect of epidermal growth factor (EGF) ligand overexpression. We characterize heat-induced quiescence in detail and show that it is indeed dependent on components of EGF signaling, providing physiological relevance to the behavioral effects of EGF family ligands. We find that after noxious heat exposure, quiescence-defective animals show elevated expression of cellular stress reporter genes and are impaired for survival, demonstrating the benefit of stress-induced behavioral quiescence. These data provide evidence that cellular stress can induce a protective sleep-like state in C. elegans and suggest that a deeply conserved function of sleep is to mitigate disruptions of cellular homeostasis.

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

    PubMed Central

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

    1991-01-01

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

  8. Robustness of CDK2 in Triggering Cellular Senescence based on Probability of DNA-damaged Cells Passing G1/S Checkpoint

    NASA Astrophysics Data System (ADS)

    Ling, Hong; Samarasinghe, Sandhya; Kulasiri, Don

    2011-06-01

    Recent experiments have shown that cellular senescence, a mechanism employed by cells for thwarting cell proliferation, plays an important role in protecting cells against cancer; therefore, a deeper understanding of cellular senescence can lead to effective cancer treatment. Inhibition of CDK2 is thought to be the critical trigger for cellular senescence. In this study, we first implement a mathematical model of G1/S transition involving the DNA-damage pathway and show that cellular senescence can be achieved by lowering CDK2. The robustness of CDK2 in triggering cellular senescence is determined from the probability (β) of DNA-damaged cells passing G1/S checkpoint for normal CDK2 and CDK2-deficient situations based on different thresholds of the peak time of two important biomarkers, CycE and E2F. The comparison of the values of β under the normal CDK2 and lower CDK2 levels reveals that reducing CDK2 levels can decrease the percentage of damaged cells passing G1/S checkpoint; more importantly, 50% reduction of CDK2 achieves 65% reduction in the percentage of damaged cells passing the G1/S checkpoint. These results point out that the developed model can highlight the possibility of lowering the bar for cellular senescence by reducing CDK2 levels. The results of investigation of β for the different thresholds of the peak times of other biomarkers show that β is insensitive to these perturbations of the peak time indicating that CDK2 activity is robust in lowering the senescence bar for low and high levels of DNA-damage. Furthermore, a mathematical formulation of robustness indicates that the robustness of CDK2-triggered senescence increases with decreasing levels of CDK2, and is slightly greater for low-level DNA damage condition.

  9. Chemical conjugate TMV-peptide bivalent fusion vaccines improve cellular immunity and tumor protection.

    PubMed

    McCormick, Alison A; Corbo, Tina A; Wykoff-Clary, Sherri; Palmer, Kenneth E; Pogue, Gregory P

    2006-01-01

    Chemical conjugation of CTL peptides to tobacco mosaic virus (TMV) has shown promise as a molecular adjuvant scaffold for augmentation of cellular immune responses to peptide vaccines. This study demonstrates the ease of generating complex multipeptide vaccine formulations using chemical conjugation to TMV for improved vaccine efficacy. We have tested a model foreign antigen target-the chicken ovalbumin-derived CTL peptide (Ova peptide), as well as mouse melanoma-associated CTL epitopes p15e and tyrosinase-related protein 2 (Trp2) peptides that are self-antigen targets. Ova peptide fusions to TMV, as bivalent formulations with peptides encoding additional T-help or cellular uptake via the integrin-receptor binding RGD peptide, showed improved vaccine potency evidenced by significantly enhanced numbers of antigen-reactive T cells measured by in vitro IFNgamma cellular analysis. We measured the biologically relevant outcome of vaccination in protection of mice from EG.7-Ova tumor challenge, which was achieved with only two doses of vaccine ( approximately 600 ng peptide) given without adjuvant. The p15e peptide alone or Trp2 peptide alone, or as a bivalent formulation with T-help or RGD uptake epitopes, was unable to stimulate effective tumor protection. However, a vaccine with both CTL peptides fused together onto TMV generated significantly improved survival. Interestingly, different bivalent vaccine formulations were required to improve vaccine efficacy for Ova or melanoma tumor model systems.

  10. Cellular uptake and intracellular pathways of PLL-g-PEG-DNA nanoparticles.

    PubMed

    Lühmann, Tessa; Rimann, Markus; Bittermann, Anne Greet; Hall, Heike

    2008-09-01

    Polycationic molecules form condensates with DNA and are used for gene therapy as an alternative to viral vectors. As clinical efficacy corresponds to cellular uptake, intracellular stability of the condensates, and bioavailability of the DNA, it is crucial to analyze uptake mechanisms and trafficking pathways. Here, a detailed study of uptake, stability, and localization of PLL-g-PEG-DNA nanoparticles within COS-7 cells is presented, using FACS analysis to assess the involvement of different uptake mechanisms, colocalization studies with markers indicative for different endocytotic pathways, and immunofluorescence staining to analyze colocalization with intracellular compartments. PLL-g-PEG-DNA nanoparticles were internalized in an energy-dependent manner after 2 h and accumulated in the perinuclear region after >6 h. The nanoparticles were found to be stable within the cytoplasm for at least 24 h and did not colocalize with the endosomal pathway. Nanoparticle uptake was approximately 50% inhibited by genistein, an inhibitor of the caveolae-mediated pathway. However, genistein did not inhibit gene expression, and PLL-g-PEG-DNA nanoparticles were not colocalized with caveolin-1 indicating that caveolae-mediated endocytosis is not decisive for DNA delivery. Clathrin-mediated endocytosis and macropinocytosis pathways were reduced by 17 and 24%, respectively, in the presence of the respective inhibitors. When cells were transfected in the presence of double and triple inhibitors, transfection efficiencies were increasingly reduced by 40 and 70%, respectively; however, no differences were found between the different uptake mechanisms. These findings suggest that PLL-g-PEG-DNA nanoparticles enter by several pathways and might therefore be an efficient and versatile tool to deliver therapeutic DNA.

  11. Stimulation of lactate receptor (HCAR1) affects cellular DNA repair capacity.

    PubMed

    Wagner, Waldemar; Kania, Katarzyna D; Ciszewski, Wojciech M

    2017-04-01

    Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. The catalytic subunit of DNA-dependent protein kinase is required for cellular resistance to oxidative stress independent of DNA double-strand break repair.

    PubMed

    Li, Mengxia; Lin, Yu-Fen; Palchik, Guillermo A; Matsunaga, Shinji; Wang, Dong; Chen, Benjamin P C

    2014-11-01

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) are the two major kinases involved in DNA double-strand break (DSB) repair, and are required for cellular resistance to ionizing radiation. Whereas ATM is the key upstream kinase for DSB signaling, DNA-PKcs is primarily involved in DSB repair through the nonhomologous end-joining (NHEJ) mechanism. In addition to DSB repair, ATM has been shown to be involved in the oxidative stress response and could be activated directly in vitro on hydrogen peroxide (H2O2) treatment. However, the role of DNA-PKcs in cellular response to oxidative stress is not clear. We hypothesize that DNA-PKcs may participate in the regulation of ATM activation in response to oxidative stress, and that this regulatory role is independent of its role in DNA double-strand break repair. Our findings reveal that H2O2 induces hyperactivation of ATM signaling in DNA-PKcs-deficient, but not Ligase 4-deficient cells, suggesting an NHEJ-independent role for DNA-PKcs. Furthermore, DNA-PKcs deficiency leads to the elevation of reactive oxygen species (ROS) production, and to a decrease in cellular survival against H2O2. For the first time, our results reveal that DNA-PKcs plays a noncanonical role in the cellular response to oxidative stress, which is independent from its role in NHEJ. In addition, DNA-PKcs is a critical regulator of the oxidative stress response and contributes to the maintenance of redox homeostasis. Our findings reveal that DNA-PKcs is required for cellular resistance to oxidative stress and suppression of ROS buildup independently of its function in DSB repair.

  13. Phylogenetic evidence for extensive lateral acquisition of cellular genes by Nucleocytoplasmic large DNA viruses

    PubMed Central

    2008-01-01

    Background Nucleo-Cytoplasmic Large DNA viruses (NCLDV), a diverse group that infects a wide range of eukaryotic hosts, exhibit a large heterogeneity in genome size (between 100 kb and 1.2 Mb) but have been suggested to form a monophyletic group on the basis of a small subset of approximately 30 conserved genes. NCLDV were proposed to have evolved by simplification from cellular organism although some of the giant NCLDV have clearly grown by gene accretion from a bacterial origin. Results We demonstrate here that many NCLDV lineages appear to have undergone frequent gene exchange in two different ways. Viruses which infect protists directly (Mimivirus) or algae which exist as intracellular protists symbionts (Phycodnaviruses) acquire genes from a bacterial source. Metazoan viruses such as the Poxviruses show a predominant acquisition of host genes. In both cases, the laterally acquired genes show a strong tendency to be positioned at the tip of the genome. Surprisingly, several core genes believed to be ancestral in the family appear to have undergone lateral gene transfers, suggesting that the NCLDV ancestor might have had a smaller genome than previously believed. Moreover, our data show that the larger the genome, the higher is the number of laterally acquired genes. This pattern is incompatible with a genome reduction from a cellular ancestor. Conclusion We propose that the NCLDV viruses have evolved by significant growth of a simple DNA virus by gene acquisition from cellular sources. PMID:19036122

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

    PubMed Central

    2015-01-01

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

  15. The interplay among chromatin dynamics, cell cycle checkpoints and repair mechanisms modulates the cellular response to DNA damage.

    PubMed

    Lazzaro, Federico; Giannattasio, Michele; Muzi-Falconi, Marco; Plevani, Paolo

    2007-06-01

    Cells are continuously under the assault of endogenous and exogenous genotoxic stress that challenges the integrity of DNA. To cope with such a formidable task cells have evolved surveillance mechanisms, known as checkpoints, and a variety of DNA repair systems responding to different types of DNA lesions. These lesions occur in the context of the chromatin structure and, as expected for all DNA transactions, the cellular response to DNA damage is going to be influenced by the chromatin enviroment. In this review, we will discuss recent studies implicating chromatin remodelling factors and histone modifications in the response to DNA double-strand breaks (DSBs) and in checkpoint activation in response to UV lesions.

  16. HK2/hexokinase-II integrates glycolysis and autophagy to confer cellular protection.

    PubMed

    Tan, Valerie P; Miyamoto, Shigeki

    2015-01-01

    Hexokinases (HKs) catalyze the first step of glucose metabolism, phosphorylating glucose to glucose 6-phosphate (G6P). HK2/hexokinase-II is a predominant isoform in insulin-sensitive tissues such as heart, skeletal muscle, and adipose tissues and is also upregulated in many types of tumors associated with enhanced aerobic glycolysis (the Warburg effect). Accumulating evidence indicates that HK2 plays an important role not only in glycolysis but also in cell survival. Although there is increasing recognition that cellular metabolism and cell survival are closely related, the molecular link between metabolism and autophagic pathways has not been fully elucidated. We recently discovered that HK2 facilitates autophagy in response to glucose deprivation (HK substrate deprivation) to protect cardiomyocytes, and suggest that HK2 functions as a molecular switch from glycolysis to autophagy to ensure cellular energy homeostasis under starvation conditions.

  17. Assessment of the cellular internalization of thermolytic phosphorothioate DNA oligonucleotide prodrugs.

    PubMed

    Jain, Harsh V; Takeda, Kazuyo; Tami, Cecilia; Verthelyi, Daniela; Beaucage, Serge L

    2013-10-15

    The bioactivity of a CpG-containing phosphorothioate DNA oligonucleotide with thermolytic 2-(N-formyl-N-methylamino)ethyl (fma) thiophosphate groups in mice led us to investigate the parameters affecting the internalization of these thermosensitive DNA prodrugs in various cell lines. Flow cytometry and confocal microscopy analyses indicate that 5'-fluoresceinated fma-phosphorothioate DNA sequences are poorly internalized in Vero, HeLa and GC-2 cells. However, when four fma-thiophosphate groups of a 15-nucleotide long oligothymidylate prodrug are replaced with 3-(N,N-dimethylamino)prop-1-yl thiophosphate functions, internalization of the positively charged prodrug, under physiological conditions, increased fourfold in HeLa and 40-fold in Vero or GC-2 cells. No cytotoxic effects are observed in Vero cells even at an extracellular prodrug concentration of 50 μM over a period of 72 h. Confocal microscopy studies show that internalization of the positively charged oligothymidylate prodrug in Vero cells is time-dependent with early trafficking of the DNA sequence through endosomal vesicles and, eventually, to the nucleus of the cells. Thus, the incorporation of four 3-(N,N-dimethylamino)prop-1-yl thiophosphate groups into thermosentive fma-phosphorothioate DNA prodrugs is an attractive strategy for efficient cellular internalization of these nucleic acid-based drugs for potential therapeutic indications.

  18. Cellular uptake of fluorophore-labeled glyco-DNA-gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Witten, Katrin G.; Ruff, Julie; Mohr, Anne; Görtz, Dieter; Recker, Tobias; Rinis, Natalie; Rech, Claudia; Elling, Lothar; Müller-Newen, Gerhard; Simon, Ulrich

    2013-10-01

    DNA-functionalized gold nanoparticles (AuNP-DNA) were hybridized with complementary di- N-acetyllactosamine-( di-LacNAc, [3Gal(β1-4)GlcNAc(β1-]2)-modified oligonucleotides to form glycol-functionalized particles, AuNP-DNA- di-LacNAc. While AuNP-DNA are known to be taken up by cells via scavenger receptors, glycol-functionalized particles have shown to be taken up via asialoglycoprotein receptors (ASGP-R). In this work, the interaction of these new particles with HepG2 cells was analyzed, which express scavenger receptors class B type I (SR-BI) and ASGP-R. To study the contribution of these receptors as potential mediators for cellular uptake, receptor-blocking experiments were performed with d-lactose, a ligand for ASGP-R, Fucoidan, a putative ligand for SR-BI, and a SR-BI blocking antibody. Labeling with Cy5-modified DNA ligands enabled us to monitor the particle uptake by confocal fluorescence microscopy and flow cytometry, in order to discriminate the two putative pathways by competitive binding studies. While SR-BI-antibody and d-lactose had no inhibiting effects on particle uptake Fucoidan led to a complete inhibition. Thus, a receptor-mediated uptake by the two receptors studied could not be proven and therefore other uptake mechanisms have to be considered.

  19. Cellular DNA ligase I is recruited to cytoplasmic vaccinia virus factories and masks the role of the vaccinia ligase in viral DNA replication.

    PubMed

    Paran, Nir; De Silva, Frank S; Senkevich, Tatiana G; Moss, Bernard

    2009-12-17

    Vaccinia virus (VACV) encodes DNA polymerase and additional proteins that enable cytoplasmic replication. We confirmed the ability of VACV DNA ligase mutants to replicate and tested the hypothesis that cellular ligases compensate for loss of viral gene expression. RNA silencing of human DNA ligase I expression and a small molecule inhibitor of human DNA ligase I [corrected] severely reduced replication of viral DNA in cells infected with VACV ligase-deficient mutants, indicating that the cellular enzyme plays a complementary role. Replication of ligase-deficient VACV was greatly reduced and delayed in resting primary cells, correlating with initial low levels of ligase I and subsequent viral induction and localization of ligase I in virus factories. These studies indicate that DNA ligation is essential for poxvirus replication and explain the ability of ligase deletion mutants to replicate in dividing cells but exhibit decreased pathogenicity in mice. Encoding its own ligase might allow VACV to "jump-start" DNA synthesis.

  20. Trichothiodystrophy, a human DNA repair disorder with heterogeneity in the cellular response to ultraviolet light

    SciTech Connect

    Lehmann, A.R.; Arlett, C.F.; Broughton, B.C.; Harcourt, S.A.; Steingrimsdottir, H.; Stefanini, M.; Malcolm, A.; Taylor, R.; Natarajan, A.T.; Green, S.

    1988-11-01

    Trichothiodystrophy (TTD) is an autosomal recessive disorder characterized by brittle hair with reduced sulfur content, ichthyosis, peculiar face, and mental and physical retardation. Some patients are photosensitive. A previous study by Stefanini et al. showed that cells from four photosensitive patients with TTD had a molecular defect in DNA repair, which was not complemented by cells from xeroderma pigmentosum, complementation group D. In a detailed molecular and cellular study of the effects of UV light on cells cultured from three further TTD patients who did not exhibit photosensitivity we have found an array of different responses. In cells from the first patient, survival, excision repair, and DNA and RNA synthesis following UV irradiation were all normal, whereas in cells from the second patient all these responses were similar to those of excision-defective xeroderma pigmentosum (group D) cells. With the third patient, cell survival measured by colony-forming ability was normal following UV irradiation, even though repair synthesis was only 50% of normal and RNA synthesis was severely reduced. The excision-repair defect in these cells was not complemented by other TTD cell strains. These cellular characteristics of patient 3 have not been described previously for any other cell line. The normal survival may be attributed to the finding that the deficiency in excision-repair is confined to early times after irradiation. Our results pose a number of questions about the relationship between the molecular defect in DNA repair and the clinical symptoms of xeroderma pigmentosum and TTD.

  1. Intranasal DNA Vaccination Induces Potent Mucosal and Systemic Immune Responses and Cross-protective Immunity Against Influenza Viruses

    PubMed Central

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

    2011-01-01

    The induction of potent virus-specific immune responses at mucosal surfaces where virus transmission occurs is a major challenge for vaccination strategies. In the case of influenza vaccination, this has been achieved only by intranasal delivery of live-attenuated vaccines that otherwise pose safety problems. Here, we demonstrate that potent mucosal and systemic immune responses, both cellular and humoral, are induced by intranasal immunization using formulated DNA. We show that formulation with the DNA carrier polyethylenimine (PEI) improved by a 1,000-fold the efficiency of gene transfer in the respiratory track following intranasal administration of luciferase-coding DNA. Using PEI formulation, intranasal vaccination with DNA-encoding hemagglutinin (HA) from influenza A H5N1 or (H1N1)2009 viruses induced high levels of HA-specific immunoglobulin A (IgA) antibodies that were detected in bronchoalveolar lavages (BALs) and the serum. No mucosal responses could be detected after parenteral or intranasal immunization with naked-DNA. Furthermore, intranasal DNA vaccination with HA from a given H5N1 virus elicited full protection against the parental strain and partial cross-protection against a distinct highly pathogenic H5N1 strain that could be improved by adding neuraminidase (NA) DNA plasmids. Our observations warrant further investigation of intranasal DNA as an effective vaccination route. PMID:20959813

  2. FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5'-DNA end.

    PubMed

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

    2016-12-15

    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.

  3. Protective immunity induced by a DNA vaccine encoding Eimeria tenella rhomboid against homologous challenge.

    PubMed

    Liu, Yingli; Zheng, Jun; Li, Jianhua; Gong, Pengtao; Zhang, Xichen

    2013-01-01

    Rhomboid protein in Apicomplexa was associated with the process of host cell invasion. To evaluate the potential of the protein in eliciting protective immunity against challenge, a DNA vaccine pVAX1-Rho encoding Eimeria tenella rhomboid was constructed. Recombinant protein was expressed in Hela cells and verified by indirect immunofluorescence and western blotting analysis. In vivo experiments, 1-week-old chickens were randomly divided into three groups. Experimental group of chickens were immunized with DNA vaccines while control group of chickens were injected with pVAX1 plasmid alone or sterile water. Two weeks following the booster dose, all chickens were inoculated orally with 5 × 10(4) sporulated oocysts of E. tenella. The host immunity and protective efficacy of this vaccine against E. tenella challenge in broilers were evaluated. Results showed that specific antibody, the levels of interleukin-2 (IL-2), interferon-γ (IFN-γ), and the percentages of CD4(+) and CD8(+) T lymphocyte cells were significantly increased in the pVAX1-Rho group. Challenge experiments demonstrated that pVAX1-Rho vaccination could reduce oocyst excretion, decrease cecal lesion, increase bodyweight gains and provide chickens with oocysts decrease ratio around 75.8 %. These results suggest that the pVAX1-Rho was able to induce humoral and cellular responses and generate protective immunity against E. tenella infection.

  4. Differential Activation of Cellular DNA Damage Responses by Replication-Defective and Replication-Competent Adenovirus Mutants

    PubMed Central

    Prakash, Anand; Jayaram, Sumithra

    2012-01-01

    Adenovirus (Ad) mutants that lack early region 4 (E4) activate the phosphorylation of cellular DNA damage response proteins. In wild-type Ad type 5 (Ad5) infections, E1b and E4 proteins target the cellular DNA repair protein Mre11 for redistribution and degradation, thereby interfering with its ability to activate phosphorylation cascades important during DNA repair. The characteristics of Ad infection that activate cellular DNA repair processes are not yet well understood. We investigated the activation of DNA damage responses by a replication-defective Ad vector (AdRSVβgal) that lacks E1 and fails to produce the immediate-early E1a protein. E1a is important for activating early gene expression from the other viral early transcription units, including E4. AdRSVβgal can deliver its genome to the cell, but it is subsequently deficient for viral early gene expression and DNA replication. We studied the ability of AdRSVβgal-infected cells to induce cellular DNA damage responses. AdRSVβgal infection does activate formation of foci containing the Mdc1 protein. However, AdRSVβgal fails to activate phosphorylation of the damage response proteins Nbs1 and Chk1. We found that viral DNA replication is important for Nbs1 phosphorylation, suggesting that this step in the viral life cycle may provide an important trigger for activating at least some DNA repair proteins. PMID:23015708

  5. Recursive DNA Assembly Using Protected Oligonucleotide Duplex Assisted Cloning (PODAC).

    PubMed

    Van Hove, Bob; Guidi, Chiara; De Wannemaeker, Lien; Maertens, Jo; De Mey, Marjan

    2017-06-16

    A problem rarely tackled by current DNA assembly methods is the issue of cloning additional parts into an already assembled construct. Costly PCR workflows are often hindered by repeated sequences, and restriction based strategies impose design constraints for each enzyme used. Here we present Protected Oligonucleotide Duplex Assisted Cloning (PODAC), a novel technique that makes use of an oligonucleotide duplex for iterative Golden Gate cloning using only one restriction enzyme. Methylated bases confer protection from digestion during the assembly reaction and are removed during replication in vivo, unveiling a new cloning site in the process. We used this method to efficiently and accurately assemble a biosynthetic pathway and demonstrated its robustness toward sequence repeats by constructing artificial CRISPR arrays. As PODAC is readily amenable to standardization, it would make a useful addition to the synthetic biology toolkit.

  6. 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.

  7. Molecular and biochemical evidence on the protective role of ellagic acid and silybin against oxidative stress-induced cellular aging.

    PubMed

    Baeeri, Maryam; Mohammadi-Nejad, Solmaz; Rahimifard, Mahban; Navaei-Nigjeh, Mona; Moeini-Nodeh, Shermineh; Khorasani, Reza; Abdollahi, Mohammad

    2017-09-08

    Aging is a natural process in living organisms that is defined by some molecular and cellular changes with time. Various causes such as mitochondrial DNA aberrations, aggregation of proteins, telomere shortening, and oxidative stress have an influential role in aging of the cells. Natural antioxidants are compounds that are potent to protect the body from detrimental effects of molecules such as free radicals. The aim of this study was to evaluate the anti-aging properties of ellagic acid (EA) and silybin (SIL), as natural antioxidant compounds on rat embryonic fibroblast (REF) cells. These cells were pre-incubated with EA and SIL, thereafter were exposed to hydrogen peroxide (H2O2). Then, the cell viability, SA-β-GAL activity, distribution of cell cycle, NF-κB, and mitochondrial complex I, II/IV enzyme activity were measured. The results of this study revealed the protective effects of EA and SIL in H2O2-treated REF cells, which confirm the previous achieved data on antioxidant and anti-inflammatory characteristics of EA and SIL against H2O2 in the treated REF cells. However, more new in vivo experiments are required to discover the anti-aging effects and mechanism of action of such compounds.

  8. 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

  9. Role of DNA repair enzymes in the cellular resistance to oxidative stress.

    PubMed

    Laval, J

    1996-01-01

    Oxidative stress occurs in cells when the equilibrium between prooxidant and antioxidant species is broken in favor of the prooxidant state. It is due to reactive oxygen species (ROS) generated either by the cellular metabolism such as phagocytosis, mitochondrial respiration, xenobiotic detoxification, or by exogenous factors such as ionizing radiation or chemical compounds performing red-ox reactions. Some ROS are extremely reactive and interact with all the macromolecules including lipids, nucleic acids and proteins. Cells have numerous defence systems to counteract the deleterious effects of ROS. Proteins and small molecules specifically eliminate ROS when they are formed. There are three species of superoxyde dismutases which transform the superoxyde anion O2- in hydrogen peroxyde H2O2 which in turn will be destroyed by peroxysomal catalase or by various peroxydases. There are numerous small molecules in the cell such as glutathion, alpha-tocopherol, vitamines A and C, melanine, etc. which are antioxydant molecules. ROS escaping destruction generate various lesions in DNA such as base modifications, degradation products of deoxyribose, chain breaks. These various lesions have been characterized and it is possible to quantitate them in the DNA of cells which have been irradiated or treated by free radical generating systems. The biological properties of the bases modified by ROS have been established. For example C8-hydroxyguanine (8-oxoG) is promutagenic since, if present in DNA during replication, it leads to incorporation of dAMP residues, leading to transversion mutation (GC-->TA). Purines whose imidazole ring is opened (Fapy residues) are stops for the DNA polymerase during DNA replication and are therefore potentially lethal lesions for the cell. Oxidized pyrimidines have comparable coding properties. Efficient DNA repair mechanisms remove these oxidized bases. In Escherichia coli cells, endonuclease III (NTH protein) and endonuclease VIII (NEI protein

  10. Improvement influenza HA2 DNA vaccine cellular and humoral immune responses with Mx bio adjuvant.

    PubMed

    Soleimani, Sina; Shahsavandi, Shahla; Maddadgar, Omid

    2017-03-01

    Immunization with DNA vaccines as a novel alternative to conventional vaccination strategy requires adjuvant for improving vaccine efficacy. The conserved immunogenic HA2 subunit, which harbors neutralizing epitopes is a promising vaccine candidate against influenza viruses. In this study, for the first time we explore the idea of using host interferon inducible Mx protein to increase the immunogenicity of HA2 H9N2 influenza DNA vaccine. The potency and safety of the Mx adjuvanted-HA2 vaccine was evaluated in BALB/c mice by different prime-boost strategies. To assess the effect of the vaccination on the virus clearance rate, mice were challenged with homologous influenza virus. Administration of the adjuvanted vaccine and boosting with the same regimen could effectively enhance both humoral and cellular immune responses in treated mice. These data demonstrated that Mx as host defense peptide can be potentiated for improving influenza vaccine efficacy.

  11. A possible mechanism for combined arsenic and fluoride induced cellular and DNA damage in mice.

    PubMed

    Flora, Swaran J S; Mittal, Megha; Pachauri, Vidhu; Dwivedi, Nidhi

    2012-01-01

    Arsenic and fluoride are major contaminants of drinking water. Mechanisms of toxicity following individual exposure to arsenic or fluoride are well known. However, it is not explicit how combined exposure to arsenic and fluoride leads to cellular and/or DNA damage. The present study was planned to assess (i) oxidative stress during combined chronic exposure to arsenic and fluoride in drinking water, (ii) correlation of oxidative stress with cellular and DNA damage and (iii) mechanism of cellular damage using IR spectroscopy. Mice were exposed to arsenic and fluoride (50 ppm) either individually or in combination for 28 weeks. Arsenic or fluoride exposure individually led to a significant increase in reactive oxygen species (ROS) generation and associated oxidative stress in blood, liver and brain. Individual exposure to the two toxicants showed significant depletion of blood glutathione (GSH) and glucose 6-phosphate dehydrogenase (G6PD) activity, and single-stranded DNA damage using a comet assay in lymphocytes. We also observed an increase in the activity of ATPase, thiobarbituric acid reactive substance (TBARS) and a decreased, reduced and oxidized glutathione (GSH : GSSG) ratio in the liver and brain. Antioxidant enzymes like superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were decreased and increased in liver and brain respectively. The changes were more pronounced in liver compared to brain suggesting liver to be more susceptible to the toxic effects of arsenic and fluoride. Interestingly, combined exposure to arsenic and fluoride resulted in less pronounced toxic effects compared to their individual effects based on biochemical variables, IR spectra, DNA damage (TUNEL and comet assays) and histopathological observations. IR spectra suggested that arsenic or fluoride perturbs the strength of protein and amide groups; however, the shifts in peaks were not pronounced during combined exposure. These results thus highlight the role of

  12. The DNA-bending protein HMGB1 is a cellular cofactor of Sleeping Beauty transposition.

    PubMed

    Zayed, Hatem; Izsvák, Zsuzsanna; Khare, Dheeraj; Heinemann, Udo; Ivics, Zoltán

    2003-05-01

    Sleeping Beauty (SB) is the most active Tc1/ mariner-type transposon in vertebrates. SB contains two transposase-binding sites (DRs) at the end of each terminal inverted repeat (IR), a feature termed the IR/DR structure. We investigated the involvement of cellular proteins in the regulation of SB transposition. Here, we establish that the DNA-bending, high-mobility group protein, HMGB1 is a host-encoded cofactor of SB transposition. Transposition was severely reduced in mouse cells deficient in HMGB1. This effect was rescued by transient over-expression of HMGB1, and was partially complemented by HMGB2, but not with the HMGA1 protein. Over-expression of HMGB1 in wild-type mouse cells enhanced transposition, indicating that HMGB1 can be a limiting factor of transposition. SB transposase was found to interact with HMGB1 in vivo, suggesting that the transposase may recruit HMGB1 to transposon DNA. HMGB1 stimulated preferential binding of the transposase to the DR further from the cleavage site, and promoted bending of DNA fragments containing the transposon IR. We propose that the role of HMGB1 is to ensure that transposase-transposon complexes are first formed at the internal DRs, and subsequently to promote juxtaposition of functional sites in transposon DNA, thereby assisting the formation of synaptic complexes.

  13. DNA methylation control of tissue polarity and cellular differentiation in the mammary epithelium.

    PubMed

    Plachot, Cedric; Lelièvre, Sophie A

    2004-08-01

    Alterations in gene expression accompany cell-type-specific differentiation. In complex systems where functional differentiation depends on the organization of specific cell types into highly specialized structures (tissue morphogenesis), it is not known how epigenetic mechanisms that control gene expression influence this stepwise differentiation process. We have investigated the effect of DNA methylation, a major epigenetic pathway of gene silencing, on the regulation of mammary acinar differentiation. Our in vitro model of differentiation encompasses human mammary epithelial cells that form polarized and hollow tissue structures (acini) when cultured in the presence of basement membrane components. We found that acinar morphogenesis was accompanied with chromatin remodeling, as shown by alterations in histone 4 acetylation, heterochromatin 1 protein, and histone 3 methylated on lysine 9, and with an increase in expression of MeCP2, a mediator of DNA-methylation-induced gene silencing. DNA hypomethylation induced by treatment with 5-aza-2' deoxycytidine during acinar differentiation essentially prevented the formation of apical tissue polarity. This treatment also induced the expression of CK19, a marker of cells that are in a transitional differentiation stage. These results suggest that DNA methylation is a mechanism by which mammary epithelial differentiation is coordinated both at the tissue and cellular levels.

  14. Germ-line variant of human NTH1 DNA glycosylase induces genomic instability and cellular transformation.

    PubMed

    Galick, Heather A; Kathe, Scott; Liu, Minmin; Robey-Bond, Susan; Kidane, Dawit; Wallace, Susan S; Sweasy, Joann B

    2013-08-27

    Base excision repair (BER) removes at least 20,000 DNA lesions per human cell per day and is critical for the maintenance of genomic stability. We hypothesize that aberrant BER, resulting from mutations in BER genes, can lead to genomic instability and cancer. The first step in BER is catalyzed by DNA N-glycosylases. One of these, n(th) endonuclease III-like (NTH1), removes oxidized pyrimidines from DNA, including thymine glycol. The rs3087468 single nucleotide polymorphism of the NTH1 gene is a G-to-T base substitution that results in the NTH1 D239Y variant protein that occurs in ∼6.2% of the global population and is found in Europeans, Asians, and sub-Saharan Africans. In this study, we functionally characterize the effect of the D239Y variant expressed in immortal but nontransformed human and mouse mammary epithelial cells. We demonstrate that expression of the D239Y variant in cells also expressing wild-type NTH1 leads to genomic instability and cellular transformation as assessed by anchorage-independent growth, focus formation, invasion, and chromosomal aberrations. We also show that cells expressing the D239Y variant are sensitive to ionizing radiation and hydrogen peroxide and accumulate double strand breaks after treatment with these agents. The DNA damage response is also activated in D239Y-expressing cells. In combination, our data suggest that individuals possessing the D239Y variant are at risk for genomic instability and cancer.

  15. Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences (a review).

    PubMed

    Bertin, G; Averbeck, D

    2006-11-01

    Cadmium is an important toxic environmental heavy metal. Occupational and environmental pollution with cadmium results mainly from mining, metallurgy industry and manufactures of nickel-cadmium batteries, pigments and plastic stabilizers. Important sources of human intoxication are cigarette smoke as well as food, water and air contaminations. In humans, cadmium exposures have been associated with cancers of the prostate, lungs and testes. Acute exposures are responsible for damage to these organs. Chronic intoxication is associated with obstructive airway disease, emphysema, irreversible renal failure, bone disorders and immuno-suppression. At the cellular level, cadmium affects proliferation, differentiation and causes apoptosis. It has been classified as a carcinogen by the International Agency for Research on Cancer (IARC). However, it is weakly genotoxic. Indirect effects of cadmium provoke generation of reactive oxygen species (ROS) and DNA damage. Cadmium modulates also gene expression and signal transduction, reduces activities of proteins involved in antioxidant defenses. Several studies have shown that it interferes with DNA repair. The present review focuses on the effects of cadmium in mammalian cells with special emphasis on the induction of damage to DNA, membranes and proteins, the inhibition of different types of DNA repair and the induction of apoptosis. Current data and hypotheses on the mechanisms involved in cadmium genotoxicity and carcinogenesis are outlined.

  16. DNA-protected silver emitters: charge dependent switching of fluorescence.

    PubMed

    Berdakin, Matías; Taccone, Martin I; Pino, Gustavo A; Sánchez, Cristián G

    2017-02-22

    The relationship between the state of charge and spectroscopy of DNA-protected silver emitters is not yet well understood. This remains one of the major issues to unveil in order to fully disentangle the spectroscopic features of these novel systems. It is a well known fact that a fluorescence response arises upon chemical reduction of silver cations attached to DNA, leading to neutral (or partially oxidized) "bright" clusters. It is important to note that the absence of fluorescence in completely ionic complexes is universal in the sense that it does not depend on any experimental variable. This suggests that its origin may be founded on the nature of the interaction between DNA bases and silver cations. Nevertheless, to the best of our knowledge, no explanation exists for this charge dependent switching between dark completely ionic complexes and bright (neutral or partially oxidized) clusters. In this brief report we address this experimental fact on the basis of the electronic structure of the complex as a function of its charge and quantum dynamical simulations of the processes following photoexcitation. These data provide a dynamical picture of the correlation between charge and fluorescence.

  17. Coupled actin-lamin biopolymer networks and protecting DNA

    NASA Astrophysics Data System (ADS)

    Zhang, Tao; Rocklin, D. Zeb; Mao, Xiaoming; Schwarz, J. M.

    The mechanical properties of cells are largely determined by networks of semiflexible biopolymers forming the cytoskeleton. Similarly, the mechanical properties of cell nuclei are also largely determined by networks of semiflexible biopolymers forming the nuclear cytoskeleton. In particular, a network of filamentous lamin sits just inside the inner nuclear membrane to presumably protect the heart of the cell nucleus--the DNA. It has been demonstrated over the past decade that the actin cytoskeletal biopolymer network and the lamin biopolymer network are coupled via a sequence of proteins bridging the outer and inner nuclear membranes, known as the LINC complex. We, therefore, probe the consequences of such a coupling in a model biopolymer network system via numerical simulations to understand the resulting deformations in the lamin network in response to perturbations in the actin cytoskeletal network. We find, for example, that the force transmission across the coupled system can depend sensitively on the concentration of LINC complexes. Such study could have implications for mechanical mechanisms of the regulation of transcription since DNA couples to lamin via lamin-binding domains so that deformations in the lamin network may result in deformations in the DNA.

  18. Radiation-induced damage to cellular DNA: Chemical nature and mechanisms of lesion formation

    NASA Astrophysics Data System (ADS)

    Cadet, Jean; Wagner, J. Richard

    2016-11-01

    This mini-review focuses on the recent identification of several novel radiation-induced single and tandem modifications in cellular DNA. For this purpose accurate high-performance electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was applied allowing their quantitative measurement and unambiguous characterization. Exposure of human cells to gamma rays led to the formation of several modified bases arising from the rearrangement of the pyrimidine ring of thymine, cytosine and 5-methylcytosine subsequent to initial addition of an hydroxyl radical (•OH) to the 5,6-ethylenic bond. In addition, 5-hydroxymethylcytosine, an novel epigenetic mark, and 5-formylcytosine, were found to be generated consecutively to •OH-mediated hydrogen abstraction from the methyl group of 5-methylcytosine. Relevant mechanistic information on one-oxidation reactions of cellular DNA was also gained from the detection of 5-hydroxycytosine and guanine-thymine intra-strand adducts whose formation is rationalized by the generation of related base radical cation. Attempts to search for the radiation-induced formation of purine 5‧,8-cyclo-2‧-deoxyribonucleosides were unsuccessful with the exception of trace amounts of (5‧S)-5‧,8-cyclo-2‧-deoxyadenosine.

  19. SIRT6 Depletion Suppresses Tumor Growth by Promoting Cellular Senescence Induced by DNA Damage in HCC.

    PubMed

    Lee, Namgyu; Ryu, Hye Guk; Kwon, Jung-Hee; Kim, Dae-Kyum; Kim, Sae Rom; Wang, Hee Jung; Kim, Kyong-Tai; Choi, Kwan Yong

    2016-01-01

    The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC.

  20. SIRT6 Depletion Suppresses Tumor Growth by Promoting Cellular Senescence Induced by DNA Damage in HCC

    PubMed Central

    Lee, Namgyu; Ryu, Hye Guk; Kwon, Jung-Hee; Kim, Dae-Kyum; Kim, Sae Rom; Wang, Hee Jung; Kim, Kyong-Tai; Choi, Kwan Yong

    2016-01-01

    The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC. PMID:27824900

  1. DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis.

    PubMed

    Schmitt, Estelle; Paquet, Claudie; Beauchemin, Myriam; Bertrand, Richard

    2007-06-01

    Tissue homeostasis requires a carefully-orchestrated balance between cell proliferation, cellular senescence and cell death. Cells proliferate through a cell cycle that is tightly regulated by cyclin-dependent kinase activities. Cellular senescence is a safeguard program limiting the proliferative competence of cells in living organisms. Apoptosis eliminates unwanted cells by the coordinated activity of gene products that regulate and effect cell death. The intimate link between the cell cycle, cellular senescence, apoptosis regulation, cancer development and tumor responses to cancer treatment has become eminently apparent. Extensive research on tumor suppressor genes, oncogenes, the cell cycle and apoptosis regulatory genes has revealed how the DNA damage-sensing and -signaling pathways, referred to as the DNA-damage response network, are tied to cell proliferation, cell-cycle arrest, cellular senescence and apoptosis. DNA-damage responses are complex, involving "sensor" proteins that sense the damage, and transmit signals to "transducer" proteins, which, in turn, convey the signals to numerous "effector" proteins implicated in specific cellular pathways, including DNA repair mechanisms, cell-cycle checkpoints, cellular senescence and apoptosis. The Bcl-2 family of proteins stands among the most crucial regulators of apoptosis and performs vital functions in deciding whether a cell will live or die after cancer chemotherapy and irradiation. In addition, several studies have now revealed that members of the Bcl-2 family also interface with the cell cycle, DNA repair/recombination and cellular senescence, effects that are generally distinct from their function in apoptosis. In this review, we report progress in understanding the molecular networks that regulate cell-cycle checkpoints, cellular senescence and apoptosis after DNA damage, and discuss the influence of some Bcl-2 family members on cell-cycle checkpoint regulation.

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

    PubMed

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

    2014-10-03

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

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

    PubMed Central

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

    2013-01-01

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

  4. Insulin Protects Pancreatic Acinar Cells from Palmitoleic Acid-induced Cellular Injury*

    PubMed Central

    Samad, Aysha; James, Andrew; Wong, James; Mankad, Parini; Whitehouse, John; Patel, Waseema; Alves-Simoes, Marta; Siriwardena, Ajith K.; Bruce, Jason I. E.

    2014-01-01

    Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca2+ ([Ca2+]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca2+]i are linked critically by the ATP-driven plasma membrane Ca2+-ATPase (PMCA) important for maintaining low resting [Ca2+]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca2+]i was measured by fura-2 imaging. An in situ [Ca2+]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca2+ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca2+ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca2+ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca2+ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis. PMID:24993827

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

    PubMed

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

    1997-01-01

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

  6. A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR.

    PubMed

    Norton, S E; Lechner, J M; Williams, T; Fernando, M R

    2013-10-01

    To study the ability of a stabilizing reagent to prevent cellular DNA contamination of cell-free DNA (cfDNA) in plasma during whole blood sample storage and shipping. Samples were drawn from healthy donors into K₃EDTA and Cell-Free DNA BCTs (BCT) and stored at room temperature (RT). Aliquots were removed at specified time points and cfDNA was purified from the plasma. A Droplet Digital PCR (ddPCR) assay that amplifies a short β-actin gene fragment (136 bp) was used to measure the total plasma cfDNA (pDNA) concentration while a longer β-actin fragment (420 bp) was used to quantify genomic DNA (gDNA). In a follow-up experiment, blood samples drawn into the same types of tubes were shipped round trip by overnight air before cfDNA was isolated and analyzed. Blood stored in K₃EDTA tubes at RT showed increases in pDNA and gDNA concentrations over time. However, both pDNA and gDNA levels remained stable in BCT for at least seven days. On day 14, there was a 4.5-fold increase in pDNA in BCT as compared to >200-fold increase in K₃EDTA tubes. Likewise, gDNA increased <2-fold on day 14 in BCT as opposed to a 456-fold increase in K₃EDTA tubes. Similar results were observed after samples were shipped. Cell-Free DNA BCTs prevent gDNA contamination that may occur due to nucleated cell disruption during sample storage and shipping. This novel blood collection tube provides a method for obtaining stable cfDNA samples for rare target detection and accurate analysis while mitigating the threat of gDNA contamination. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

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

    SciTech Connect

    Not Available

    1988-01-01

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

  8. The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia

    PubMed Central

    Danilova, Nadia; Bibikova, Elena; Covey, Todd M.; Nathanson, David; Dimitrova, Elizabeth; Konto, Yoan; Lindgren, Anne; Glader, Bertil; Radu, Caius G.; Sakamoto, Kathleen M.; Lin, Shuo

    2014-01-01

    Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis – a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA. PMID:24812435

  9. DNA vaccine encoding Middle East respiratory syndrome coronavirus S1 protein induces protective immune responses in mice.

    PubMed

    Chi, Hang; Zheng, Xuexing; Wang, Xiwen; Wang, Chong; Wang, Hualei; Gai, Weiwei; Perlman, Stanley; Yang, Songtao; Zhao, Jincun; Xia, Xianzhu

    2017-04-11

    The Middle East respiratory syndrome coronavirus (MERS-CoV), is an emerging pathogen that continues to cause outbreaks in the Arabian peninsula and in travelers from this region, raising the concern that a global pandemic could occur. Here, we show that a DNA vaccine encoding the first 725 amino acids (S1) of MERS-CoV spike (S) protein induces antigen-specific humoral and cellular immune responses in mice. With three immunizations, high titers of neutralizing antibodies (up to 1: 10(4)) were generated without adjuvant. DNA vaccination with the MERS-CoV S1 gene markedly increased the frequencies of antigen-specific CD4(+) and CD8(+) T cells secreting IFN-γ and other cytokines. Both pcDNA3.1-S1 DNA vaccine immunization and passive transfer of immune serum from pcDNA3.1-S1 vaccinated mice protected Ad5-hDPP4-transduced mice from MERS-CoV challenge. These results demonstrate that a DNA vaccine encoding MERS-CoV S1 protein induces strong protective immune responses against MERS-CoV infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Hydroxytyrosol protects against oxidative DNA damage in human breast cells.

    PubMed

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

    2011-10-01

    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.

  11. 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

  12. Protective effect of N-acetylcysteine against radiation induced DNA damage and hepatic toxicity in rats.

    PubMed

    Mansour, Heba H; Hafez, Hafez F; Fahmy, Nadia M; Hanafi, Nemat

    2008-02-01

    The present study was designed to evaluate the radioprotective effect of N- acetylcysteine (NAC) on gamma-radiation induced toxicity in hepatic tissue in rat. The cellular changes were estimated using malondialdehyde (MDA, an index of lipid peroxidation), superoxide dismutase (SOD), glutathione peroxidase (GSHPx), reduced glutathione (GSH), and total nitrate/nitrite (NO(x)) as markers of hepatic oxidative stress in rats following gamma-irradiation. The DNA damage was determined by agarose gel electrophoresis. To achieve the ultimate goal of this study, 40 adult rats were randomly divided into 4 groups of 10 animals each. Group I was injected intraperitoneally with saline solution for 7 consecutive days and served as control group. Group II was irradiated with a single dose of 6Gy gamma-radiation. Group III was daily injected with NAC (1g/kg, i.p.) for 7 consecutive days. Group IV received a daily i.p. injection of NAC (1g/kg, i.p.) for 7 consecutive days and 1h after the last dose, rats were irradiated with a single dose (6Gy) gamma-radiation. The animals were sacrificed after 24h. DNA damage was observed in tissue after total body irradiation with a single dose of 6Gy. Malondialdehyde and total nitrate/nitrite were increased significantly whereas the levels of GSH and antioxidant enzymes were significantly decreased in gamma-irradiated group. Pretreatment with NAC showed a significant decrease in the levels of MDA, NO(x) and DNA damage. The antioxidant enzymes increased significantly along with the levels of GSH. Moreover, histopathological examination of liver tissues confirmed the biochemical data. Thus, our results show that pretreatment with N-acetylcysteine offers protection against gamma-radiation induced cellular damage.

  13. Induction of a Cellular DNA Damage Response by Porcine Circovirus Type 2 Facilitates Viral Replication and Mediates Apoptotic Responses

    PubMed Central

    Wei, Li; Zhu, Shanshan; Wang, Jing; Quan, Rong; Yan, Xu; Li, Zixue; Hou, Lei; Wang, Naidong; Yang, Yi; Jiang, Haijun; Liu, Jue

    2016-01-01

    Cellular DNA damage response (DDR) triggered by infection of DNA viruses mediate cell cycle checkpoint activation, DNA repair, or apoptosis induction. In the present study, infection of porcine circovirus type 2 (PCV2), which serves as a major etiological agent of PCV2-associated diseases (PCVAD), was found to elicit a DNA damage response (DDR) as observed by the phosphorylation of H2AX and RPA32 following infection. The response requires active viral replication, and all the ATM (ataxia telangiectasia-mutated kinase), ATR (ATM- and Rad3-related kinase), and DNA-PK (DNA-dependent protein kinase) are the transducers of the DDR signaling events in the PCV2-infected cells as demonstrated by the phosphorylation of ATM, ATR, and DNA-PK signalings as well as reductions in their activations after treatment with specific kinase inhibitors. Inhibitions of ATM, ATR, and DNA-PK activations block viral replication and prevent apoptotic responses as observed by decreases in cleaved poly-ADP ribose polymerase (PARP) and caspase-3 as well as fragmented DNA following PCV2 infection. These results reveal that PCV2 is able to exploit the cellular DNA damage response machinery for its own efficient replication and for apoptosis induction, further extending our understanding for the molecular mechanism of PCV2 infection. PMID:27982097

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

    SciTech Connect

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

    1991-01-01

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

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

    SciTech Connect

    Roper, Katherine; Coverley, Dawn

    2012-03-10

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

  16. Protective mechanism of morin against ultraviolet B-induced cellular senescence in human keratinocyte stem cells.

    PubMed

    Lee, Jienny; Shin, Yeun-Kyung; Song, Jae-Young; Lee, Kyung-Woo

    2014-01-01

    Ultraviolet-B (UVB) irradiation is a major inducer of DNA damage in the epidermis. Here we investigated the protective mechanism of polyphenolic phytonutrient, morin against UVB-induced DNA damage in human keratinocyte stem cells (KSC). After confirming the characteristics of the KSC, we examined the protective ability of morin against the cell damage of KSC under UVB irradiation condition. As a result, morin significantly inhibited the UVB-induced damage to KSC. These inhibitory effects by morin were also confirmed by the senescence-associated beta-galactosidase and alkaline comet assays. Next, we monitored the effects of morin on the UVB-induced production of inflammatory cytokines. Morin significantly decreased the production of tumor necrosis factor-α, interleukin-1β, and interleukin-6 in the UVB-irradiated KSC. Also, morin significantly inhibited the UVB-induced phosphorylation of ataxia telangiectasia mutated (ATM), serine threonine kinase checkpoint kinase 2, tumor suppressor protein 53 (p53), c-Jun N-terminal kinase/stress-activated protein kinase, p38/mitogen-activated protein kinase, S6 ribosomal protein, and histone 2A family member X in KSC. Furthermore, while UVB irradiation induced p53 reporter activation in KSC, morin significantly inhibited UVB-induced p53 reporter activation in KSC. In addition, mouse double minute 2 homolog (MDM2, p53 E3 ubiquitin protein ligase) inhibitor significantly increased the p53 reporter activation in the UVB-irradiated KSC, but morin decreased the MDM2 inhibitor-mediated increase in p53 reporter activation. On the contrary, ATM inhibitor did not affect the protective effect of morin in UVB irradiation-induced p53 reporter activation. Collectively, these findings suggest that morin could effectively enrich the p53 specific ligasing ability of MDM2 in UVB irradiation-induced p53 activation.

  17. Erythropoietin Protects against Murine Cerebral Malaria through Actions on Host Cellular Immunity

    PubMed Central

    Wei, Xu; Li, Ying; Sun, Xiaodan; Zhu, Xiaotong; Feng, Yonghui; Liu, Jun; Jiang, Yongjun; Shang, Hong

    2014-01-01

    Cerebral malaria (CM) is associated with excessive host proinflammatory responses and endothelial activation. The hematopoietic hormone erythropoietin (EPO) possesses neuroprotective functions in animal models of ischemic-hypoxic, traumatic, and inflammatory injuries. In the Plasmodium berghei ANKA model of experimental CM (ECM), recombinant human EPO (rhEPO) has shown evident protection against ECM. To elucidate the mechanism of EPO in this ECM model, we investigated the effect of rhEPO on host cellular immune responses. We demonstrated that improved survival of mice with ECM after rhEPO treatment was associated with reduced endothelial activation and improved integrity of the blood-brain barrier. Our results revealed that rhEPO downregulated the inflammatory responses by directly inhibiting the levels and functions of splenic dendritic cells. Conversely, rhEPO treatment led to significant expansion of regulatory T cells and increased expression of the receptor cytotoxic T lymphocyte antigen 4 (CTLA-4). The data presented here provide evidence of the direct effect of rhEPO on host cellular immunity during ECM. PMID:24126529

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Hani, Saleem Bani; Bayachou, Mekki

    2014-01-01

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

  20. Protective immune response in mice induced by a suicidal DNA vaccine encoding NTPase-II gene of Toxoplasma gondii.

    PubMed

    Zheng, Lina; Hu, Yue; Hua, Qianqian; Luo, Fangjun; Xie, Guizhen; Li, Xiangzhi; Lin, Jiaxin; Wan, Yujing; Ren, Shoufeng; Pan, Changwang; Tan, Feng

    2017-02-01

    DNA-based alphaviral RNA replicon vectors, also called suicidal DNA vectors, have been employed to alleviate biosafety concerns attribution to its ability to induce apoptotic cell death of the transfected cells. Toxoplasma gondii nucleoside triphosphate hydrolase-II (TgNTPase-II), which facilitates the parasite to salvage purines from the host cell for survival and replication, have been demonstrated to be a potential vaccine candidate for toxoplasmosis. Herein, we evaluated the immunogenic potential of a suicidal DNA vaccine encoding TgNTPase-II gene, pDREP-TgNTPase-II, delivered intramuscularly in combination with electroporation. Immunization of mice with pDREP-TgNTPase-II elicited specific humoral responses, with high IgG antibody titers and a mixed IgG1/IgG2a response. The cellular immune response was associated with high level production of IFN-γ, IL-2, IL-10 cytokines and low level IL-4 production as well as the increase of the percentage of CD8+ T cells, indicating that a Th1 predominant response was elicited. Furthermore, mice vaccinated with this suicidal DNA vaccine displayed partial protection against acute infection with the virulent RH strain as well as chronic infection with PRU cyst, which shows 77.7% and 71.4% reduction in brain cyst burden in comparison to PBS and pDREP-eGFP control group, respectively. Based on the cellular and antibody responses, the suicidal DNA vaccine elicited a Th1-predominant immune response against T. gondii challenge.

  1. Comparison of food antioxidants and iron chelators in two cellular free radical assays: strong protection by luteolin.

    PubMed

    Hofer, Tim; Jørgensen, Trond Ø; Olsen, Ragnar L

    2014-08-20

    Liver (HepG2) cells were incubated with 21 edible flavonoids, carotenoids, polyunsaturated fatty acid (PUFA) chromones, and metal chelators for 1 h, washed in PBS, and challenged in the cellular antioxidant activity (CAA) and the cellular lipid peroxidation antioxidant activity (CLPAA) assays. These microplate format assays assess the compounds' ability to protect against cytosolic peroxyl radicals (CAA) and induced membrane lipid peroxidation (CLPAA), respectively. Incubation encompassing a broad compound concentration range determined half-maximal inhibitory concentrations (IC(50)) by using sigmoidal curve fits. Overall, considering both assays, luteolin offered the greatest protection. The carotenoid astaxanthin offered only modest protection, whereas β-carotene was ineffective. Subtle structural differences between flavonoids were found to have amplified effects on protective abilities, and mechanisms of flavonoid antioxidant action are discussed. Membrane-permeable iron chelators (deferasirox and SIH) offered strong protective effects in CLPAA, but not in CAA, suggesting that CLPAA is dependent on membrane-associated free iron ions.

  2. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

    PubMed Central

    Macurek, Libor; Benada, Jan; Müllers, Erik; Halim, Vincentius A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Sona; Hodný, Zdeněk; Lindqvist, Arne; Medema, René H.; Bartek, Jiri

    2013-01-01

    Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G1 phase to G2 and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G1 cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression. PMID:23255129

  3. pH-sensitive carbonate apatite nanoparticles as DNA vaccine carriers enhance humoral and cellular immunity.

    PubMed

    He, Pan; Takeshima, Shin-nosuke; Tada, Seiichi; Akaike, Toshihiro; Ito, Yoshihiro; Aida, Yoko

    2014-10-29

    To demonstrate the potential of pH-sensitive carbonate apatite (CO₃Ap) nanoparticles as DNA vaccine carriers to enhance vaccination efficacy, we examined the humoral and cellular immune responses of C57BL/6 mice immunized with the plasmid expression vector pCI-neo encoding the full-length soluble ovalbumin (OVA) (pCI-neo-sOVA), pCI-neo-sOVA/CO₃Ap complexes, or pCI-neo/CO₃Ap complexes as a control. Mice immunized with a low dose of pCI-neo-sOVA-loaded CO₃Ap (10 μg) produced ex vivo splenocyte proliferation after stimulation with CD8 T-cell but not CD4 T-cell epitopes and a delayed-type-hypersensitivity reaction more efficiently than mice in the other groups. Furthermore, mice receiving this immunization generated the same levels of OVA-specific antibodies and interferon (IFN)-γ secretion after CD8 T-cell and CD4 T-cell epitope challenges as those in mice treated with 100 μg of free pCI-neo-sOVA, whereas mice injected with a high dose of pCI-neo-sOVA-loaded CO₃Ap (100 μg) or with control plasmids produced negligible levels of OVA-specific antibodies or IFN-γ. Therefore, our results showed that 10 μg of pCI-neo-sOVA delivered by CO₃Ap strongly elicited humoral and cellular immune responses. This study is the first to demonstrate the promising potential of CO₃Ap nanoparticles for DNA vaccine delivery.

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

    SciTech Connect

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

    2014-10-03

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

  5. Melatonin enhances thermotolerance by promoting cellular protein protection in tomato plants.

    PubMed

    Xu, Wen; Cai, Shu-Yu; Zhang, Yun; Wang, Yu; Ahammed, Golam Jalal; Xia, Xiao-Jian; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Reiter, Russel J; Zhou, Jie

    2016-11-01

    Melatonin is a pleiotropic signaling molecule that provides physiological protection against diverse environmental stresses in plants. Nonetheless, the mechanisms for melatonin-mediated thermotolerance remain largely unknown. Here, we report that endogenous melatonin levels increased with a rise in ambient temperature and that peaked at 40°C. Foliar pretreatment with an optimal dose of melatonin (10 μmol/L) or the overexpression of N-acetylserotonin methyltransferase (ASMT) gene effectively ameliorated heat-induced photoinhibition and electrolyte leakage in tomato plants. Both exogenous melatonin treatment and endogenous melatonin manipulation by overexpression of ASMT decreased the levels of insoluble and ubiquitinated proteins, but enhanced the expression of heat-shock proteins (HSPs) to refold denatured and unfolded proteins under heat stress. Meanwhile, melatonin also induced expression of several ATG genes and formation of autophagosomes to degrade aggregated proteins under the same stress. Proteomic profile analyses revealed that protein aggregates for a large number of biological processes accumulated in wild-type plants. However, exogenous melatonin treatment or overexpression of ASMT reduced the accumulation of aggregated proteins. Aggregation responsive proteins such as HSP70 and Rubisco activase were preferentially accumulated and ubiquitinated in wild-type plants under heat stress, while melatonin mitigated heat stress-induced accumulation and ubiquitination of aggregated proteins. These results suggest that melatonin promotes cellular protein protection through induction of HSPs and autophagy to refold or degrade denatured proteins under heat stress in tomato plants. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Early warning of illegal development for protected areas by integrating cellular automata with neural networks.

    PubMed

    Li, Xia; Lao, Chunhua; Liu, Yilun; Liu, Xiaoping; Chen, Yimin; Li, Shaoying; Ai, Bing; He, Zijian

    2013-11-30

    Ecological security has become a major issue under fast urbanization in China. As the first two cities in this country, Shenzhen and Dongguan issued the ordinance of Eco-designated Line of Control (ELC) to "wire" ecologically important areas for strict protection in 2005 and 2009 respectively. Early warning systems (EWS) are a useful tool for assisting the implementation ELC. In this study, a multi-model approach is proposed for the early warning of illegal development by integrating cellular automata (CA) and artificial neural networks (ANN). The objective is to prevent the ecological risks or catastrophe caused by such development at an early stage. The integrated model is calibrated by using the empirical information from both remote sensing and handheld GPS (global positioning systems). The MAR indicator which is the ratio of missing alarms to all the warnings is proposed for better assessment of the model performance. It is found that the fast urban development has caused significant threats to natural-area protection in the study area. The integration of CA, ANN and GPS provides a powerful tool for describing and predicting illegal development which is in highly non-linear and fragmented forms. The comparison shows that this multi-model approach has much better performances than the single-model approach for the early warning. Compared with the single models of CA and ANN, this integrated multi-model can improve the value of MAR by 65.48% and 5.17% respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Physicochemical properties and cellular protection against oxidation of degraded Konjac glucomannan prepared by γ-irradiation.

    PubMed

    Jian, Wenjie; Tu, Liying; Wu, Lanlan; Xiong, Hejian; Pang, Jie; Sun, Yuan-Ming

    2017-09-15

    Konjac glucomannan (KGM) is an important functional polysaccharide in food research. However, unstable dispersibility of KGM inhibits its in-depth study and wide application. In this study, a degraded KGM (100kGy-KGM), which showed excellent dispersibility and specific physicochemical properties, were obtained by γ-irradiation in a dosage of 100kGy. We investigated the protective effect of 100kGy-KGM against H2O2 induced oxidative damage in LO2 cells. Our results demonstrated that pretreatment of LO2 cells with 100kGy-KGM not only significantly increased cellular survivals and activities of GSH-Px and CAT, but also reduced levels of LDH, MDA and intracellular accumulation of ROS. The marked protective effect against oxidative damage and excellent dispersibility in 100kGy-KGM allowed its possible use as an antioxidant. Our study provided fundamental knowledge to understand the structure-functions relationships of degraded-KGM, which could result in a theoretical guidance for the future application of KGM. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Cellular glutathione peroxidase protects mice against lethal oxidative stress induced by various doses of diquat.

    PubMed

    Fu, Y; Cheng, W H; Ross, D A; Lei, X g

    1999-11-01

    This study was to determine if cellular glutathione peroxidase (GPX1) protects against acute oxidative stress induced by diquat. Lethality and hepatic biochemical indicators in GPX1 knockout mice [GPX1(-/-)] were compared with those of wild-type mice (WT) after an intraperitoneal injection of diquat at 6, 12, 24, or 48 mg/kg of body weight. Although the WT survived all the doses, the GPX1(-/-) survived only 6 mg diquat/kg and were killed by 12, 24, and 48 mg diquat/kg at 52, 4.4 and 3.9 hr, respectively. Compared with those of surviving mice that were sacrificed on Day 7, the dead GPX1(-/-) had diquat dose-dependent increases (P < 0.05) in plasma alanine aminotransferase (ALT) activities. The GPX1(-/-) also had higher (P < 0.05) liver carbonyl contents than those of the WT, but the differences were irrespective of diquat doses. Whereas hepatic total GPX and phospholipid hydroperoxide glutathione peroxidase activities or hepatic GPX1 protein was not significantly affected by the diquat treatment, liver thioredoxin reductase and catalase activities were lower (P < 0.05) in the GPX1(-/-) injected with 12 mg diquat/kg than those of other groups. In conclusion, normal GPX1 expression is necessary to protect mice against the lethality, hepatic protein oxidation, and elevation of plasma ALT activity induced by 12-48 mg diquat/kg.

  9. Estrogens protect against hydrogen peroxide and arachidonic acid induced DNA damage.

    PubMed

    Tang, M; Subbiah, M T

    1996-01-19

    The ability of estrogens to protect against DNA damage induced by either hydrogen peroxide or arachidonic acid alone or in combination with Cu2+ was investigated. DNA strand breaks were determined by conversion of double stranded supercoiled OX-174 RFI DNA to double stranded open circular DNA and linear single stranded DNA. Estradiol-17 beta significantly decreased the formation of single and double strand breaks in DNA induced by H2O2 alone or with Cu2+. Equilin (an equine estrogen) was more effective than estradiol-17 beta at the doses tested. Arachidonic acid in the presence of Cu2+ caused the formation of high levels of linear DNA which was protected by estrogen with equilen being more effective. These studies suggest that estrogens through this protective effect on DNA damage might contribute to cardioprotection.

  10. Plasmid DNA Vaccine Co-Immunisation Modulates Cellular and Humoral Immune Responses Induced by Intranasal Inoculation in Mice.

    PubMed

    King, Deborah F L; McKay, Paul F; Mann, Jamie F S; Jones, C Bryn; Shattock, Robin J

    2015-01-01

    An effective HIV vaccine will likely require induction of both mucosal and systemic cellular and humoral immune responses. We investigated whether intramuscular (IM) delivery of electroporated plasmid DNA vaccine and simultaneous protein vaccinations by intranasal (IN) and IM routes could be combined to induce mucosal and systemic cellular and humoral immune responses to a model HIV-1 CN54 gp140 antigen in mice. Co-immunisation of DNA with intranasal protein successfully elicited both serum and vaginal IgG and IgA responses, whereas DNA and IM protein co-delivery did not induce systemic or mucosal IgA responses. Cellular IFNγ responses were preserved in co-immunisation protocols compared to protein-only vaccination groups. The addition of DNA to IN protein vaccination reduced the strong Th2 bias observed with IN protein vaccination alone. Luminex analysis also revealed that co-immunisation with DNA and IN protein induced expression of cytokines that promote B-cell function, generation of TFH cells and CCR5 ligands that can reduce HIV infectivity. These data suggest that while IN inoculation alone elicits both cellular and humoral responses, co-administration with homologous DNA vaccination can tailor these towards a more balanced Th1/Th2 phenotype modulating the cellular cytokine profile while eliciting high-levels of antigen-specific antibody. This work provides insights on how to generate differential immune responses within the same vaccination visit, and supports co-immunisation with DNA and protein by a mucosal route as a potential delivery strategy for HIV vaccines.

  11. Plasmid DNA Vaccine Co-Immunisation Modulates Cellular and Humoral Immune Responses Induced by Intranasal Inoculation in Mice

    PubMed Central

    King, Deborah F. L.; McKay, Paul F.; Mann, Jamie F. S.; Jones, C. Bryn; Shattock, Robin J.

    2015-01-01

    Background An effective HIV vaccine will likely require induction of both mucosal and systemic cellular and humoral immune responses. We investigated whether intramuscular (IM) delivery of electroporated plasmid DNA vaccine and simultaneous protein vaccinations by intranasal (IN) and IM routes could be combined to induce mucosal and systemic cellular and humoral immune responses to a model HIV-1 CN54 gp140 antigen in mice. Results Co-immunisation of DNA with intranasal protein successfully elicited both serum and vaginal IgG and IgA responses, whereas DNA and IM protein co-delivery did not induce systemic or mucosal IgA responses. Cellular IFNγ responses were preserved in co-immunisation protocols compared to protein-only vaccination groups. The addition of DNA to IN protein vaccination reduced the strong Th2 bias observed with IN protein vaccination alone. Luminex analysis also revealed that co-immunisation with DNA and IN protein induced expression of cytokines that promote B-cell function, generation of TFH cells and CCR5 ligands that can reduce HIV infectivity. Significance These data suggest that while IN inoculation alone elicits both cellular and humoral responses, co-administration with homologous DNA vaccination can tailor these towards a more balanced Th1/Th2 phenotype modulating the cellular cytokine profile while eliciting high-levels of antigen-specific antibody. This work provides insights on how to generate differential immune responses within the same vaccination visit, and supports co-immunisation with DNA and protein by a mucosal route as a potential delivery strategy for HIV vaccines. PMID:26544970

  12. Effects of trypsin on cellular, chromosomal and DNA damage induced by X-rays

    NASA Astrophysics Data System (ADS)

    Sprunt, Elizabeth A.

    When cells are trypsinized before irradiation, potentiation of cell killing is seen; this is known as the 'trypsin effect'. The trypsin effect is re-examined here in the light of experiments in which enzymatic modifications of DNA in permeabilized cells has become a powerful experimental tool (Bryant et al, 1978, Ahnstrom and Bryant,1982; Natarajan et al, 1980; Bryant, 1984, 1985; Natarajan and Obe, 1984) and where in some cases it is suspected that trypsinization as part of the technique could significantly alter cell membrane permeability and chromatin structure (Obe et al, 1985; Obe and Winkel, 1985; Bryant and Christie, 1989). The trypsin effect was investigated at various cellular levels, assaying for cell survival (to verify the potentiation), anaphase chromosomal aberrations, DNA damage and repair and lastly using a nucleoid assay to investigate the effect of trypsin on DNA-nuclear matrix interactions. Each of these are considered in separate chapters as individual studies, then all compared in the final discussion. A small potentiation effect of X-ray damage on cell killing was seen when using Chinese Hamster Ovary (CHO) cells but no potentiating effect was found in the murine Ehrlich ascites tumour (EAT) cell line. Trypsinization was found to increase the number of X-ray induced chromosomal anaphase abnormalities in EAT cells. To investigate the possibility that the basis of the trypsin effect lies in its action at the DNA level, further experiments were performed to monitor DNA damage and repair using the DNA unwinding and neutral elution techniques. No difference was seen in the unwinding kinetics or in the DNA unwinding dose-effect curves for induction of DNA single strand breakage (ssb); when using neutral elution however. treatment of cells with trypsin or buffer alone increased the incidence of X-ray induced double strand breaks (dsb) at higher doses. Trypsinized EAT cells were found to repair ssb after 12 Gy less rapidly than those treated with

  13. Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice.

    PubMed

    Liu, Shan; Shi, Lin; Cheng, Yan-bin; Fan, Gui-xiang; Ren, Hui-xun; Yuan, Yu-kang

    2009-07-01

    To investigate the vaccine potential of multi-epitope vaccines against toxoplasmosis, a multi-epitope DNA vaccine, eukaryotic plasmid pcDNA3.1/T-ME expressing six antigen segments (SAG1(238-256), SAG1(281-320), GRA1(170-193), GRA4(331-345), GRA4(229-245), and GRA2(171-185)) of Toxoplasma gondii was constructed. We investigated the efficacy of pcDNA3.1/T-ME with or without co-administration of a CpG-oligodeoxynucleotide (CpG-ODN) as an adjuvant to protect mice (BALB/c and C57BL/6) against toxoplasmosis. High survival rates were observed in mice immunized with pcDNA3.1/T-ME when challenged with T. gondii RH strain. Lymphocyte proliferation assays, cytokine, and antibody determinations show that mice immunized with pcDNA3.1/T-ME produced stronger humoral and Th1-type cellular immune responses compared to untreated mice or those immunized with empty plasmids. However, co-immunization with CpG-ODN resulted in impaired immune responses. Our data demonstrates that multi-epitope DNA vaccination is a potential strategy for the control of toxoplasmosis and paves the way for further investigations into producing a multi-epitope anti-T. gondii DNA vaccine.

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

    PubMed

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

    2014-10-01

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

  15. Tuning the cellular uptake properties of luminescent heterobimetallic iridium(III)-ruthenium(II) DNA imaging probes.

    PubMed

    Wragg, Ashley; Gill, Martin R; Turton, David; Adams, Harry; Roseveare, Thomas M; Smythe, Carl; Su, Xiaodi; Thomas, Jim A

    2014-10-20

    The synthesis of two new luminescent dinuclear Ir(III)-Ru(II) complexes containing tetrapyrido[3,2-a:2',3'-c:3'',2''-h:2''',3'''-j]phenazine (tpphz) as the bridging ligand is reported. Unlike many other complexes incorporating cyclometalated Ir(III) moieties, these complexes display good water solubility, allowing the first cell-based study on Ir(III)-Ru(II) bioprobes to be carried out. Photophysical studies indicate that emission from each complex is from a Ru(II) excited state and both complexes display significant in vitro DNA-binding affinities. Cellular studies show that each complex is rapidly internalised by HeLa cells, in which they function as luminescent nuclear DNA-imaging agents for confocal microscopy. Furthermore, the uptake and nuclear targeting properties of the complex incorporating cyclometalating 2-(4-fluorophenyl)pyridine ligands around its Ir(III) centre is enhanced in comparison to the non-fluorinated analogue, indicating that fluorination may provide a route to promote cell uptake of transition-metal bioprobes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed Central

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

    2015-01-01

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

  17. Preparation of astaxanthin-loaded DNA/chitosan nanoparticles for improved cellular uptake and antioxidation capability.

    PubMed

    Wang, Qian; Zhao, Yingyuan; Guan, Lei; Zhang, Yaping; Dang, Qifeng; Dong, Ping; Li, Jing; Liang, Xingguo

    2017-07-15

    DNA/chitosan co-assemblies were initially used as nanocarriers for efficient astaxanthin encapsulation and delivery. The obtained astaxanthin-loaded DNA/chitosan (ADC) colloidal system was transparent and homogenous, with astaxanthin content up to 65μg/ml. Compared to free astaxanthin, ADC nanoparticles with an astaxanthin concentration as low as 3.35nM still showed a more powerful cytoprotective effect on H2O2-induced oxidative cell damage, and improved cell viability from 49.9% to 61.9%. The ROS scavenging efficiency of ADC nanoparticles was as high as 54.3%, which was 2-fold higher than that of free astaxanthin. Besides this, ADC nanoparticles were easily engulfed by Caco-2 cells in a short time, indicating that the encapsulated astaxanthin could be absorbed through endocytosis by intestinal epithelial cells. The improved antioxidation capability and facilitated cellular uptake enabled the ADC nanoparticles to be good candidates for efficient delivery and absorption of astaxanthin. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. DNA-binding determinants and cellular thresholds for human telomerase repeat addition processivity.

    PubMed

    Wu, Robert Alexander; Tam, Jane; Collins, Kathleen

    2017-07-03

    The reverse transcriptase telomerase adds telomeric repeats to chromosome ends. Purified human telomerase catalyzes processive repeat synthesis, which could restore the full ~100 nucleotides of (T2AG3)n lost from replicated chromosome ends as a single elongation event. Processivity inhibition is proposed to be a basis of human disease, but the impacts of different levels of processivity on telomere maintenance have not been examined. Here, we delineate side chains in the telomerase active-site cavity important for repeat addition processivity, determine how they contribute to duplex and single-stranded DNA handling, and test the cellular consequences of partial or complete loss of repeat addition processivity for telomere maintenance. Biochemical findings oblige a new model for DNA and RNA handling dynamics in processive repeat synthesis. Biological analyses implicate repeat addition processivity as essential for telomerase function. However, telomeres can be maintained by telomerases with lower than wild-type processivity. Furthermore, telomerases with low processivity dramatically elongate telomeres when overexpressed. These studies reveal distinct consequences of changes in telomerase repeat addition processivity and expression level on telomere elongation and length maintenance. © 2017 The Authors.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  1. Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging

    PubMed Central

    Guo, Jia; Wang, Shenliang; Dai, Nan; Teo, Yin Nah; Kool, Eric T.

    2011-01-01

    Most current approaches to multiantigen fluorescent imaging require overlaying of multiple images taken with separate filter sets as a result of differing dye excitation requirements. This requirement for false-color composite imaging prevents the user from visualizing multiple species in real time and disallows imaging of rapidly moving specimens. To address this limitation, here we investigate the use of oligodeoxyfluoroside (ODF) fluorophores as labels for antibodies. ODFs are short DNA-like oligomers with fluorophores replacing the DNA bases and can be assembled in many colors with excitation at a single wavelength. A DNA synthesizer was used to construct several short ODFs carrying a terminal alkyne group and having emission maxima of 410–670 nm. We developed a new approach to antibody conjugation, using Huisgen–Sharpless cycloaddition, which was used to react the alkynes on ODFs with azide groups added to secondary antibodies. Multiple ODF-tagged secondary antibodies were then used to mark primary antibodies. The set of antibodies was tested for spectral characteristics in labeling tubulin in HeLa cells and revealed a wide spectrum of colors, ranging from violet-blue to red with excitation through a single filter (340–380 nm). Selected sets of the differently labeled secondary antibodies were then used to simultaneously mark four antigens in fixed cells, using a single image and filter set. We also imaged different surface tumor markers on two live cell lines. Experiments showed that all colors could be visualized simultaneously by eye under the microscope, yielding multicolor images of multiple cellular antigens in real time. PMID:21321224

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

    PubMed

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

    2014-01-01

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

  3. DNA prime Listeria boost induces a cellular immune response to SIV antigens in the rhesus macaque model that is capable of limited suppression of SIV239 viral replication.

    PubMed

    Boyer, Jean D; Robinson, Tara M; Maciag, Paulo C; Peng, Xiaohui; Johnson, Ross S; Pavlakis, George; Lewis, Mark G; Shen, Anding; Siliciano, Robert; Brown, Charles R; Weiner, David B; Paterson, Yvonne

    2005-03-01

    DNA vaccines and recombinant Listeria monocytogenes that express and secrete SIV Gag and Env antigens were combined in a nonhuman primate prime-boost immunogenicity study followed by a challenge with SIV239. We report that recombinant DNA vaccine delivered intramuscularly, and recombinant L. monocytogenes delivered orally each individually have the ability to induce CD8+ and CD4+ T cell immune responses in a nonhuman primate. Four rhesus monkeys were immunized at weeks 0, 4, 8, and 12 with the pCSIVgag and pCSIVenv DNA plasmids and boosted with SIV expressing L. monocytogenes vaccines at weeks 16, 20, and 28. Four rhesus monkeys received only the L. monocytogenes vaccines at weeks 16, 20, and 28. A final group of monkeys served as a control group. Blood samples were taken before vaccination and 2 weeks post each injection and analyzed by ELISPOT for CD4+ and CD8+ T cell responses. Moderate vaccine induced SIV-specific cellular immune responses were observed following immunization with either DNA or L. monocytogenes vectors. However, the SIV antigen-specific immune responses were significantly increased when Rhesus macaques were primed with SIV DNA vaccines and boosted with the SIV expressing L. monocytogenes vectors. In addition, the combined vaccine was able to impact SIV239 viral replication following an intrarectal challenge. This study demonstrates for the first time that oral L. monocytogenes can induce a cellular immune response in a nonhuman primate and is able to enhance the efficacy of a DNA vaccine as well as provide modest protection against SIV239 challenge.

  4. Protective Effect of Folic Acid on Oxidative DNA Damage

    PubMed Central

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

    2015-01-01

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

  5. Bystander effects as manifestation of intercellular communication of DNA damage and of the cellular oxidative status.

    PubMed

    Klammer, Holger; Mladenov, Emil; Li, Fanghua; Iliakis, George

    2015-01-01

    It is becoming increasingly clear that cells exposed to ionizing radiation (IR) and other genotoxic agents (targeted cells) can communicate their DNA damage response (DDR) status to cells that have not been directly irradiated (bystander cells). The term radiation-induced bystander effects (RIBE) describes facets of this phenomenon, but its molecular underpinnings are incompletely characterized. Consequences of DDR in bystander cells have been extensively studied and include transformation and mutation induction; micronuclei, chromosome aberration and sister chromatid exchange formation; as well as modulations in gene expression, proliferation and differentiation patterns. A fundamental question arising from such observations is why targeted cells induce DNA damage in non-targeted, bystander cells threatening thus their genomic stability and risking the induction of cancer. Here, we review and synthesize available literature to gather support for a model according to which targeted cells modulate as part of DDR their redox status and use it as a source to generate signals for neighboring cells. Such signals can be either small molecules transported to adjacent non-targeted cells via gap-junction intercellular communication (GJIC), or secreted factors that can reach remote, non-targeted cells by diffusion or through the circulation. We review evidence that such signals can induce in the recipient cell modulations of redox status similar to those seen in the originating targeted cell - occasionally though self-amplifying feedback loops. The resulting increase of oxidative stress in bystander cells induces, often in conjunction with DNA replication, the observed DDR-like responses that are at times strong enough to cause apoptosis. We reason that RIBE reflect the function of intercellular communication mechanisms designed to spread within tissues, or the entire organism, information about DNA damage inflicted to individual, constituent cells. Such responses are

  6. IFI16, an amplifier of DNA-damage response: Role in cellular senescence and aging-associated inflammatory diseases.

    PubMed

    Choubey, Divaker; Panchanathan, Ravichandran

    2016-07-01

    DNA-damage induces a DNA-damage response (DDR) in mammalian cells. The response, depending upon the cell-type and the extent of DNA-damage, ultimately results in cell death or cellular senescence. DDR-induced signaling in cells activates the ATM-p53 and ATM-IKKα/β-interferon (IFN)-β signaling pathways, thus leading to an induction of the p53 and IFN-inducible IFI16 gene. Further, upon DNA-damage, DNA accumulates in the cytoplasm, thereby inducing the IFI16 protein and STING-dependent IFN-β production and activation of the IFI16 inflammasome, resulting in the production of proinflammatory cytokines (e.g., IL-1β and IL-18). Increased expression of IFI16 protein in a variety of cell-types promotes cellular senescence. However, reduced expression of IFI16 in cells promotes cell proliferation. Because expression of the IFI16 gene is induced by activation of DNA-damage response in cells and increased levels of IFI16 protein in cells potentiate the p53-mediated transcriptional activation of genes and p53 and pRb-mediated cell cycle arrest, we discuss how an improved understanding of the role of IFI16 protein in cellular senescence and associated inflammatory secretory phenotype is likely to identify the molecular mechanisms that contribute to the development of aging-associated human inflammatory diseases and a failure to cancer therapy. Copyright © 2016. Published by Elsevier B.V.

  7. Probing the Dynamic Interaction between Damaged DNA and a Cellular Responsive Protein Using a Piezoelectric Mass Biosensor.

    PubMed

    Jin, Yulong; Xie, Yunfeng; Wu, Kui; Huang, Yanyan; Wang, Fuyi; Zhao, Rui

    2017-03-15

    The binding events between damaged DNA and recognition biomolecules are of great interest for understanding the activity of DNA-damaging drugs and the related DNA repair networks. Herein, a simple and sensitive sensor system was tailored for real-time probing of the dynamic molecular recognition between cisplatin-damaged-DNA (cisPt-DNA) and a cellular responsive protein, high-mobility-group box 1 (HMGB1). By integration of flow injection analysis (FIA) with quartz crystal microbalance (QCM), the interaction time-course of cisPt-DNA and HMGB1 domain A (HMGB1a) was investigated. The highly specific sensing interface was carefully designed and fabricated using cisPt-DNA as recognition element. A hybrid self-assembled monolayer consisting of cysteamine and mercaptohexanol was introduced to resist nonspecific adsorption. The calculated kinetic parameters (kass and kdiss) and the dissociation constant (KD) demonstrated the rapid recognition and tight binding of HMGB1a toward cisPt-DNA. Molecular docking was employed to simulate the complex formed by cisPt-DNA and HMGB1a. The tight binding of such a DNA-damage responsive complex is appealing for the downstream molecular recognition event related to the resistance to DNA repair. This continuous-flow QCM biosensor is an ideal tool for studying specific interactions between drug-damaged-DNAs and their recognition proteins in a physiological-relevant environment, and will provide a potential sensor platform for rapid screening and evaluating metal anticancer drugs.

  8. Protection of DNA and microsomal membranes in vitro by Glycyrrhiza glabra L. against gamma irradiation.

    PubMed

    Shetty, T K; Satav, J G; Nair, C K K

    2002-09-01

    The radioprotective effect of the root extract of Glycyrrhiza glabra L on lipid peroxidation in rat liver microsomes and plasmid pBR322 DNA was investigated. The extract was found to protect microsomal membranes, as evident from reduction in lipid peroxidation, and could also protect plasmid DNA from radiation-induced strand breaks. Copyright 2002 John Wiley & Sons, Ltd.

  9. Radiation protection of DNA and membrane in vitro by extract of Hemidesmus indicus.

    PubMed

    Shetty, T K; Satav, J G; Nair, C K K

    2005-05-01

    Radioprotective effect of H. indicus root extract on lipid peroxidation in rat liver microsomes and plasmid DNA was examined. Hemidesmus indicus (HI) root extract was found to protect microsomal membranes as evident from reduction in lipid peroxidation values. The extract could also protect DNA from radiation induced strand breaks. Copyright (c) 2005 John Wiley & Sons, Ltd.

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

    PubMed

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

    2015-11-13

    Maternal diabetes-induced birth defects remain a significant health problem. Studying the effect of natural compounds with antioxidant properties and minimal toxicities on diabetic embryopathy may lead to the development of new and safe dietary supplements. Punicalagin is a primary polyphenol found in pomegranate juice, which possesses antioxidant, anti-inflammatory and anti-tumorigenic properties, suggesting a protective effect of punicalagin on diabetic embryopathy. Here, we examined whether punicalagin could reduce high glucose-induced neural tube defects (NTDs), and if this rescue occurs through blockage of cellular stress and caspase activation. Embryonic day 8.5 (E8.5) mouse embryos were cultured for 24 or 36 h with normal (5 mM) glucose or high glucose (16.7 mM), in presence or absence of 10 or 20 μM punicalagin. 10 μM punicalagin slightly reduced NTD formation under high glucose conditions; however, 20 μM punicalagin significantly inhibited high glucose-induced NTD formation. Punicalagin suppressed high glucose-induced lipid peroxidation marker 4-hydroxynonenal, nitrotyrosine-modified proteins, and lipid peroxides. Moreover, punicalagin abrogated endoplasmic reticulum stress by inhibiting phosphorylated protein kinase ribonucleic acid (RNA)-like ER kinase (p-PERK), phosphorylated inositol-requiring protein-1α (p-IRE1α), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), C/EBP-homologous protein (CHOP), binding immunoglobulin protein (BiP) and x-box binding protein 1 (XBP1) mRNA splicing. Additionally, punicalagin suppressed high glucose-induced caspase 3 and caspase 8 cleavage. Punicalagin reduces high glucose-induced NTD formation by blocking cellular stress and caspase activation. These observations suggest punicalagin supplements could mitigate the teratogenic effects of hyperglycemia in the developing embryo, and possibly prevent diabetes-induced NTDs. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Protection of low density lipoprotein oxidation at chemical and cellular level by the antioxidant drug dipyridamole.

    PubMed Central

    Iuliano, L.; Colavita, A. R.; Camastra, C.; Bello, V.; Quintarelli, C.; Alessandroni, M.; Piovella, F.; Violi, F.

    1996-01-01

    1. The oxidative modification of low density lipoprotein (LDL) is thought to be an important factor in the initiation and development of atherosclerosis. Natural and synthetic antioxidants have been shown to protect LDL from oxidation and to inhibit atherosclerosis development in animals. Synthetic antioxidants are currently being tested, by they are not necessarily safe for human use. 2. We have previously reported that dipyridamole, currently used in clinical practice, is a potent scavenger of free radicals. Thus, we tested whether dipyridamole could affect LDL oxidation at chemical and cellular level. 3. Chemically induced LDL oxidation was made by Cu(II), Cu(II) plus hydrogen peroxide or peroxyl radicals generated by thermolysis of 2,2'-azo-bis(2-amidino propane). Dipyridamole, (1-10 microM), inhibited LDL oxidation as monitored by diene formation, evolution of hydroperoxides and thiobarbituric acid reactive substances, apoprotein modification and by the fluorescence of cis-parinaric acid. 4. The physiological relevance of the antioxidant activity was validated by experiments at the cellular level where dipyridamole inhibited endothelial cell-mediated LDL oxidation, their degradation by monocytes, and cytotoxicity. 5. In comparison with ascorbic acid, alpha-tocopherol and probucol, dipyridamole was the more efficient antioxidant with the following order of activity: dipyridamole > probucol > ascorbic acid > alpha-tocopherol. The present study shows that dipyridamole inhibits oxidation of LDL at pharmacologically relevant concentrations. The inhibition of LDL oxidation is unequivocally confirmed by use of three different methods of chemical oxidation, by several methods of oxidation monitoring, and the pharmacological relevance is demonstrated by the superiority of dipyridamole over the naturally occurring antioxidants, ascorbic acid and alpha-tocopherol and the synthetic antioxidant probucol. Images Figure 6 PMID:8968553

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

    PubMed Central

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

    2015-01-01

    Maternal diabetes-induced birth defects remain a significant health problem. Studying the effect of natural compounds with antioxidant properties and minimal toxicities on diabetic embryopathy may lead to the development of new and safe dietary supplements. Punicalagin is a primary polyphenol found in pomegranate juice, which possesses antioxidant, anti-inflammatory and anti-tumorigenic properties, suggesting a protective effect of punicalagin on diabetic embryopathy. Here, we examined whether punicalagin could reduce high glucose-induced neural tube defects (NTDs), and if this rescue occurs through blockage of cellular stress and caspase activation. Embryonic day 8.5 (E8.5) mouse embryos were cultured for 24 or 36 hours with normal (5 mM) glucose or high glucose (16.7 mM), in presence or absence of 10 or 20 µM punicalagin. 10 µM punicalagin slightly reduced NTD formation under high glucose conditions; however, 20 µM punicalagin significantly inhibited high glucose-induced NTD formation. Punicalagin suppressed high glucose-induced lipid peroxidation marker 4-hydroxynonenal, nitrotyrosine-modified proteins, and lipid peroxides. Moreover, punicalagin abrogated endoplasmic reticulum stress by inhibiting phosphorylated protein kinase ribonucleic acid (RNA)-like ER kinase (p-PERK), phosphorylated inositol-requiring protein-1α (p-IRE1α), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), C/EBP-homologous protein (CHOP), binding immunoglobulin protein (BiP) and x-box binding protein 1 (XBP1) mRNA splicing. Additionally, punicalagin suppressed high glucose-induced caspase 3 and caspase 8 cleavage. Punicalagin reduces high glucose-induced NTD formation by blocking cellular stress and caspase activation. These observations suggest punicalagin supplements could mitigate the teratogenic effects of hyperglycemia in the developing embryo, and possibly prevent diabetesinduced NTDs. PMID:26453010

  13. Mitochondrial respiration protects against oxygen-associated DNA damage

    PubMed Central

    Sung, Ho Joong; Ma, Wenzhe; Wang, Ping-yuan; Hynes, James; O'Riordan, Tomas C.; Combs, Christian A.; McCoy, J. Philip; Bunz, Fred; Kang, Ju-Gyeong; Hwang, Paul M.

    2012-01-01

    Oxygen is not only required for oxidative phosphorylation but also serves as the essential substrate for the formation of reactive oxygen species (ROS), which is implicated in ageing and tumorigenesis. Although the mitochondrion is known for its bioenergetic function, the symbiotic theory originally proposed that it provided protection against the toxicity of increasing oxygen in the primordial atmosphere. Using human cells lacking Synthesis of Cytochrome c Oxidase 2 (SCO2 –/–), we have tested the oxygen toxicity hypothesis. These cells are oxidative phosphorylation defective and glycolysis dependent; they exhibit increased viability under hypoxia and feature an inverted growth response to oxygen compared with wild-type cells. SCO2 –/– cells have increased intracellular oxygen and nicotinamide adenine dinucleotide (NADH) levels, which result in increased ROS and oxidative DNA damage. Using this isogenic cell line, we have revealed the genotoxicity of ambient oxygen. Our study highlights the importance of mitochondrial respiration both for bioenergetic benefits and for maintaining genomic stability in an oxygen-rich environment. PMID:20975668

  14. Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response

    PubMed Central

    Edmonds, Matthew J.; Carter, Rachel J.; Nickson, Catherine M.; Williams, Sarah C.; Parsons, Jason L.

    2017-01-01

    Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo. We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response. PMID:27924031

  15. Vaccines Displaying Mycobacterial Proteins on Biopolyester Beads Stimulate Cellular Immunity and Induce Protection against Tuberculosis

    PubMed Central

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

    2012-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  17. DNA vaccination partially protects against African swine fever virus lethal challenge in the absence of antibodies.

    PubMed

    Argilaguet, Jordi M; Pérez-Martín, Eva; Nofrarías, Miquel; Gallardo, Carmina; Accensi, Francesc; Lacasta, Anna; Mora, Mercedes; Ballester, Maria; Galindo-Cardiel, Ivan; López-Soria, Sergio; Escribano, José M; Reche, Pedro A; Rodríguez, Fernando

    2012-01-01

    The lack of available vaccines against African swine fever virus (ASFV) means that the evaluation of new immunization strategies is required. Here we show that fusion of the extracellular domain of the ASFV Hemagglutinin (sHA) to p54 and p30, two immunodominant structural viral antigens, exponentially improved both the humoral and the cellular responses induced in pigs after DNA immunization. However, immunization with the resulting plasmid (pCMV-sHAPQ) did not confer protection against lethal challenge with the virulent E75 ASFV-strain. Due to the fact that CD8(+) T-cell responses are emerging as key components for ASFV protection, we designed a new plasmid construct, pCMV-UbsHAPQ, encoding the three viral determinants above mentioned (sHA, p54 and p30) fused to ubiquitin, aiming to improve Class I antigen presentation and to enhance the CTL responses induced. As expected, immunization with pCMV-UbsHAPQ induced specific T-cell responses in the absence of antibodies and, more important, protected a proportion of immunized-pigs from lethal challenge with ASFV. In contrast with control pigs, survivor animals showed a peak of CD8(+) T-cells at day 3 post-infection, coinciding with the absence of viremia at this time point. Finally, an in silico prediction of CTL peptides has allowed the identification of two SLA I-restricted 9-mer peptides within the hemagglutinin of the virus, capable of in vitro stimulating the specific secretion of IFNγ when using PBMCs from survivor pigs. Our results confirm the relevance of T-cell responses in protection against ASF and open new expectations for the future development of more efficient recombinant vaccines against this disease.

  18. DNA Vaccination Partially Protects against African Swine Fever Virus Lethal Challenge in the Absence of Antibodies

    PubMed Central

    Argilaguet, Jordi M.; Pérez-Martín, Eva; Nofrarías, Miquel; Gallardo, Carmina; Accensi, Francesc; Lacasta, Anna; Mora, Mercedes; Ballester, Maria; Galindo-Cardiel, Ivan; López-Soria, Sergio; Escribano, José M.; Reche, Pedro A.; Rodríguez, Fernando

    2012-01-01

    The lack of available vaccines against African swine fever virus (ASFV) means that the evaluation of new immunization strategies is required. Here we show that fusion of the extracellular domain of the ASFV Hemagglutinin (sHA) to p54 and p30, two immunodominant structural viral antigens, exponentially improved both the humoral and the cellular responses induced in pigs after DNA immunization. However, immunization with the resulting plasmid (pCMV-sHAPQ) did not confer protection against lethal challenge with the virulent E75 ASFV-strain. Due to the fact that CD8+ T-cell responses are emerging as key components for ASFV protection, we designed a new plasmid construct, pCMV-UbsHAPQ, encoding the three viral determinants above mentioned (sHA, p54 and p30) fused to ubiquitin, aiming to improve Class I antigen presentation and to enhance the CTL responses induced. As expected, immunization with pCMV-UbsHAPQ induced specific T-cell responses in the absence of antibodies and, more important, protected a proportion of immunized-pigs from lethal challenge with ASFV. In contrast with control pigs, survivor animals showed a peak of CD8+ T-cells at day 3 post-infection, coinciding with the absence of viremia at this time point. Finally, an in silico prediction of CTL peptides has allowed the identification of two SLA I-restricted 9-mer peptides within the hemagglutinin of the virus, capable of in vitro stimulating the specific secretion of IFNγ when using PBMCs from survivor pigs. Our results confirm the relevance of T-cell responses in protection against ASF and open new expectations for the future development of more efficient recombinant vaccines against this disease. PMID:23049728

  19. Increased proportions of C1 truncated prion protein protect against cellular M1000 prion infection.

    PubMed

    Lewis, Victoria; Hill, Andrew F; Haigh, Cathryn L; Klug, Genevieve M; Masters, Colin L; Lawson, Victoria A; Collins, Steven J

    2009-10-01

    Prion disease pathogenesis is linked to the cell-associated propagation of misfolded protease-resistant conformers (PrP) of the normal cellular prion protein (PrP). Ongoing PrP expression is the only known absolute requirement for successful prion disease transmission and PrP propagation. Further typifying prion disease is selective neuronal dysfunction and loss, although the precise mechanisms underlying this are undefined. We utilized a single prion strain (M1000) and a range of neuronal and nonneuronal, PrP endogenously expressing and transgenically modified overexpressing cell lines, to evaluate whether PrP glycosylation patterns or constitutive N-terminal cleavage events may be determinants of sustained PrP propagation. Our data demonstrates that relative proportions of full-length and C1 truncated PrP are the most important characteristics influencing susceptibility to sustained M1000 prion infection, supporting PrP alpha-cleavage as a protective event, which may contribute to the selective neuronal vulnerability observed in vivo.

  20. Whole genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains.

    PubMed

    Jensen, Taylor J; Kim, Sung K; Zhu, Zhanyang; Chin, Christine; Gebhard, Claudia; Lu, Tim; Deciu, Cosmin; van den Boom, Dirk; Ehrich, Mathias

    2015-04-15

    Circulating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors. We perform whole genome bisulfite sequencing on a set of unmatched samples including circulating cell-free DNA from non-pregnant and pregnant female donors and genomic DNA from maternal buffy coat and placenta samples. We find CpG cytosines within longer fragments are more likely to be methylated. Comparison of the methylomes of placenta and non-pregnant circulating cell-free DNA reveal many of the 51,259 identified differentially methylated regions are located in domains exhibiting consistent placenta hypomethylation across millions of consecutive bases. We find these placenta hypomethylated domains are consistently located within regions exhibiting low CpG and gene density. Differentially methylated regions identified when comparing placenta to non-pregnant circulating cell-free DNA are recapitulated in pregnant circulating cell-free DNA, confirming the ability to detect differential methylation in circulating cell-free DNA mixtures. We generate methylome maps for four sample types at single-base resolution, identify a link between DNA methylation and fragment length in circulating cell-free DNA, identify differentially methylated regions between sample groups, and uncover the presence of megabase-size placenta hypomethylated domains.

  1. Protection of the genome and central protein-coding sequences by non-coding DNA against DNA damage from radiation.

    PubMed

    Qiu, Guo-Hua

    2015-01-01

    Non-coding DNA comprises a very large proportion of the total genomic content in higher organisms, but its function remains largely unclear. Non-coding DNA sequences constitute the majority of peripheral heterochromatin, which has been hypothesized to be the genome's 'bodyguard' against DNA damage from chemicals and radiation for almost four decades. The bodyguard protective function of peripheral heterochromatin in genome defense has been strengthened by the results from numerous recent studies, which are summarized in this review. These data have suggested that cells and/or organisms with a higher level of heterochromatin and more non-coding DNA sequences, including longer telomeric DNA and rDNAs, exhibit a lower frequency of DNA damage, higher radioresistance and longer lifespan after IR exposure. In addition, the majority of heterochromatin is peripherally located in the three-dimensional structure of genome organization. Therefore, the peripheral heterochromatin with non-coding DNA could play a protective role in genome defense against DNA damage from ionizing radiation by both absorbing the radicals from water radiolysis in the cytosol and reducing the energy of IR. However, the bodyguard protection by heterochromatin has been challenged by the observation that DNA damage is less frequently detected in peripheral heterochromatin than in euchromatin, which is inconsistent with the expectation and simulation results. Previous studies have also shown that the DNA damage in peripheral heterochromatin is rarely repaired and moves more quickly, broadly and outwardly to approach the nuclear pore complex (NPC). Additionally, it has been shown that extrachromosomal circular DNAs (eccDNAs) are formed in the nucleus, highly detectable in the cytoplasm (particularly under stress conditions) and shuttle between the nucleus and the cytoplasm. Based on these studies, this review speculates that the sites of DNA damage in peripheral heterochromatin could occur more

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

    PubMed

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

    2011-01-01

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

  3. SIRT4 has tumor suppressive activity and regulates the cellular metabolic response to DNA damage by inhibiting mitochondrial glutamine metabolism

    PubMed Central

    Jeong, Seung Min; Xiao, Cuiying; Finley, Lydia W.S; Lahusen, Tyler; Souza, Amanda L.; Pierce, Kerry; Li, Ying-Hua; Wang, Xiaoxu; Laurent, Gaëlle; German, Natalie J.; Xu, Xiaoling; Li, Cuiling; Wang, Rui-Hong; Lee, Jaewon; Csibi, Alfredo; Cerione, Richard; Blenis, John; Clish, Clary B.; Kimmelman, Alec; Deng, Chu-Xia; Haigis, Marcia C.

    2013-01-01

    SUMMARY DNA damage elicits a cellular signaling response that initiates cell cycle arrest and DNA repair. Here we find that DNA damage triggers a critical block in glutamine metabolism, which is required for proper DNA damage responses. This block requires the mitochondrial SIRT4, which is induced by numerous genotoxic agents and represses the metabolism of glutamine into TCA cycle. SIRT4 loss leads to both increased glutamine-dependent proliferation and stress-induced genomic instability, resulting in tumorigenic phenotypes. Moreover, SIRT4 knockout mice spontaneously develop lung tumors. Our data uncover SIRT4 as an important component of the DNA damage response pathway that orchestrates a metabolic block in glutamine metabolism, cell cycle arrest and tumor suppression. PMID:23562301

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

    PubMed

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

    2016-10-01

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

  5. Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c mice.

    PubMed

    Luo, Deyan; Ni, Bing; Li, Peng; Shi, Wei; Zhang, Songle; Han, Yue; Mao, Liwei; He, Yangdong; Wu, Yuzhang; Wang, Xiliang

    2006-05-01

    This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. Intramuscular injection of this divalent DNA vaccine into BALB/c mice elicited markedly both humoral and cellular immune responses. The specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the dual-gene DNA vaccine elicited a strong T-cell proliferative response and induced a large amount of gamma interferon-producing T cells upon restimulation in vitro with recombinant fusion protein L7/L12-Omp16, suggesting the induction of a typical T-helper-1-dominated immune response in vivo. This divalent DNA vaccine could also induce a significant level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice. Furthermore, the protection level induced by the divalent DNA vaccine was significantly higher than that induced by the univalent DNA vaccines pcDNA3.1-L7/L12 or pcDNA3.1-Omp16. Taken together, the results of this study verify for the first time that the Omp16 gene can be a candidate target for a DNA vaccine against brucellosis. Additionally, a divalent genetic vaccine based on the L7/L12 and Omp16 genes can elicit a stronger cellular immune response and better immunoprotection than the relevant univalent vaccines can.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2007-01-02

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

  8. The ATM cofactor ATMIN protects against oxidative stress and accumulation of DNA damage in the aging brain.

    PubMed

    Kanu, Nnennaya; Penicud, Kay; Hristova, Mariya; Wong, Barnaby; Irvine, Elaine; Plattner, Florian; Raivich, Gennadij; Behrens, Axel

    2010-12-03

    Progressive accumulation of DNA damage is causally involved in cellular senescence and organismal aging. The DNA damage kinase ATM plays a central role in maintaining genomic stability. ATM mutations cause the genetic disorder ataxia telangiectasia, which is primarily characterized by progressive neurodegeneration and cancer susceptibility. Although the importance of ATM function to protect against oxidative DNA damage and during aging is well described, the mechanism of ATM activation by these stimuli is not known. Here we identify ATM interactor (ATMIN) as an essential component of the ATM signaling pathway in response to oxidative stress and aging. Embryos lacking ATMIN (atmin(Δ/Δ)) died in utero and showed increased numbers of cells positive for phosphorylated histone H2aX, indicative of increased DNA damage. atmin(Δ/Δ) mouse embryonic fibroblasts accumulated DNA damage and prematurely entered senescence when cultured at atmospheric oxygen levels (20%), but this defect was rescued by addition of an antioxidant and also by culturing cells at physiological oxygen levels (3%). In response to acute oxidative stress, atmin(Δ/Δ) mouse embryonic fibroblasts showed slightly lower levels of ATM phosphorylation and reduced ATM substrate phosphorylation. Conditional deletion of ATMIN in the murine nervous system (atmin(ΔN)) resulted in reduced numbers of dopaminergic neurons, as does ATM deficiency. ATM activity was observed in old, but not in young, control mice, but aging-induced ATM signaling was impaired by ATMIN deficiency. Consequently, old atmin(ΔN) mice showed accumulation of DNA damage in the cortex accompanied by gliosis, resulting in increased mortality of aging mutant mice. These results suggest that ATMIN mediates ATM activation by oxidative stress, and thereby ATMIN protects the aging brain by preventing accumulation of DNA damage.

  9. The cellular immune response plays an important role in protecting against dengue virus in the mouse encephalitis model.

    PubMed

    Gil, Lázaro; López, Carlos; Blanco, Aracelys; Lazo, Laura; Martín, Jorge; Valdés, Iris; Romero, Yaremis; Figueroa, Yassel; Guillén, Gerardo; Hermida, Lisset

    2009-02-01

    For several years, researchers have known that the generation of neutralizing antibodies is a prerequisite for attaining adequate protection against dengue virus. Nevertheless, the cellular immune response is the principal arm of the adaptive immune system against non-cytopathic viruses such as dengue, as once the virus enters into the cell it is necessary to destroy it to eliminate the virus. To define the role of the cellular immune response in the protection against dengue, we selected the mouse encephalitis model. Mice were immunized with a single dose of infective dengue 2 virus and different markers of both branches of the induced adaptive immunity were measured. Animals elicited a broad antibody response against the four dengue virus serotypes, but neutralizing activity was only detected against the homologous serotype. On the other hand, the splenocytes of the infected animals strongly proliferated after in vitro stimulation with the homologous virus, and specifically the CD8 T-cell subset was responsible for the secretion of the cytokine IFN-gamma. Finally, to define the role of T cells in in vivo protection, groups of animals were inoculated with the depleting monoclonal antibodies anti-CD4 or anti-CD8. Only depletion with anti-CD8 decreased to 50% the level of protection reached in the non-depleted mice. The present work constitutes the first report defining the role of the cellular immune response in protection against dengue virus in the mouse model.

  10. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration

    PubMed Central

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-01-01

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk. PMID:27705914

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

    PubMed Central

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

    2016-01-01

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

  12. Inhibition of Cellular Entry of Lymphocytic Choriomeningitis Virus by Amphipathic DNA Polymers

    PubMed Central

    Lee, Andrew M.; Rojek, Jillian M.; Gundersen, Anette; Ströher, Ute; Juteau, Jean-Marc; Vaillant, Andrew; Kunz, Stefan

    2008-01-01

    The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) represents a powerful experimental model for the study of the basic virology and pathogenesis of arenaviruses. In the present study, we used the LCMV model to evaluate the anti-viral potential of phosphorothioate oligonucleotides against arenaviruses. Our findings indicate that amphipathic DNA polymers (APs) are potent inhibitors of infection with a series of LCMV isolates with IC50 in the low nanomolar range. APs target the surface glycoprotein (GP) of LCMV and block viral entry and cell-cell propagation of the virus, without affecting later steps in replication or release of progeny virus from infected cells. The anti-viral action of APs is sequence-independent but is critically dependent on their size and hydrophobicity. Mechanistically, we provide evidence that APs disrupt the interaction between LCMVGP and its cellular receptor, α-dystroglycan. Exposure of LCMV to APs does not affect the stability of the GP virion spike and has no effect on the conformation of a neutralizing antibody epitope, suggesting rather subtle changes in the conformation and/or conformational dynamics of the viral GP. PMID:18022208

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

    PubMed

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

    2014-09-01

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

  14. Broad humoral and cellular immunity elicited by a bivalent DNA vaccine encoding HA and NP genes from an H5N1 virus.

    PubMed

    Xu, Ke; Ling, Zhi-Yang; Sun, Liang; Xu, Ying; Bian, Chao; He, Yuan; Lu, Wei; Chen, Ze; Sun, Bing

    2011-02-01

    Influenza A virus is highly variable and a major viral respiratory pathogen that can cause severe illness in humans. Therefore it is important to induce a sufficient immune response specific to current strains and to heterosubtypic viruses with vaccines. In this study, we developed a dual-promoter-based bivalent DNA vaccine that encodes both hemagglutinin (HA) and nucleoprotein (NP) proteins from a highly pathogenic A/Chicken/Henan/12/2004 (H5N1) virus. Our results show that the expression levels of HA and NP genes from the dual-promoter plasmid are similar to those seen when they are expressed individually in independent plasmids. When the bivalent DNA vaccine was inoculated via intramuscular injection and in vivo electroporation, high levels of both humoral and cellular immune responses were elicited against homologous H5N1 virus and heterosubtypic H9N2 virus. Furthermore, no obvious antigenic competition was observed between HA and NP proteins in the dual-promoter-based bivalent vaccine compared to monovalent vaccines. Our data suggest that a combination of influenza surface and internal viral genes in a dual-promoter-expressing plasmid may provide a new approach for developing a DNA vaccine that may protect not only specifically against a currently circulating strain, but also may cross-protect broadly against new heterosubtypic viruses.

  15. Structure of the DNA-SspC Complex: Implications for DNA Packaging, Protection, and Repair in Bacterial Spores

    PubMed Central

    Frenkiel-Krispin, Daphna; Sack, Rinat; Englander, Joseph; Shimoni, Eyal; Eisenstein, Miriam; Bullitt, Esther; Horowitz-Scherer, Rachel; Hayes, Christopher S.; Setlow, Peter; Minsky, Abraham; Wolf, Sharon Grayer

    2004-01-01

    Bacterial spores have long been recognized as the sturdiest known life forms on earth, revealing extraordinary resistance to a broad range of environmental assaults. A family of highly conserved spore-specific DNA-binding proteins, termed α/β-type small, acid-soluble spore proteins (SASP), plays a major role in mediating spore resistance. The mechanism by which these proteins exert their protective activity remains poorly understood, in part due to the lack of structural data on the DNA-SASP complex. By using cryoelectron microscopy, we have determined the structure of the helical complex formed between DNA and SspC, a characteristic member of the α/β-type SASP family. The protein is found to fully coat the DNA, forming distinct protruding domains, and to modify DNA structure such that it adopts a 3.2-nm pitch. The protruding SspC motifs allow for interdigitation of adjacent DNA-SspC filaments into a tightly packed assembly of nucleoprotein helices. By effectively sequestering DNA molecules, this dense assembly of filaments is proposed to enhance and complement DNA protection obtained by DNA saturation with the α/β-type SASP. PMID:15150240

  16. Potential mechanisms for cancer resistance in elephants and comparative cellular response to DNA damage in humans

    DOE PAGES

    Abegglen, Lisa M.; Caulin, Aleah F.; Chan, Ashley; ...

    2015-10-08

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

  17. Potential mechanisms for cancer resistance in elephants and comparative cellular response to DNA damage in humans

    SciTech Connect

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

    2015-10-08

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

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

    PubMed

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

    2015-11-03

    Evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology. To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS). A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n = 644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n = 8), patients with LFS (n = 10), and age-matched human controls (n = 11). Human samples were collected at the University of Utah between June 2014 and July 2015. Ionizing radiation and doxorubicin. Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes. Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95% CI, 0%-5%]; African wild dog, 8% [95% CI, 0%-16%]; lion, 2% [95% CI, 0%-7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%), compared with humans, who have 11% to 25% cancer mortality. While humans have 1 copy (2 alleles) of TP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain reaction. In response to DNA damage, elephant lymphocytes underwent p53-mediated apoptosis

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

    PubMed Central

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

    2016-01-01

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

  20. Induction of Cellular Immune Response by DNA Vaccine Coexpressing E. acervulina 3-1E Gene and Mature CHIl-15 Gene

    PubMed Central

    Ma, Dexing; Ma, Chunli; Gao, Mingyang; Li, Guangxing; Niu, Ze; Huang, Xiaodan

    2012-01-01

    We previously reported that the chimeric DNA vaccine pcDNA-3-1E-linker-mChIL-15, fused through linking Eimeria acervulina 3-1E encoding gene and mature chicken IL-15 (mChIL-15) gene with four flexible amino acid SPGS, could significantly offer protection against homologous challenge. In the present study, the induction of cellular immune response induced by the chimeric DNA vaccine pcDNA-3-1E-linker-mChIL-15 was investigated. Spleen lymphocyte subpopulations were characterized by flow cytometric analysis. The spleen lymphocyte proliferation assays were measured by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide (MTT) method. The mRNA profiles of ChIL-2 and ChIFN-γ in spleen were characterized by means of real-time PCR. Chickens immunized with pcDNA-3-1E-linker-mChIL-15 exhibited significant upregulated level of ChIL-2 and ChIFN-γ transcripts in spleen following two immunizations compared with chickens in other groups (P < 0.01). In comparison with pcDNA3.1-immunized and control groups, lymphocyte proliferation, percentage of CD8α+ cell, and levels of ChIL-2 and ChIFN-γ transcripts in the group immunized with pcDNA-3-1E-linker-mChIL-15 were significantly increased on day 6 following challenge (P < 0.05, P < 0.01, and P < 0.01, resp.). Our data suggested that the fusion antigen 3-1E-linker-mChIL-15 could be a potential candidate for E. acervulina vaccine development. PMID:22754694

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

    PubMed

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

    2013-01-01

    Diet may induce colon carcinogenesis through oxidative or alkylating DNA damage. However, diet may also contain anticarcinogenic compounds that contribute to cancer prevention. DNA damage prevention and/or induction of repair are two important mechanisms involved in cancer chemoprevention by dietary compounds. Hypericum sps. are widely used in traditional medicine to prepare infusions due to their beneficial digestive and neurologic effects. In this study, we investigated the potential of water extracts from three Hypericum sps. and some of their main phenolic compounds to prevent and repair oxidative and alkylating DNA damage in colon cells. The results showed that water extracts of Hypericum perforatum, Hypericum androsaemum, Hypericum undulatum, quercetin and rutin have protective effect against oxidative DNA damage in HT29 cells. Protective effect was also observed against alkylating DNA damage induced by methyl-methanesulfonate, except for H. androsaemum. With regard to alkylating damage repair H. perforatum, H. androsaemum and chlorogenic acid increased repair of alkylating DNA damage by base excision repair pathway. No effect was observed on nucleotide excision repair pathway. Antigenotoxic effects of Hypericum sps. may contribute to colon cancer prevention and the high amount of phenolic compounds present in Hypericum sps. play an important role in DNA protective effects. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Protection of mammalian cells by o-phenanthroline from lethal and DNA-damaging effects produced by active oxygen species.

    PubMed

    de Mello Filho, A C; Meneghini, R

    1985-10-30

    Active oxygen species are suspected as being a cause of the cellular damage that occurs at the site of inflammation. Phagocytic cells accumulate at these sites and produce superoxide ion, hydrogen peroxide and hydroxyl radical. The ultimate killing species, the cellular target and the mechanism whereby the lethal injury is produced are unknown. We exposed mouse fibroblasts to xanthine oxidase and acetaldehyde, a system which mimics the membrane of phagocytic cells in terms of production of oxygen species. We observed that the generation of these species produced DNA strand breaks and cellular death. The metal chelator o-phenanthroline completely abolished the former effect, and at the same time it effectively protected the cells from lethal injuries. Because complexing iron o-phenanthroline prevents the formation of hydroxyl radical by the Fendon reaction (Fe(II) + H2O2----Fe(III) + OH- + OH.), it is proposed that most of the cell death and DNA damage are brought about by OH radical, produced from other species by iron-mediated reactions.

  3. Midkine secretion protects Hep3B cells from cadmium induced cellular damage

    PubMed Central

    Yazihan, Nuray; Ataoglu, Haluk; Akcil, Ethem; Yener, Burcu; Salman, Bulent; Aydin, Cengiz

    2008-01-01

    AIM: To evaluate role of midkine secretion during Cadmium (Cd) exposure in the human hepatocyte cell line Hep3B cells. METHODS: Different dosages of Cd (0.5-1-5-10 μg/mL) were applied to Hep3B cells and their effects to apoptosis, lactate dehydrogenase (LDH) leakage and midkine secretion were evaluated as time dependent manner. Same experiments were repeated with exogenously applied midkine (250-5000 pg/mL) and/or 5 μg/mL Cd. RESULTS: Cd exposure induced prominent apoptosis and LDH leakage beginning from lower dosages at the 48th h. Cd induced midkine secretion with higher dosages (P < 0.001), (control, Cd 0.5-1-5-10 μg/mL respectively: 1123 ± 73, 1157 ± 63, 1242 ± 90, 1886 ± 175, 1712 ± 166 pg/mL). Exogenous 500-5000 pg/mL midkine application during 5 μg/mL Cd toxicity prevented caspase-3 activation (control, Cd toxicity, 250, 500, 1000, 2500, 5000 pg/mL midkine+ Cd toxicity, respectively: 374 ± 64, 1786 ± 156, 1545 ± 179, 1203 ± 113, 974 ± 116, 646 ± 56, 556 ± 63 cfu) LDH leakage and cell death in Hep3B cells (P < 0.001). CONCLUSION: Our results showed that midkine secretion from Hep3B cells during Cd exposure protects liver cells from Cd induced cellular damage. Midkine has anti-apoptotic and cytoprotective role during Cd toxicity. Further studies are needed to explain the mechanism of midkine secretion and cytoprotective role of midkine during Cd exposure. Midkine may be a promising therapeutic agent in different toxic hepatic diseases. PMID:18176965

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

    PubMed

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

    2011-09-13

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

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

    SciTech Connect

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

    2009-08-15

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

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

    PubMed

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

    2009-08-15

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

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

    PubMed

    Tinker, Andrew; Aziz, Qadeer; Thomas, Alison

    2014-01-01

    ATP-sensitive potassium channels (K(ATP)) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated K(ATP) channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system. © 2013 The British Pharmacological Society.

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

    PubMed Central

    Tinker, Andrew; Aziz, Qadeer; Thomas, Alison

    2014-01-01

    ATP-sensitive potassium channels (KATP) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated KATP channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system. PMID:24102106

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

    SciTech Connect

    Kiran, Shashi; Oddi, Vineesha; Ramakrishna, Gayatri

    2015-02-01

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

  10. A cellular defense pathway regulating transcription through poly(ADP-ribosyl)ation in response to DNA damage

    PubMed Central

    Vispé, Stéphane; Yung, Tetsu M. C.; Ritchot, Janelle; Serizawa, Hiroaki; Satoh, Masahiko S.

    2000-01-01

    DNA damage is known to trigger key cellular defense pathways such as those involved in DNA repair. Here we provide evidence for a previously unrecognized pathway regulating transcription in response to DNA damage and show that this regulation is mediated by the abundant nuclear enzyme poly(ADP-ribose) polymerase. We found that poly(ADP-ribose) polymerase reduced the rate of transcription elongation by RNA polymerase II, suggesting that poly(ADP-ribose) polymerase negatively regulates transcription, possibly through the formation of poly(ADP-ribose) polymerase–RNA complexes. In damaged cells, poly(ADP-ribose) polymerase binds to DNA breaks and automodifies itself in the presence of NAD+, resulting in poly(ADP-ribose) polymerase inactivation. We found that automodification of poly(ADP-ribose) polymerase in response to DNA damage resulted in the up-regulation of transcription, presumably because automodified poly(ADP-ribose) polymerase molecules were released from transcripts, thereby relieving the block on transcription. Because agents that damage DNA damage RNA as well, up-regulation of RNA synthesis in response to DNA damage may provide cells with a mechanism to compensate for the loss of damaged transcripts and may be critical for cell survival after exposure to DNA-damaging agents. PMID:10944198

  11. A cellular defense pathway regulating transcription through poly(ADP-ribosyl)ation in response to DNA damage.

    PubMed

    Vispe, S; Yung, T M; Ritchot, J; Serizawa, H; Satoh, M S

    2000-08-29

    DNA damage is known to trigger key cellular defense pathways such as those involved in DNA repair. Here we provide evidence for a previously unrecognized pathway regulating transcription in response to DNA damage and show that this regulation is mediated by the abundant nuclear enzyme poly(ADP-ribose) polymerase. We found that poly(ADP-ribose) polymerase reduced the rate of transcription elongation by RNA polymerase II, suggesting that poly(ADP-ribose) polymerase negatively regulates transcription, possibly through the formation of poly(ADP-ribose) polymerase-RNA complexes. In damaged cells, poly(ADP-ribose) polymerase binds to DNA breaks and automodifies itself in the presence of NAD(+), resulting in poly(ADP-ribose) polymerase inactivation. We found that automodification of poly(ADP-ribose) polymerase in response to DNA damage resulted in the up-regulation of transcription, presumably because automodified poly(ADP-ribose) polymerase molecules were released from transcripts, thereby relieving the block on transcription. Because agents that damage DNA damage RNA as well, up-regulation of RNA synthesis in response to DNA damage may provide cells with a mechanism to compensate for the loss of damaged transcripts and may be critical for cell survival after exposure to DNA-damaging agents.

  12. DNA vaccination with a gene encoding Toxoplasma gondii Rhoptry Protein 17 induces partial protective immunity against lethal challenge in mice

    PubMed Central

    Wang, Hai-Long; Wang, Yu-Jing; Pei, Yan-Jiang; Bai, Ji-Zhong; Yin, Li-Tian; Guo, Rui; Yin, Guo-Rong

    2016-01-01

    Toxoplasma gondii is an obligate intracellular apicomplexan parasite that affects humans and various vertebrate livestock and causes serious economic losses. To develop an effective vaccine against T. gondii infection, we constructed a DNA vaccine encoding the T. gondii rhoptry protein 17 (TgROP17) and evaluated its immune protective efficacy against acute T. gondii infection in mice. The DNA vaccine (p3×Flag-CMV-14-ROP17) was intramuscularly injected to BALB/c mice and the immune responses of the vaccinated mice were determined. Compared to control mice treated with empty vector or PBS, mice immunized with the ROP17 vaccine showed a relatively high level of specific anti-T. gondii antibodies, and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a specific lymphocyte proliferative response, a Th1-type cellular immune response with production of IFN-γ and interleukin-2, and increased number of CD8+ T cells. Immunization with the ROP17 DNA significantly prolonged the survival time (15.6 ± 5.4 days, P < 0.05) of mice after challenge infection with the virulent T. gondii RH strain (Type I), compared with the control groups which died within 8 days. Therefore, our data suggest that DNA vaccination with TgROP17 triggers significant humoral and cellular responses and induces effective protection in mice against acute T. gondii infection, indicating that TgROP17 is a promising vaccine candidate against acute toxoplasmosis. PMID:26842927

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

    PubMed

    Cao, Zhuoxiao; Li, Yunbo

    2004-04-05

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

  14. 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

  15. 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.

  16. Role of TRPM2 and TRPV1 cation channels in cellular responses to radiation-induced DNA damage.

    PubMed

    Masumoto, Kanako; Tsukimoto, Mitsutoshi; Kojima, Shuji

    2013-06-01

    Radiation exposure causes DNA damage, and DNA repair systems are essential to rescue damaged cells. Although DNA damage or oxidative stress activates transient receptor potential melastatin 2 (TRPM2) and vanilloid 1 (TRPV1) cation channels, it has not been established whether these TRP channels are involved in cellular responses to radiation-induced DNA damage. Here, we investigated the contribution of TRPM2 and TRPV1 channels to γ-irradiation- and UVB-induced DNA damage responses in human lung cancer A549 cells. A549 cells were irradiated with γ-rays (2.0Gy) or UVB (5-10mJ/cm(2)). γH2AX foci, ATM activation, 53BP1 accumulation and EGFR expression were evaluated by immunofluorescence staining. Extracellular ATP concentration was measured by luciferin-luciferase assay. Knockdown of TRPM2 and TRPV1 expression was done by siRNA transfection. γ-Irradiation-induced γH2AX focus formation, ATM activation, 53BP1 accumulation and EGFR nuclear translocation, which are all associated with DNA repair, were suppressed by knockdown of TRPM2 and TRPV1 channels in A549 cells. Release of ATP, which mediates DNA damage response-associated activation of P2Y receptors, was suppressed by pre-treatment with catalase or knockdown of TRPM2 channel, but not TRPV1 channel. Similarly, UVB-induced γH2AX focus formation was suppressed in TRPM2- and TRPV1-knockdown cells, while UVB-induced ATP release was blocked in TRPM2- but not TRPV1-knockdown cells. Our results suggest that the activation of TRPM2 channel, which mediates ATP release, and TRPV1 channel plays significant roles in the cellular responses to DNA damage induced by γ-irradiation and UVB irradiation. Our results provide a new insight into the function of TRP channels from the viewpoint of radiation biology. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. A DNA vaccine encoding the E protein of West Nile virus is protective and can be boosted by recombinant domain DIII.

    PubMed

    Schneeweiss, Anne; Chabierski, Stefan; Salomo, Mathias; Delaroque, Nicolas; Al-Robaiy, Samiya; Grunwald, Thomas; Bürki, Kurt; Liebert, Uwe G; Ulbert, Sebastian

    2011-08-26

    West Nile Virus (WNV) is an emerging pathogenic flavivirus with increasing distribution worldwide. Birds are the natural host of the virus, but also mammals, including humans, can be infected. In some cases, a WNV infection can be associated with severe neurological symptoms. All currently available WNV vaccines are in the veterinary sector, and there is a need to develop safe and effective immunization technologies, which can also be used in humans. An alternative to current vaccination methods is DNA immunization. Most current DNA vaccine candidates against flaviviruses simultaneously express the viral envelope (E) and membrane (prM) proteins, which leads to the formation of virus-like particles. Here we generated a DNA plasmid, which expresses only the E-protein ectodomain. Vaccination of mice stimulated anti-WNV T-cell responses and neutralizing antibodies that were higher than those obtained after immunizing with a recombinant protein previously shown to be a protective WNV vaccine. A single dose of the plasmid was sufficient to protect animals from a lethal challenge with the virus. Moreover, immunogenicity could be boosted when DNA injection was followed by immunization with recombinant domain DIII of the E-protein. This resulted in significantly enhanced neutralizing antibody titers and a more prominent cellular immune response. The results suggest that the WNV E-protein is sufficient as a protective antigen in DNA vaccines and that protection can be significantly improved by adding a recombinant protein boost to the DNA prime.

  18. 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.

  19. Nucleosomes protect DNA from DNA methylation in vivo and in vitro

    PubMed Central

    Felle, Max; Hoffmeister, Helen; Rothammer, Julia; Fuchs, Andreas; Exler, Josef H.; Längst, Gernot

    2011-01-01

    Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo. PMID:21622955

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

    2013-01-01

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

  2. Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks.

    PubMed

    Levy-Barda, Adva; Lerenthal, Yaniv; Davis, Anthony J; Chung, Young Min; Essers, Jeroen; Shao, Zhengping; van Vliet, Nicole; Chen, David J; Hu, Mickey C-T; Kanaar, Roland; Ziv, Yael; Shiloh, Yosef

    2011-12-15

    The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs), a highly cytotoxic DNA lesion, activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28γ (REGγ; PSME3) as a novel DDR player. PA28γ depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28γ deficiency abrogates the balance between the two major DSB repair pathways--nonhomologous end-joining and homologous recombination repair. Furthermore, PA28γ is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28γ-proteasome axis of the DDR that is required for timely coordination of DSB repair.

  3. Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks

    PubMed Central

    Levy-Barda, Adva; Lerenthal, Yaniv; Davis, Anthony J; Chung, Young Min; Essers, Jeroen; Shao, Zhengping; van Vliet, Nicole; Chen, David J; Hu, Mickey C-T; Kanaar, Roland

    2011-01-01

    The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs), a highly cytotoxic DNA lesion, activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28γ (REGγ; PSME3) as a novel DDR player. PA28γ depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28γ deficiency abrogates the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair. Furthermore, PA28γ is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28γ-proteasome axis of the DDR that is required for timely coordination of DSB repair. PMID:22134242

  4. Nimotuzumab abrogates acquired radioresistance of KYSE-150R esophageal cancer cells by inhibiting EGFR signaling and cellular DNA repair

    PubMed Central

    Liu, Hai; Yang, Weifang; Gao, Huaping; Jiang, Tingting; Gu, Bengxin; Dong, Qinghua; Xu, Wenhong; Wu, Shixiu; Sun, Xiaonan

    2015-01-01

    Background Acquired radioresistance of cancer is common after repeated irradiation and often leads to treatment failure. This study aimed to examine the effects of nimotuzumab on acquired radioresistance in human esophageal carcinoma cells and to investigate its underlying mechanisms. Methods The radioresistant human esophageal carcinoma cell line KYSE-150R was generated by using fractionated irradiation. KYSE-150R cells were pretreated with or without nimotuzumab before ionizing radiation. Cell growth and colony formation were measured to quantitate the effects of radiation. The γ-H2AX foci assay was employed to determine cellular DNA-repairing capacity. The phosphorylation of key molecules involved in the epidermal growth factor receptor (EGFR) signaling pathway and cellular DNA repair was measured by Western blot analysis. Results Nimotuzumab enhanced radiation-induced inhibition on cell growth and clonogenic survival in KYSE-150R cells. The average number of γ-H2AX foci increased in the irradiated cells treated with nimotuzumab. Nimotuzumab inhibited phosphorylation of the EGFR and its downstream molecules AKT and ERK. Phosphorylation of the DNA repair-related proteins DNA-PKcs, ATM, and RAD51 was also inhibited by nimotuzumab. Conclusions These results indicate that nimotuzumab can inhibit key cancer survival mechanisms, the EGFR signaling pathway, and DNA repair and thereby reverse acquired radioresistance in KYSE-150R cell line. PMID:25750543

  5. Nimotuzumab abrogates acquired radioresistance of KYSE-150R esophageal cancer cells by inhibiting EGFR signaling and cellular DNA repair.

    PubMed

    Liu, Hai; Yang, Weifang; Gao, Huaping; Jiang, Tingting; Gu, Bengxin; Dong, Qinghua; Xu, Wenhong; Wu, Shixiu; Sun, Xiaonan

    2015-01-01

    Acquired radioresistance of cancer is common after repeated irradiation and often leads to treatment failure. This study aimed to examine the effects of nimotuzumab on acquired radioresistance in human esophageal carcinoma cells and to investigate its underlying mechanisms. The radioresistant human esophageal carcinoma cell line KYSE-150R was generated by using fractionated irradiation. KYSE-150R cells were pretreated with or without nimotuzumab before ionizing radiation. Cell growth and colony formation were measured to quantitate the effects of radiation. The γ-H2AX foci assay was employed to determine cellular DNA-repairing capacity. The phosphorylation of key molecules involved in the epidermal growth factor receptor (EGFR) signaling pathway and cellular DNA repair was measured by Western blot analysis. Nimotuzumab enhanced radiation-induced inhibition on cell growth and clonogenic survival in KYSE-150R cells. The average number of γ-H2AX foci increased in the irradiated cells treated with nimotuzumab. Nimotuzumab inhibited phosphorylation of the EGFR and its downstream molecules AKT and ERK. Phosphorylation of the DNA repair-related proteins DNA-PKcs, ATM, and RAD51 was also inhibited by nimotuzumab. These results indicate that nimotuzumab can inhibit key cancer survival mechanisms, the EGFR signaling pathway, and DNA repair and thereby reverse acquired radioresistance in KYSE-150R cell line.

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

    PubMed Central

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

    2016-01-01

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

  7. Oxidatively generated base damage to cellular DNA by hydroxyl radical and one-electron oxidants: similarities and differences.

    PubMed

    Cadet, Jean; Wagner, J Richard

    2014-09-01

    Hydroxyl radical (OH) and one-electron oxidants that may be endogenously formed through oxidative metabolism, phagocytosis, inflammation and pathological conditions constitute the main sources of oxidatively generated damage to cellular DNA. It is worth mentioning that exposure of cells to exogenous physical agents (UV light, high intensity UV laser, ionizing radiation) and chemicals may also induce oxidatively generated damage to DNA. Emphasis is placed in this short review article on the mechanistic aspects of OH and one-electron oxidant-mediated formation of single and more complex damage (tandem lesions, intra- and interstrand cross-links, DNA-protein cross-links) in cellular DNA arising from one radical hit. This concerns DNA modifications that have been accurately measured using suitable analytical methods such as high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Evidence is provided that OH and one-electron oxidants after generating neutral radicals and base radical cations respectively may partly induce common degradation pathways. In addition, selective oxidative reactions giving rise to specific degradation products of OH and one-electron oxidation reactions that can be used as representative biomarkers of these oxidants have been identified.

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

    SciTech Connect

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

    2008-02-01

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

  9. Protection of pigs against genital Chlamydia trachomatis challenge by parenteral or mucosal DNA immunization.

    PubMed

    Schautteet, Katelijn; De Clercq, Evelien; Jönsson, Yannick; Lagae, Stefanie; Chiers, Koen; Cox, Eric; Vanrompay, Daisy

    2012-04-16

    The current study evaluates combined aerosol-vaginal delivery of a MOMP-based Chlamydia trachomatis (serovar E) DNA vaccine in a pig genital challenge model. Most non-replicating antigens are rather poor mucosal immunogens in comparison to replicating antigens. Therefore, a mucosal administered DNA vaccine, which actually mimics a live vaccine, could be promising. Protection was promoted by plasmids encoding the porcine granulocyte macrophage-colony stimulating factor (pcDNA3.1zeo::GM-CSF), the Escherichia coli thermo-labile enterotoxin (LT) subunit A (plasmid PJV2004::LTa) and subunit B (plasmid PJV2005::LTb). Mucosal C. trachomatis DNA vaccination induced significant protection against genital C. trachomatis challenge although the infection could not be eradicated. Intradermal immunization was significantly less efficient in protecting experimentally infected pigs. Protection was correlated with efficient T cell priming and significantly higher serum IgA titers following primo vaccination. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    PubMed

    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; Cacchione, Stefano; Raffa, Grazia Daniela

    2017-04-07

    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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Evidence for mitochondrial DNA polymorphism and uniparental inheritance in the cellular slime mold Polysphondylium pallidum: effect of intraspecies mating on mitochondrial DNA transmission.

    PubMed

    Mirfakhrai, M; Tanaka, Y; Yanagisawa, K

    1990-03-01

    Restriction fragment length polymorphisms (RFLPs) were used as markers to monitor mitochondrial inheritance in the cellular slime mold, Polysphondylium pallidum. When two opposite mating types (mat1 and mat2) of closely related strains were crossed, all the haploid progeny regardless of mating type inherited their mitochondrial DNA from the mat2 parent only. When opposite mating types from more distantly related strains were crossed, most of the progeny also inherited their mitochondrial DNA from the mat2 parent, but some inherited their mitochondrial DNA from the mat1 parent. In both cases however, the transmission of mitochondrial DNA was uniparental, since in every individual progeny only one type of mitochondrial DNA exists. Moreover, in crosses involving more distantly related strains all the progeny of a single macrocyst were shown to contain the same type of mitochondrial DNA. These findings are discussed in regard to mechanisms of transmission and the possible involvement of nuclear genes in the control of transmission of mitochondrial DNA in Polysphondylium.

  12. A DNA Vaccine Formulated with Chemical Adjuvant Provides Partial Protection against Bovine Herpes Virus Infection in Cattle.

    PubMed

    Quattrocchi, Valeria; Soria, Ivana; Langellotti, Cecilia Ana; Gnazzo, Victoria; Gammella, Mariela; Moore, Dadin P; Zamorano, Patricia I

    2017-01-01

    Bovine herpesvirus-1 (BoHV-1) is the causative agent of bovine infectious rhinotracheitis, an important disease worldwide. Although conventional BoHV-1 vaccines, including those based on the use of modified live virus and also inactivated vaccines, are currently used in many countries, they have several disadvantages. DNA vaccines have emerged as an attractive approach since they have the potential to induce both humoral and cellular immune response; nevertheless, it is largely known that potency of naked DNA vaccines is limited. We demonstrated previously, in the murine model, that the use of adjuvants in combination with a DNA vaccine against BoHV-1 is immunologically beneficial. In this study, we evaluate the immune response and protection against challenge elicited in bovines, by a DNA vaccine carrying the sequence of secreted version of glycoprotein D (gD) of BoHV-1 formulated with chemical adjuvants. Bovines were vaccinated with formulations containing the sequence of gD alone or in combination with adjuvants ESSAI 903110 or Montanide™ 1113101PR. After prime vaccination and two boosters, animals were challenged with infectious BoHV-1. Formulations containing adjuvants Montanide™ 1113101PR and ESSAI 903110 were both, capable of increasing humoral immune response against the virus and diminishing clinical symptoms. Nevertheless, only formulations containing adjuvant Montanide™ 1113101PR was capable of improving cellular immune response and diminishing viral excretion. To our knowledge, it is the first time that a BoHV-1 DNA vaccine is combined with adjuvants and tested in cattle. These results could be useful to design a vaccine for the control of bovine rhinotracheitis.

  13. Vaccination with plasmid DNA encoding KMPII, TRYP, LACK and GP63 does not protect dogs against Leishmania infantum experimental challenge.

    PubMed

    Rodríguez-Cortés, Alhelí; Ojeda, Ana; López-Fuertes, Laura; Timón, Marcos; Altet, Laura; Solano-Gallego, Laia; Sánchez-Robert, Elisenda; Francino, Olga; Alberola, Jordi

    2007-11-14

    Vaccination of dogs, the domestic reservoir of Leishmania infantum, is the best method for controlling zoonotic visceral leishmaniasis. This strategy would reduce the incidence of disease in both the canine and, indirectly, the human population. Different vaccination approaches have been investigated against canine leishmaniasis (CaL) but to date there is only one licensed vaccine against this disease in dogs, in Brazil. DNA immunization is a promising method for inducing both humoral and cellular immune responses against this parasitic disease. Here, we report the results of a multiantigenic plasmid DNA vaccine encoding KMPII, TRYP, LACK and GP63 L. infantum antigens against experimentally induced CaL. Twelve dogs were randomly assigned to two groups receiving, at a 15 days interval, either four doses of plasmid DNA or similar injections of PBS. After vaccination, dogs were intravenously challenged with 5 x 10(7) promastigotes of L. infantum. The vaccine showed to be safe and well-tolerated. Neither cellular immune response nor antibodies directed against whole Leishmania antigen were detected after immunization in vaccinated dogs, although anti-LACK-specific antibodies were sporadically detected in two vaccinated dogs before challenge, thus suggesting that antigens were indeed expressed. A delay in the development of detectable specific immune response and parasite multiplication in vaccinated dogs was observed after challenge. Nevertheless, the multiantigenic Leishmania DNA vaccine was unable to induce protection against parasite dissemination or disease. This study emphasizes the need to strengthen DNA vaccines in order to obtain effective immune responses in models other than the murine.

  14. A DNA Vaccine Formulated with Chemical Adjuvant Provides Partial Protection against Bovine Herpes Virus Infection in Cattle

    PubMed Central

    Quattrocchi, Valeria; Soria, Ivana; Langellotti, Cecilia Ana; Gnazzo, Victoria; Gammella, Mariela; Moore, Dadin P.; Zamorano, Patricia I.

    2017-01-01

    Bovine herpesvirus-1 (BoHV-1) is the causative agent of bovine infectious rhinotracheitis, an important disease worldwide. Although conventional BoHV-1 vaccines, including those based on the use of modified live virus and also inactivated vaccines, are currently used in many countries, they have several disadvantages. DNA vaccines have emerged as an attractive approach since they have the potential to induce both humoral and cellular immune response; nevertheless, it is largely known that potency of naked DNA vaccines is limited. We demonstrated previously, in the murine model, that the use of adjuvants in combination with a DNA vaccine against BoHV-1 is immunologically beneficial. In this study, we evaluate the immune response and protection against challenge elicited in bovines, by a DNA vaccine carrying the sequence of secreted version of glycoprotein D (gD) of BoHV-1 formulated with chemical adjuvants. Bovines were vaccinated with formulations containing the sequence of gD alone or in combination with adjuvants ESSAI 903110 or Montanide™ 1113101PR. After prime vaccination and two boosters, animals were challenged with infectious BoHV-1. Formulations containing adjuvants Montanide™ 1113101PR and ESSAI 903110 were both, capable of increasing humoral immune response against the virus and diminishing clinical symptoms. Nevertheless, only formulations containing adjuvant Montanide™ 1113101PR was capable of improving cellular immune response and diminishing viral excretion. To our knowledge, it is the first time that a BoHV-1 DNA vaccine is combined with adjuvants and tested in cattle. These results could be useful to design a vaccine for the control of bovine rhinotracheitis. PMID:28179907

  15. A preparative suspension culture system permitting quantitation of anchorage-independent growth by direct radiolabeling of cellular DNA.

    PubMed

    Assoian, R K; Boardman, L A; Drosinos, S

    1989-02-15

    We have developed a hybrid methylcellulose/agar suspension culture system which permits long-term colony formation of transformed mesenchymal cells. In contrast to traditional agar suspensions, our system allows for recovery of cells and direct biochemical analysis of anchorage-independent growth. The ability to readily radiolabel cellular macromolecules in these preparative cultures permits a quantitative and objective analysis of colony formation by incorporation of [3H]thymidine into newly synthesized DNA.

  16. 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

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

    PubMed

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

    2016-06-14

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

  18. Epitope analysis and protection by a ROP19 DNA vaccine against Toxoplasma gondii

    PubMed Central

    Zhou, Jian; Wang, Lin; Lu, Gang; Zhou, Aihua; Zhu, Meiyan; Li, Qihang; Wang, Zhilin; Arken, Miradel; Wang, Ao; He, Shenyi

    2016-01-01

    We used bioinformatics approaches to identify B-cell and T-cell epitopes on the ROP19 protein of Toxoplasma gondii. Then, we constructed plasmids with ROP19 (pEGFP-C1-ROP19) and injected them into BALB/c mice to test the immunoprotection induced by this vaccine candidate. The results showed that immunization with pEGFP-C1-ROP19 induced effective cellular and humoral immune responses in mice; specifically, high serum levels of T. gondii-specific IgG and increased interferon-gamma production by splenocytes. Furthermore, the mice vaccinated with pROP19 had significantly fewer brain cysts (583 ± 160) than the mice injected with phosphate-buffered saline (1350 ± 243) or with the control plasmid, pEGFP-C1 (1300 ± 167). Compared with PBS-treated mice, those immunized with pROP19 had only 43% of the number of brain cysts. These results suggest that the DNA vaccine encoding ROP19 induced a significant immune response and provided protection against a challenge with T. gondii strain PRU cysts. PMID:27055564

  19. Targeted cellular process profiling approach for uterine leiomyoma using cDNA microarray, proteomics and gene ontology analysis

    PubMed Central

    Ahn, Woong Shick; Kim, Ko-Woon; Bae, Su Mi; Yoon, Joo Hee; Lee, Joon Mo; Namkoong, Sung Eun; Kim, Jin Hong; Kim, Chong Kook; Lee, Young Joo; Kim, Yong-Wan

    2003-01-01

    This study utilized both cDNA microarray and two-dimensional protein gel electrophoresis technology to investigate the multiple interactions of genes and proteins involved in uterine leiomyoma pathophysiology. Also, the gene ontology analysis was used to systematically characterize the global expression profiles at cellular process levels. We profiled differentially expressed transcriptome and proteome in six-paired leiomyoma and normal myometrium. Screening up to 17 000 genes identified 21 upregulated and 50 downregulated genes. The gene-expression profiles were classified into mutually dependent 420 functional sets, resulting in 611 cellular processes according to the gene ontology. Also, protein analysis using two-dimensional gel electrophoresis identified 33 proteins (17 upregulated and 16 downregulated) of more than 500 total spots, which was classified into 302 cellular processes. Of these functional profilings, downregulations of transcriptomes and proteoms were shown in cell adhesion, cell motility, organogenesis, enzyme regulator, structural molecule activity and response to external stimulus functional activities that are supposed to play important roles in pathophysiology. In contrast, the upregulation was only shown in nucleic acid-binding activity. Taken together, potentially significant pathogenetic cellular processes were identified and showed that the downregulated functional profiling has a significant impact on the discovery of pathogenic pathway in leiomyoma. Also, the gene ontology analysis can overcome the complexity of expression profiles of cDNA microarray and two-dimensional protein analysis via its cellular process-level approach. Therefore, a valuable prognostic candidate gene with relevance to disease-specific pathogenesis can be found at cellular process levels. PMID:14748746

  20. The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication

    SciTech Connect

    Mathew, Shomita S.; Bridge, Eileen

    2007-09-01

    Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci.

  1. Novel synthetic plasmid and Doggybone™ DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice

    PubMed Central

    Scott, Veronica L; Patel, Ami; Villarreal, Daniel O; Hensley, Scott E; Ragwan, Edwin; Yan, Jian; Sardesai, Niranjan Y; Rothwell, Paul J; Extance, Jonathan P; Caproni, Lisa J; Weiner, David B

    2015-01-01

    Nucleic acid-based vaccines (NAVs) are a promising alternative to conventional influenza vaccines with the potential to increase influenza vaccine availability due to their simplicity in design and rapid speed of production. NAVs can also target multiple influenza antigens and control flu variants. Traditionally NAVs have been DNA plasmids however, we are continuing to explore new methods that may enhance vaccine efficacy. Recently new focus has been on RNA cassettes as NAVs. RNA vaccines combine conceptual advantages in that they focus on delivery of only the coding cassette. However, RNA vaccines have a short half-life and cause interferon-induced fevers. Here we describe a new NAV approach where we study delivery of a linear DNA cassette [Doggybone™ linear closed DNA [(dbDNA™)] produced by an enzymatic process that yields an antigen expression cassette comprising a promoter, DNA antigen, poly A tail, and telomeric ends. This focused approach has many of the advantages of plasmid DNA as well as a minimal cassette size similar to RNA strategies. For this study, we characterized the specific CD4+ and CD8+ T cell responses and determined the hemagglutination inhibition (HI) titers induced by dbDNA™ and compared the responses with those of an optimized plasmid DNA (pDNA) vaccine encoding the same H1N1 influenza A/PR/8/34 HA gene. Immunizations with the constructs resulted in similar humoral and cellular immune responses. Both constructs induced high-titer HI antibodies and fully protected animals from lethal viral challenge. The data obtained from this study provides important validation for further development of novel vector approaches. PMID:26091432

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

    PubMed

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

    2016-01-01

    Viral infections have caused numerous diseases and deaths worldwide. Due to the emergence of new viruses and frequent virus variation, conventional antiviral strategies that directly target viral or cellular proteins are limited because of the specificity, drug resistance and rapid clearance from the human body. Therefore, developing safe and potent antiviral agents with activity against viral infection at multiple points in the viral life cycle remains a major challenge. In this report, we propose a new modality to inhibit viral infection by fabricating DNA conjugated gold nanoparticle (DNA-AuNP) networks on cell membranes as a protective barrier. The DNA-AuNPs networks were found, via a plaque formation assay and viral titers, to have potent antiviral ability and protect host cells from human respiratory syncytial virus (RSV). Confocal immunofluorescence image analysis showed 80 ± 3.8% of viral attachment, 91.1 ± 0.9% of viral entry and 87.9 ± 2.8% of viral budding were inhibited by the DNA-AuNP networks, which were further confirmed by real-time fluorescence imaging of the RSV infection process. The antiviral activity of the networks may be attributed to steric effects, the disruption of membrane glycoproteins and limited fusion of cell membrane bilayers, all of which play important roles in viral infection. Therefore, our results suggest that the DNA-AuNP networks have not only prophylactic effects to inhibit virus attachment and entry, but also therapeutic effects to inhibit viral budding and cell-to-cell spread. More importantly, this proof-of-principle study provides a pathway for the development of a universal, broad-spectrum antiviral therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Protective DNA vaccination against experimental autoimmune encephalomyelitis is associated with induction of IFNbeta.

    PubMed

    Wefer, Judit; Harris, Robert A; Lobell, Anna

    2004-04-01

    DNA vaccines encoding encephalitogenic peptides protect against subsequent development of rat experimental autoimmune encephalomyelitis (EAE) through unknown mechanisms. We investigated immune cell phenotypes at different time points after DNA vaccination with vaccine encoding myelin oligodendrocyte glycoprotein peptide 91-108 and subsequent induction of EAE. In protected rats, we observed (i) no alterations in antigen-specific Th2 or Th3 responses, (ii) reduced MHC II expression on splenocytes early after EAE induction, (iii) antigen-specific upregulation of IFNbeta upon recall stimulation and (iv) reduced IL-12Rbeta2 on lymphocytes. We suggest that the underlying mechanism of DNA vaccination is associated with immunomodulation exerted by induced IFNbeta.

  4. Characterization of the protective and therapeutic efficiency of a DNA vaccine encoding the major birch pollen allergen Bet v 1a.

    PubMed

    Hartl, A; Hochreiter, R; Stepanoska, T; Ferreira, F; Thalhamer, J

    2004-01-01

    An estimated 100 million individuals suffer from birch pollen allergy. More than 95% of birch pollen-allergic subjects react with the major birch pollen allergen Bet v 1a, and almost 60% of them are sensitized exclusively to this allergen. DNA immunization using the Bet v 1a gene was evaluated with respect to its prophylactic and therapeutic efficacy. A DNA vaccine containing the entire Bet v 1a cDNA under the control of a CMV-promoter was constructed. In order to estimate the protective efficiency, animals received three injections of this vaccine prior to sensitization with recombinant Bet v 1a. Vice versa, in a therapeutic approach, sensitization was followed by treatment with the DNA vaccine. The Bet v 1a DNA vaccine induced strong Bet v 1-specific antibody responses with a Th1-biased response type. Animals which received the DNA vaccine were protected against a following allergic sensitization with Bet v 1a. The protective effect was characterized by suppression of Bet v 1-specific immunoglobulin (Ig)E production, lack of basophil activation and enhanced interferon (IFN)-gamma expression. In a therapeutic situation, treatment of sensitized animals with DNA vaccines decreased IgE production, IgE-mediated basophil release and drastically reduced anaphylactic activity as measured by passive cutaneous anaphylaxis assays. Concerning the cellular immune response, DNA immunization induced a sustaining and dominant shift from a Th2 type response towards a balanced Th1/Th2 type response as indicated by increased IFN-gamma but unchanged IL-5 levels in lymphoproliferation assays. The results demonstrate the allergen-specific protective and therapeutic efficacy of a DNA vaccine encoding the clinically highly relevant allergen Bet v 1a indicating the suitability of this concept for the treatment of allergic diseases.

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

    PubMed

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

    2015-08-15

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

  6. Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA.

    PubMed Central

    Khan, A S; Repaske, R; Garon, C F; Chan, H W; Rowe, W P; Martin, M A

    1982-01-01

    Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA. Images PMID:6281459

  7. Compromised cellular responses to DNA damage accelerate chronological aging by incurring cell wall fragility in Saccharomyces cerevisiae.

    PubMed

    Yu, Shanshan; Zhang, Xian-En; Chen, Guanjun; Liu, Weifeng

    2012-04-01

    Elevated levels of reactive oxygen species (ROS) can attack almost all cell components including genomic DNA to induce many types of DNA damage. In this study, we used Saccharomyces cerevisiae with various mutations in a biological network supposed to prevent deleterious effects of endogenous ROS to test the effect of such a network on yeast chronological aging. Our results showed that cells with defects in cellular antioxidation, DNA repair and DNA damage checkpoints displayed a mutation rate higher than that of wild-type strain. Moreover, the chronological life span of most mutants as determined by colony formation was found to be shorter than that of wild-type cells, especially for the mutants defective in DNA replication and DNA damage checkpoints, although the observed cell number was almost the same for wild-type and mutant strains. The mutants were finally found to be more sensitive to SDS and lysing enzyme treatment, and that the degree of sensitivity was correlated with their chronological life span.

  8. Label-free and real-time imaging of dehydration-induced DNA conformational changes in cellular nucleus using second harmonic microscopy

    NASA Astrophysics Data System (ADS)

    Zhuo, Shuangmu; Ni, Ming

    2014-12-01

    Dehydration-induced DNA conformational changes have been probed for the first time with the use of second harmonic microscopy. Unlike conventional approaches, second harmonic microscopy provides a label-free and real-time approach to detect DNA conformational changes. Upon dehydration, cellular DNA undergoes a transition from B- to A-form, whereas cellular nuclei change from invisible to visible under second harmonic microscopy. These results showed that DNA is a second order nonlinear optical material. We further confirmed this by characterizing the nonlinear optical properties of extracted DNA from human cells. Our findings open a new path for SHG imaging. DNA can change its conformations under many circumstances. For example: normal cells turning into cancerous cells and drug molecules binding with DNA. Therefore, the detection of DNA conformational changes with second harmonic microscopy will be a useful tool in cancer therapy and new drug discovery.

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

    NASA Astrophysics Data System (ADS)

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

    1993-12-01

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

  10. [Antioxidation activity and protective effection of ginger oil on DNA damage in vitro].

    PubMed

    Lu, Ping; Lai, Bing-sen; Liang, Ping; Chen, Zhen-tong; Shun, Shu-qing

    2003-09-01

    To study the antioxidation activity and protective effect of ginger oil on DNA damage. Chemical light assay was used to detect the oxygen radicals scavenging capacity of ginger oil. The erythrocyte oxidation damage was induced by H2O2. The effect of ginger oil on oxidative erythrocyte was observed by the colorimetric analysis assay, and the content of malondialdehyde (MDA) in rabit hepatocyte was measured. The anaylsis of DNA damage was made with single cell gel electrophoresis(SCGE) technique. Ginger oil might decrease light value compared with control group and inhibited erythrocyte oxidation damage. Compared with that in control group, the degress of DNA damage reduced significantly in the protected groups. Ginger oil might decrease the content of MDA remarkably and inhitibition rate was 48.16%. Ginger oil has dominantive protective effect on DNA damage induced by H2O2. Ginger oil might act as a scavenger of oxygen radical and might be used as an antioxidant.

  11. Biochemical, cellular and molecular identification of DNA polymerase α in yeast mitochondria.

    PubMed

    Lasserre, Jean-Paul; Plissonneau, Jacqueline; Velours, Christophe; Bonneu, Marc; Litvak, Simon; Laquel, Patricia; Castroviejo, Michel

    2013-04-01

    DNA replication occurs in various compartments of eukaryotic cells such as the nuclei, mitochondria and chloroplasts, the latter of which is used in plants and algae. Replication appears to be simpler in the mitochondria than in the nucleus where multiple DNA polymerases, which are key enzymes for DNA synthesis, have been characterized. In mammals, only one mitochondrial DNA polymerase (pol γ) has been described to date. However, in the mitochondria of the yeast Saccharomyces cerevisiae, we have found and characterized a second DNA polymerase. To identify this enzyme, several biochemical approaches such as proteinase K treatment of sucrose gradient purified mitochondria, analysis of mitoplasts, electron microscopy and the use of mitochondrial and cytoplasmic markers for immunoblotting demonstrated that this second DNA polymerase is neither a nuclear or cytoplasmic contaminant nor a proteolytic product of pol γ. An improved purification procedure and the use of mass spectrometry allowed us to identify this enzyme as DNA polymerase α. Moreover, tagging DNA polymerase α with a fluorescent probe demonstrated that this enzyme is localized both in the nucleus and in the organelles of intact yeast cells. The presence of two replicative DNA polymerases may shed new light on the mtDNA replication process in S. cerevisiae. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

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

    PubMed Central

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

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Baeza, Isabel; Ibáñez, Miguel; Wong, Carlos; Chávez, Pedro; Gariglio, Patricio; Oró, J.

    1992-07-01

    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 theE. 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.

  14. 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.

  15. 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.

  16. Novel Mucosal DNA-MVA HIV Vaccination in Which DNA-IL-12 Plus Cholera Toxin B Subunit (CTB) Cooperates to Enhance Cellular Systemic and Mucosal Genital Tract Immunity

    PubMed Central

    Holgado, María Pía; Falivene, Juliana; Gherardi, María Magdalena

    2014-01-01

    Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after their mucosal administration in DNA prime/MVA boost intranasal regimes, defining the cooperation of both adjuvants to enhance immune responses against the HIV-1 Env antigen. Our results demonstrated that nasal mucosal DNA/MVA immunization schemes can be effectively improved by the co-delivery of DNA-IL-12 plus CTB inducing elevated HIV-specific CD8 responses in spleen and more importantly in genital tract and genito-rectal draining lymph nodes. Remarkably, these CTL responses were of superior quality showing higher avidity, polyfunctionality and a broader cytokine profile. After IL-12+CTB co-delivery, the cellular responses induced showed an enhanced breadth recognizing with higher efficiency Env peptides from different subtypes. Even more, an in vivo CTL cytolytic assay demonstrated the higher specific CD8 T-cell performance after the IL-12+CTB immunization showing in an indirect manner its potential protective capacity. Improvements observed were maintained during the memory phase where we found higher proportions of specific central memory and T memory stem-like cells T-cell subpopulations. Together, our data show that DNA-IL-12 plus CTB can be effectively employed acting as mucosal adjuvants during DNA prime/MVA boost intranasal vaccinations, enhancing magnitude and quality of HIV-specific systemic and mucosal immune responses. PMID:25215887

  17. Synergistic and additive effects of cimetidine and levamisole on cellular immune responses to hepatitis B virus DNA vaccine in mice.

    PubMed

    Niu, X; Yang, Y; Wang, J

    2013-02-01

    We and others have previously shown that both cimetidine (CIM) and levamisole (LMS) enhance humoral and cellular responses to DNA vaccines via different mechanisms. In this study, we investigated the synergistic and additive effects of CIM and LMS on the potency of antigen-specific immunities generated by a DNA vaccine encoding the hepatitis B surface antigen (HBsAg, pVax-S2). Compared with CIM or LMS alone, the combination of CIM and LMS elicited a robust HBsAg-specific cellular response that was characterized by higher IgG2a, but did not further increase HBsAg-specific antibody IgG and IgG1 production. Consistent with these results, the combination of CIM and LMS produced the highest level of IL-2 and IFN-γ in antigen-specific CD4(+) T cells, whereas the combination of CIM and LMS did not further increase IL-4 production. Significantly, a robust HBsAg-specific cytotoxic response was also observed in the animals immunized with pVax-S2 in the presence of the combination of CIM and LMS. Further mechanistic studies demonstrated that the combination of CIM and LMS promoted dendritic cell (DC) activation and blocked anti-inflammatory cytokine IL-10 and TGF-β production in CD4(+) CD25(+) T cells. These findings suggest that CIM and LMS have the synergistic and additive ability to enhance cellular response to hepatitis B virus DNA vaccine, which may be mediated by DC activation and inhibition of anti-inflammatory cytokine expression. Thus, the combination of cimetidine and levamisole may be useful as an effective adjuvant in DNA vaccinations for chronic hepatitis B virus infection.

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

    NASA Technical Reports Server (NTRS)

    Lett, J. T.

    1992-01-01

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

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

    NASA Technical Reports Server (NTRS)

    Lett, J. T.

    1992-01-01

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

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

    PubMed

    Tuteja, Narendra; Tuteja, Renu

    2004-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

  2. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    PubMed

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-08-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene.

  3. Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein.

    PubMed Central

    Shubeita, H E; Sambrook, J F; McCormick, A M

    1987-01-01

    A recombinant cDNA clone, pCRABP-HS1, encoding cellular retinoic acid-binding protein was isolated from a bovine adrenal cDNA library. COS-7 cells transfected with pCRABP-HS1 produced a biologically active retinoic acid-binding protein molecule of the expected molecular mass (15.5 kDa). RNA blot hybridization analysis using pCRABP-HS1 as a probe revealed a single 1050-nucleotide mRNA species in bovine adrenal, uterus, and testis, tissues that contain the highest levels of retinoic acid-binding activity. No hybridization was detected in RNA extracted from ovary, spleen, kidney, or liver, which contain relatively low levels of cellular retinoic acid-binding protein activity. Analysis of genomic clones isolated from an EcoRI bovine genomic library demonstrated that the bovine cellular retinoic acid-binding protein gene is composed of four exons and three introns. Two putative promoter sequences were identified in the cloned 5' sequence of the gene. Images PMID:3039499

  4. Rapid outer-surface protein C DNA tattoo vaccination protects against Borrelia afzelii infection.

    PubMed

    Wagemakers, A; Mason, L M K; Oei, A; de Wever, B; van der Poll, T; Bins, A D; Hovius, J W R

    2014-12-01

    Borrelia afzelii is the predominant Borrelia species causing Lyme borreliosis in Europe. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines against Borrelia burgdorferi sensu stricto. DNA tattooing is a novel vaccination method that can be applied in a rapid vaccination schedule. We vaccinated C3H/HeN mice with B. afzelii strain PKo OspC (outer-surface protein C) using a codon-optimized DNA vaccine tattoo and compared this with recombinant protein vaccination in a 0-2-4 week vaccination schedule. We also assessed protection by DNA tattoo in a 0-3-6 day schedule. DNA tattoo and recombinant OspC vaccination induced comparable total IgG responses, with a lower IgG1/IgG2a ratio after DNA tattoo. Two weeks after syringe-challenge with 5 × 10(5) B. afzelii spirochetes most vaccinated mice had negative B. afzelii tissue DNA loads and all were culture negative. Furthermore, DNA tattoo vaccination in a 0-3-6 day regimen also resulted in negative Borrelia loads and cultures after challenge. To conclude, DNA vaccination by tattoo was fully protective against B. afzelii challenge in mice in a rapid vaccination protocol, and induces a favorable humoral immunity compared to recombinant protein vaccination. Rapid DNA tattoo is a promising vaccination strategy against spirochetes.

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

    PubMed Central

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

    2015-01-01

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

  6. DNA Methylation Inhibitor Zebularine Confers Stroke Protection in Ischemic Rats.

    PubMed

    Dock, Hua; Theodorsson, Annette; Theodorsson, Elvar

    2015-08-01

    5-Aza-deoxycytidine (5-aza-dC) confers neuroprotection in ischemic mice by inhibiting DNA methylation. Zebularine is another DNA methylation inhibitor, less toxic and more stable in aqueous solutions and, therefore more biologically suitable. We investigated Zebularine's effects on brain ischemia in a rat middle cerebral artery occlusion (MCAo) model in order to elucidate its therapeutic potential. Male Wistar wild-type (WT) rats were randomly allocated to three treatment groups, vehicle, Zebularine 100 μg, and Zebularine 500 μg. Saline (10 μL) or Zebularine (10 μL) was administered intracerebroventricularly 20 min before 45-min occlusion of the middle cerebral artery. Reperfusion was allowed after 45-min occlusion, and the rats were sacrificed at 24-h reperfusion. The brains were removed, sliced, and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) before measuring infarct size. Zebularine (500 μg) reduced infarct volumes significantly (p < 0.05) by 61% from 20.7 ± 4.2% in the vehicle treated to 8.1 ± 1.6% in the Zebularine treated. Zebularine (100 μg) also reduced infarct volumes dramatically by 55 to 9.4 ± 1.2%. The mechanisms behind this neuroprotection is not yet known, but the results agree with previous studies and support the notion that Zebularine-induced inhibition of DNA methyltransferase ameliorates ischemic brain injury in rats.

  7. Alpha, beta-unsaturated lactones 2-furanone and 2-pyrone induce cellular DNA damage, formation of topoisomerase I- and II-DNA complexes and cancer cell death.

    PubMed

    Calderón-Montaño, José Manuel; Burgos-Morón, Estefanía; Orta, Manuel Luis; Pastor, Nuria; Austin, Caroline A; Mateos, Santiago; López-Lázaro, Miguel

    2013-09-12

    The alpha, beta-unsaturated lactones 2-furanone and 2-pyrone are part of the chemical structure of a variety of naturally occurring compounds (e.g., cardenolides, bufadienolides, acetogenins, coumarins, and food-flavoring furanones), some of which have shown anticancer activity and/or DNA damaging effects. Here we report that 2-furanone and 2-pyrone induce cellular DNA damage (assessed by the comet assay and the gamma-H2AX focus assay) and the formation of topoisomerase I- and topoisomerase II-DNA complexes in cells (visualized and quantified in situ by the TARDIS assay). Cells mutated in BRCA2 (deficient in homologous recombination repair) were significantly hypersensitive to the cytotoxic activity of 2-pyrone, therefore suggesting that BRCA2 plays an important role in the repair of DNA damage induced by this lactone. Both lactones were cytotoxic in A549 lung cancer cells at lower concentrations than in MRC5 non-malignant lung fibroblasts. The possible involvement of 2-furanone and 2-pyrone in the anticancer and DNA-damaging activities of compounds containing these lactones is discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

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

    2015-06-01

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

  9. LKB1 is a DNA damage response protein that regulates cellular sensitivity to PARP inhibitors

    PubMed Central

    Wang, Huibo; Wang, Shuai; Hang, Wei; Zeng, Qinghua; Quan, Cheng-Shi; Zhai, Ying-Xian; Wang, Jian-Wei; Shen, Xiang-Feng; Jian, Yong-Ping; Zhao, Rui-Xun; Werle, Kaitlin D.; Cui, Rutao; Liang, Jiyong; Li, Yu-Lin; Xu, Zhi-Xiang

    2016-01-01

    Liver kinase B1 (LKB1) functions as a tumor suppressor encoded by STK11, a gene that mutated in Peutz-Jeghers syndrome and in sporadic cancers. Previous studies showed that LKB1 participates in IR- and ROS-induced DNA damage response (DDR). However, the impact of LKB1 mutations on targeted cancer therapy remains unknown. Herein, we demonstrated that LKB1 formed DNA damage-induced nuclear foci and co-localized with ataxia telangiectasia mutated kinase (ATM), γ-H2AX, and breast cancer susceptibility 1 (BRCA1). ATM mediated LKB1 phosphorylation at Thr 363 following the exposure of cells to ionizing radiation (IR). LKB1 interacted with BRCA1, a downstream effector in DDR that is recruited to sites of DNA damage and functions directly in homologous recombination (HR) DNA repair. LKB1 deficient cells exhibited delayed DNA repair due to insufficient HR. Notably, LKB1 deficiency sensitized cells to poly (ADP-ribose) polymerase (PARP) inhibitors. Thus, we have demonstrated a novel function of LKB1 in DNA damage response. Cancer cells lacking LKB1 are more susceptible to DNA damage-based therapy and, in particular, to drugs that further impair DNA repair, such as PARP inhibitors. PMID:27705915

  10. Metal-mediated DNA damage and cell death: mechanisms, detection methods, and cellular consequences.

    PubMed

    Angelé-Martínez, Carlos; Goodman, Craig; Brumaghim, Julia

    2014-08-01

    The redox activity of metal ions can lead to the formation of highly reactive species that damage DNA, producing different oxidation products and types of damage depending upon the redox potentials of the DNA bases, formation of intermediate adducts, and identity of the reactive species. Other factors are also important in determining the degree of metal-mediated DNA damage, such as localization and redox chemistry of the metal ions or complexes and lifetimes of the reactive oxygen species generated. This review examines the types of DNA damage mediated by first-row transition metals under oxidative stress conditions, with emphasis on work published in the past ten years. Similarities and differences between DNA damage mechanisms of the first-row transition metals in vitro and in E. coli and human cells are compared and their relationship to disease development are discussed. Methods to detect this metal-mediated DNA damage, including backbone breakage, base oxidation, inter- and intra-strand crosslinking, and DNA-protein crosslinking are also briefly reviewed, as well as detection methods for reactive oxygen species generated by these metal ions. Understanding the conditions that cause metal-mediated DNA damage and metal generation of reactive oxygen species in vitro and in cells is required to develop effective drugs to prevent and treat chronic disease.

  11. Remodelers Organize Cellular Chromatin by Counteracting Intrinsic Histone-DNA Sequence Preferences in a Class-Specific Manner

    PubMed Central

    Chalkley, Gillian E.; Kan, Tsung Wai; Reddy, B. Ashok; Ozgur, Zeliha; van Ijcken, Wilfred F. J.; Dekkers, Dick H. W.; Demmers, Jeroen A.; Travers, Andrew A.

    2012-01-01

    The nucleosome is the fundamental repeating unit of eukaryotic chromatin. Here, we assessed the interplay between DNA sequence and ATP-dependent chromatin-remodeling factors (remodelers) in the nucleosomal organization of a eukaryotic genome. We compared the genome-wide distribution of Drosophila NURD, (P)BAP, INO80, and ISWI, representing the four major remodeler families. Each remodeler has a unique set of genomic targets and generates distinct chromatin signatures. Remodeler loci have characteristic DNA sequence features, predicted to influence nucleosome formation. Strikingly, remodelers counteract DNA sequence-driven nucleosome distribution in two distinct ways. NURD, (P)BAP, and INO80 increase histone density at their target sequences, which intrinsically disfavor positioned nucleosome formation. In contrast, ISWI promotes open chromatin at sites that are propitious for precise nucleosome placement. Remodelers influence nucleosome organization genome-wide, reflecting their high genomic density and the propagation of nucleosome redistribution beyond remodeler binding sites. In transcriptionally silent early embryos, nucleosome organization correlates with intrinsic histone-DNA sequence preferences. Following differential expression of the genome, however, this relationship diminishes and eventually disappears. We conclude that the cellular nucleosome landscape is the result of the balance between DNA sequence-driven nucleosome placement and active nucleosome repositioning by remodelers and the transcription machinery. PMID:22124157

  12. Intradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses.

    PubMed Central

    Raz, E; Carson, D A; Parker, S E; Parr, T B; Abai, A M; Aichinger, G; Gromkowski, S H; Singh, M; Lew, D; Yankauckas, M A

    1994-01-01

    The skin and mucous membranes are the anatomical sites were most viruses are first encountered by the immune system. Previous experiments have suggested that striated muscle cells are unique among mammalian cell types in their capacity to take up and express free DNA in the absence of a viral vector or physical carrier. However, we have found that mice injected into the superficial skin with free (naked) plasmid DNA encoding the influenza nucleoprotein gene had discrete foci of epidermal and dermal cells, including cells with dendritic morphology, that contained immunoreactive nucleoprotein antigen. A single intradermal administration of 0.3-15 micrograms of free plasmid DNA induced anti-nucleoprotein-specific antibody and cytotoxic T lymphocytes that persisted for at least 68-70 weeks after vaccination. Intradermal gene administration induced higher antibody titers than did direct gene injection into skeletal muscle and did not cause local inflammation or necrosis. Compared with control animals, the gene-injected mice were resistant to challenge with a heterologous strain of influenza virus. These results indicate that the cells of the skin can take up and express free foreign DNA and induce cellular and humoral immune responses against the encoded protein. We suggest that DNA uptake by the skin-associated lymphoid tissues may play a role in the induction of cytotoxic T cells against viruses and other intracellular pathogens. Images PMID:7937799

  13. DNA vaccines as a tool for analysing the protective immune response against rhabdoviruses in rainbow trout.

    PubMed

    Lorenzen, N; Lorenzen, E; Einer-Jensen, K; LaPatra, S E

    2002-05-01

    DNA vaccines based on the glycoprotein genes of the salmonid rhabdoviruses VHSV and IHNV have been demonstrated to be very efficient in inducing a protective immune response against the respective diseases in rainbow trout. Nanogram doses of plasmid DNA delivered by intramuscular injection are sufficient to induce high levels of immunity in fingerling-size fish, whereas larger fish require more vaccine for protection. The protection is long lasting and, more surprisingly, is partly established already 4 days post vaccination. The early protection involves cross-protective anti-viral defence mechanisms, while the long duration immunity is highly specific. The nature of these immune response mechanisms is discussed and it is suggested that the efficacy of the vaccines is related to their ability to activate the innate immune system as it is activated by live virus.

  14. Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes.

    PubMed

    Manna, Sudeshna; Panse, Cornelia H; Sontakke, Vyankat A; Sangamesh, Sarangamath; Srivatsan, Seergazhi G

    2017-08-17

    The development of biophysical systems that enable an understanding of the structure and ligand-binding properties of G-quadruplex (GQ)-forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ-directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H-Telo) DNA and RNA repeats in a cell-like confined environment by using conformation-sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2-ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2'-deoxy and ribonucleoside probes, composed of a 5-benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H-Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H-Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy-to-handle RMs could provide new opportunities to study and devise screening-compatible assays in a cell-like environment to discover GQ binders of clinical potential. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Transcutaneous DNA immunization following waxing-based hair depilation elicits both humoral and cellular immune responses

    PubMed Central

    Xiao, Gang; Li, Xinran; Kumar, Amit; Cui, Zhengrong

    2012-01-01

    Previously, we showed that transcutaneous (TC) DNA immunization by applying plasmid DNA onto a mouse skin area wherein the hair follicles were induced into growth stage by plucking the hair using warm waxing induced strong and functional antigen-specific antibody responses. In the present study, using plasmids that encode β-galactosidase gene or ovalbumin (OVA) gene, we showed that this mode of TC DNA immunization not only induced specific antibody responses, but also induced antigen-specific cytotoxic T lymphocyte responses. In fact, TC DNA immunization using a plasmid that encodes OVA gene prevented the growth of OVA-expressing B16-OVA tumor cells in the immunized mice. Moreover, we provided additional evidence supporting that hair follicles are essential for this mode of TC DNA immunization. PMID:22771558

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

    PubMed

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

    2004-12-03

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

  17. 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

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

    PubMed Central

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

    2016-01-01

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

  19. Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles

    PubMed Central

    2012-01-01

    Background Gene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency. Results Nanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-γ) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression. Conclusion Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the

  20. Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles.

    PubMed

    Singh, Jagbir; Michel, Deborah; Chitanda, Jackson M; Verrall, Ronald E; Badea, Ildiko

    2012-02-01

    Gene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency. Nanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-γ) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression. Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the unsubstituted parent gemini surfactant

  1. [Role of layered double hydroxide (LDH) in the protection of herring testis DNA from heavy metals].

    PubMed

    Tang, Yi-Ni; Wu, Ping-Xiao; Zhu, Neng-Wu

    2012-10-01

    The role of layered double hydroxide (LDH) in the protection of herring testis DNA from heavy metals Cd2+ and Pb2+ was studied by X-ray diffraction ( XRD) spectra, Fourier transform infrared (FTIR) spectra, Scanning Electron Microscopy (SEM), Cyclic Voltammetry and Ultraviolet Spectrometry. Size expansion of the basal spacing (003) from 0. 76 nm in LDH to 2. 30 nm was observed in the resulting DNA-LDH nanohybrids and it gave peaks corresponding to C=O (1 534 cm(-1) and 1488 cm(-1)) in skeleton and bases, C-O stretching vibration (1228 cm(-1)), and P-O symmetrical stretching vibration (1096 cm(-1)) in functional groups of DNA, indicating that DNA were intercalated into the LDH by the ion exchange. However, the displacement of NO3(-) was not fully complete (partial intercalation of DNA). The DNA outside LDH interlayers was absorbed on the surface of LDH. The cyclic voltammetric curves showed that DNA in the composites exhibited a very similar peaks, which corresponded to the two reduction current peaks (E(P) = - 1.2 mV and E(P) = -2.4 mV) of free DNA. Also there was no cathode sag emerging in cyclic voltammetric curves, suggesting that both Cd2+ and Pb2+ cannot insert into the groove of DNA to associate with base pairs or other groups when DNA was bound on LDH. The results showed that, on the one hand, both Cd2+ and Pb2+ were absorbed on the external surface of LDH for immobilization, on the other hand, the layer of LDH provided ideal space for DNA by the action of protecting DNA molecules from Cd2+ and Pb2+.

  2. Cellular Preoxygenation Partially Attenuates the Antitumoral Effect of Cisplatin despite Highly Protective Effects on Renal Epithelial Cells.

    PubMed

    Rasoulian, Bahram; Kaeidi, Ayat; Rezaei, Maryam; Hajializadeh, Zahra

    2017-01-01

    Our previous in vitro studies demonstrated that oxygen pretreatment significantly protects human embryonic renal tubular cell against acute cisplatin- (CP-) induced cytotoxicity. The present study was designed to investigate whether this protective effect is associated with decreasing therapeutic effects of cisplatin on malignant cells. For this purpose, cultured human embryonic kidney epithelial-like (AD293), cervical carcinoma epithelial-like (Hela), and ovarian adenocarcinoma epithelial-like (OVCAR-3) cells were subjected to either 2-hour pretreatment with oxygen (≥90%) or normal air and then to a previously determined 50% lethal dose of cisplatin for 24 hours. Cellular viability was evaluated via MTT and Neutral Red assays. Also, activated caspase-3 and Bax/Bcl-2 ratio, as the biochemical markers of cell apoptosis, were determined using immunoblotting. The hyperoxic preexposure protocol significantly protects renal AD293 cells against cisplatin-induced toxicity. Oxygen pretreatment also partially attenuated the cisplatin-induced cytotoxic effects on Hela and OVCAR-3 cells. However, it did not completely protect these cells against the therapeutic cytotoxic effects of cisplatin. In summary, the protective methods for reducing cisplatin nephrotoxic side effects like oxygen pretreatment might be associated with concurrent reduction of the therapeutic cytotoxic effects of cisplatin on malignant cells like cervical carcinoma (Hela) and ovarian adenocarcinoma (OVCAR-3) cells.

  3. Cellular Preoxygenation Partially Attenuates the Antitumoral Effect of Cisplatin despite Highly Protective Effects on Renal Epithelial Cells

    PubMed Central

    Rasoulian, Bahram; Rezaei, Maryam; Hajializadeh, Zahra

    2017-01-01

    Our previous in vitro studies demonstrated that oxygen pretreatment significantly protects human embryonic renal tubular cell against acute cisplatin- (CP-) induced cytotoxicity. The present study was designed to investigate whether this protective effect is associated with decreasing therapeutic effects of cisplatin on malignant cells. For this purpose, cultured human embryonic kidney epithelial-like (AD293), cervical carcinoma epithelial-like (Hela), and ovarian adenocarcinoma epithelial-like (OVCAR-3) cells were subjected to either 2-hour pretreatment with oxygen (≥90%) or normal air and then to a previously determined 50% lethal dose of cisplatin for 24 hours. Cellular viability was evaluated via MTT and Neutral Red assays. Also, activated caspase-3 and Bax/Bcl-2 ratio, as the biochemical markers of cell apoptosis, were determined using immunoblotting. The hyperoxic preexposure protocol significantly protects renal AD293 cells against cisplatin-induced toxicity. Oxygen pretreatment also partially attenuated the cisplatin-induced cytotoxic effects on Hela and OVCAR-3 cells. However, it did not completely protect these cells against the therapeutic cytotoxic effects of cisplatin. In summary, the protective methods for reducing cisplatin nephrotoxic side effects like oxygen pretreatment might be associated with concurrent reduction of the therapeutic cytotoxic effects of cisplatin on malignant cells like cervical carcinoma (Hela) and ovarian adenocarcinoma (OVCAR-3) cells. PMID:28298953

  4. Aberrant DNA methylation of blood in schizophrenia by adjusting for estimated cellular proportions.

    PubMed

    Kinoshita, Makoto; Numata, Shusuke; Tajima, Atsushi; Ohi, Kazutaka; Hashimoto, Ryota; Shimodera, Shinji; Imoto, Issei; Takeda, Masatoshi; Ohmori, Tetsuro

    2014-12-01

    DNA methylation, which is the transference of a methyl group to the 5'-carbon position of the cytosine in a CpG dinucleotide, is one of the major mechanisms of epigenetic modifications. A number of studies have demonstrated altered DNA methylation of peripheral blood cells in schizophrenia (SCZ) in previous studies. However, most of these studies have been limited to the analysis of the CpG sites in CpG islands in gene promoter regions, and cell-type proportions of peripheral leukocytes, which may be one of the potential confounding factors for DNA methylation, have not been adjusted in these studies. In this study, we performed a genome-wide DNA methylation profiling of the peripheral leukocytes from patients with SCZ and from non-psychiatric controls (N = 105; 63 SCZ and 42 control subjects) using a quantitative high-resolution DNA methylation microarray which covered across the whole gene region (485,764 CpG dinucleotides). In the DNA methylation data analysis, we first estimated the cell-type proportions of each sample with a published algorithm. Next, we performed a surrogate variable analysis to identify potential confounding factors in our microarray data. Finally, we conducted a multiple linear regression analysis in consideration of these factors, including estimated cell-type proportions, and identified aberrant DNA methylation in SCZ at 2,552 CpG loci at a 5% false discovery rate correction. Our results suggest that altered DNA methylation may be involved in the pathophysiology of SCZ, and cell heterogeneity adjustments may be necessary for DNA methylation analysis.

  5. Recombinational DNA repair in a cellular context: a search for the homology search.

    PubMed

    Weiner, Allon; Zauberman, Nathan; Minsky, Abraham

    2009-10-01

    Double-strand DNA breaks (DSBs) are the most detrimental lesion that can be sustained by the genetic complement, and their inaccurate mending can be just as damaging. According to the consensual view, precise DSB repair relies on homologous recombination. Here, we review studies on DNA repair, chromatin diffusion and chromosome confinement, which collectively imply that a genome-wide search for a homologous template, generally thought to be a pivotal stage in all homologous DSB repair pathways, is improbable. The implications of this assertion for the scope and constraints of DSB repair pathways and for the ability of diverse organisms to cope with DNA damage are discussed.

  6. SAG4 DNA and Peptide Vaccination Provides Partial Protection against T. gondii Infection in BALB/c Mice.

    PubMed

    Zhou, Jian; Wang, Lin

    2017-01-01

    Toxoplasma gondii can lead to congenital infections in human. Surface antigen protein 4 (SAG4) of T. gondii is a potential stimulator for humoral and cellular immune responses. In the present study, a DNA vaccine encoding SAG4 from T. gondii was constructed and used to immunize BALB/c mice with peptide to evaluate the protective efficacy of the vaccine. The productions of IgG antibodies and cytokines (gamma interferon) from the vaccine (pSAG4/peptide) group were significantly higher than pSAG4 or peptide groups. After a lethal challenge by 1 × 10(4) tachyzoites from the I strain (RH), the survival time of mice immunized by pSAG4/peptide was longer than that of pSAG4 or peptide immunized mice or control mice. Moreover, after challenging by 20 cysts of the II strain (PRU) of T. gondii, the number of brain cysts from pSAG4/peptide vaccinated mice was only 31% of the number in PBS injected mice. The findings suggested the SAG4 DNA vaccine with peptide led significant immune responses and improved the protection against T. gondii challenges.

  7. SAG4 DNA and Peptide Vaccination Provides Partial Protection against T. gondii Infection in BALB/c Mice

    PubMed Central

    Zhou, Jian; Wang, Lin

    2017-01-01

    Toxoplasma gondii can lead to congenital infections in human. Surface antigen protein 4 (SAG4) of T. gondii is a potential stimulator for humoral and cellular immune responses. In the present study, a DNA vaccine encoding SAG4 from T. gondii was constructed and used to immunize BALB/c mice with peptide to evaluate the protective efficacy of the vaccine. The productions of IgG antibodies and cytokines (gamma interferon) from the vaccine (pSAG4/peptide) group were significantly higher than pSAG4 or peptide groups. After a lethal challenge by 1 × 104 tachyzoites from the I strain (RH), the survival time of mice immunized by pSAG4/peptide was longer than that of pSAG4 or peptide immunized mice or control mice. Moreover, after challenging by 20 cysts of the II strain (PRU) of T. gondii, the number of brain cysts from pSAG4/peptide vaccinated mice was only 31% of the number in PBS injected mice. The findings suggested the SAG4 DNA vaccine with peptide led significant immune responses and improved the protection against T. gondii challenges. PMID:28936207

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

    PubMed

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

    2014-01-01

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

  9. Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress

    PubMed Central

    Cheng, Kwai Wa; Agarwal, Roshan; Mitra, Shreya; Lee, Ju-Seog; Carey, Mark; Gray, Joe W; Mills, Gordon B

    2012-01-01

    Cancer cells are metabolically stressed during tumour progression due to limited tumour vascularity and resultant nutrient, growth factor and oxygen deficiency that can induce cell death and inhibit tumour growth. We demonstrate that Rab25, a small GTPase involved in endosomal recycling, that is genomically amplified in multiple tumour lineages, is a key regulator of cellular bioenergetics and autophagy. RAB25 enhanced survival during nutrient stress by preventing apoptosis and autophagy via binding and activating AKT leading to increased glucose uptake and improved cellular bioenergetics. Unexpectedly, Rab25 induced the accumulation of glycogen in epithelial cancer cells, a process not previously identified. Strikingly, an increase in basal ATP levels combined with AKT-dependent increases in glucose uptake and glycogen storage allowed maintenance of ATP levels during bioenergetic stress. The clinical relevance of these findings was validated by the ability of a Rab25-dependent expression profile enriched for bioenergetics targets to identify patients with a poor prognosis. Thus, Rab25 is an unexpected regulator of cellular bioenergetics implicated as a useful biomarker and potential therapeutic target. PMID:22253197

  10. A Review on Hemeoxygenase-2: Focus on Cellular Protection and Oxygen Response

    PubMed Central

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

    2014-01-01

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

  11. 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

  12. Inverse phosphotriester DNA synthesis using photochemically-removable dimethoxybenzoin phosphate protecting groups

    SciTech Connect

    Pirrung, M.C.; Fallon, L.; Lever, D.C.; Shuey, S.W.

    1996-03-22

    A method has been developed to prepare short DNA sequences using light to deprotect a nucleoside 3{prime}-phosphotriester, generating a phosphodiester useful for coupling with a free 5{prime}-OH-nucleotide. The dimethoxybenzoin group is used as the photochemically-removable protecting group for the 3{prime}-phosphate. Cyanoethyl is most effective as the second protecting group on the phosphodiester. Because the method is directed at the preparation and use of the DNA sequences while still bound to the support, allyl and allyloxycarbonyl protecting groups are used for the nitrogenous bases since, based on the work of Hayakawa and Noyori, they can be removed without cleaving the DNA from the support. Two simple trinucleotides have been prepared in solution using this method. It has been demonstrated that the photochemical deprotection conditions do not lead to the formation of cyclobutane dimers from adjacent T residues. 2 figs., 2 tabs.

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

    PubMed

    Hou, Yakun; Wu, Pingxiao; Zhu, Nengwu

    2014-01-01

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

  14. Infectious bursal disease DNA vaccination conferring protection by delayed appearance and rapid clearance of invading viruses.

    PubMed

    Chen, Yung-Yi; Hsieh, Ming Kun; Tung, Chun-Yu; Wu, Ching Ching; Lin, Tsang Long

    2011-12-01

    The present study was undertaken to determine the kinetics of viral load and immune response in protection against infectious bursal disease virus (IBDV) by DNA vaccination. Chickens were DNA-vaccinated and challenged with IBDV one week after the third vaccination. Tissues were collected at 12 hours postinfection (HPI), 1 day postinfection (DPI), 3, 5, 7 and 10 DPI. The vaccinated chickens had less viral RNA, with delayed appearance and shorter duration in the bursa of Fabricius, spleen, and cecal tonsil than the challenged control chickens. Their ELISA and neutralizing antibody titers were decreased at 12 HPI and significantly lower (P < 0.05) than those in the challenged control chickens at later time points. Their spleen IFNγ expression was up-regulated compared to that in the DNA-vaccinated chickens without IBDV challenge. These results indicate that DNA vaccination confers protection against IBDV challenge by delayed appearance and rapid clearance of the invading viruses.

  15. Lamin A/C-dependent interaction with 53BP1 promotes cellular responses to DNA damage

    PubMed Central

    Gibbs-Seymour, Ian; Markiewicz, Ewa; Bekker-Jensen, Simon; Mailand, Niels; Hutchison, Christopher J

    2015-01-01

    Lamins A/C have been implicated in DNA damage response pathways. We show that the DNA repair protein 53BP1 is a lamin A/C binding protein. In undamaged human dermal fibroblasts (HDF), 53BP1 is a nucleoskeleton protein. 53BP1 binds to lamins A/C via its Tudor domain, and this is abrogated by DNA damage. Lamins A/C regulate 53BP1 levels and consequently lamin A/C-null HDF display a 53BP1 null-like phenotype. Our data favour a model in which lamins A/C maintain a nucleoplasmic pool of 53BP1 in order to facilitate its rapid recruitment to sites of DNA damage and could explain why an absence of lamin A/C accelerates aging. PMID:25645366

  16. Oxidative damage to cellular and isolated DNA by metabolites of a fungicide ortho-phenylphenol.

    PubMed

    Murata, M; Moriya, K; Inoue, S; Kawanishi, S

    1999-05-01

    ortho-Phenylphenol (OPP) and its sodium salt, which are used as fungicides and antibacterial agents, have been found to cause carcinomas in the urinary tract of rats. To clarify the carcinogenic mechanism of OPP, we compared the DNA damage inducing ability of an OPP metabolite, phenyl-1,4-benzoquinone (PBQ) with that of another metabolite, phenylhydroquinone (PHQ). Pulsed field gel electrophoresis showed that PBQ and PHQ induced DNA strand breakage in cultured human cells, but PBQ did it more efficiently than PHQ. Significant increases in 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were observed in cells treated with PBQ and PHQ, and the increase of 8-oxodG induced by PBQ was significantly higher than that induced by PHQ. Using 32P-5'-end-labeled DNA fragments obtained from human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene, we showed that PBQ plus NADH, and also PHQ, induced DNA damage frequently at thymine residues, in the presence of Cu(II). The intensity of DNA damage by PBQ was stronger than that by PHQ, showing higher importance of PBQ than other OPP metabolites. Catalase and bathocuproine inhibited Cu(II)-mediated DNA damage by PBQ plus NADH and PHQ, suggesting that H2O2 reacts with Cu(I) to produce active species causing DNA damage. Electron spin resonance and UV-visible spectroscopic studies have demonstrated generation of semiquinone radical and superoxide from the reaction of PBQ with NADH or the Cu(II)-mediated autoxidation of PHQ. The present results suggest that these OPP metabolites cause oxidative DNA damage through H2O2 generation in cells, and the damage may lead to mutation and carcinogenesis. It is concluded that PBQ may play a more important role in the expression of OPP carcinogenicity than other OPP metabolites.

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

    PubMed

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

    2011-12-27

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

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

    PubMed Central

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

    2008-01-01

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

  19. From plasmids to protection: a review of DNA vaccines against infectious diseases.

    PubMed

    Laddy, Dominick J; Weiner, David B

    2006-01-01

    The field of DNA vaccine development began over 16 years ago with the observation that plasmid DNA could be injected into and expressed in vivo and drive adaptive immune responses. Since then, there has been great interest in developing this technology to create a new generation of vaccines with the ability to elicit both humoral and cellular immune responses from an inherently innocuous injection. However, DNA vaccines have yet to proceed past phase I/II clinical trials in humans--primarily due to a desire to induce more potent immune responses. This review will examine how DNA vaccines function to induce an immune response and how this information might be useful in future vaccine design.

  20. Quantification of cellular and nuclear uptake rates of polymeric gene delivery nanoparticles and DNA plasmids via flow cytometry.

    PubMed

    Bishop, Corey J; Majewski, Rebecca L; Guiriba, Toni-Rose M; Wilson, David R; Bhise, Nupura S; Quiñones-Hinojosa, Alfredo; Green, Jordan J

    2016-06-01

    Non-viral, biomaterial-mediated gene delivery has the potential to treat many diseases, but is limited by low efficacy. Elucidating the bottlenecks of plasmid mass transfer can enable an improved understanding of biomaterial structure-function relationships, leading to next-generation rationally designed non-viral gene delivery vectors. As proof of principle, we transfected human primary glioblastoma cells using a poly(beta-amino ester) complexed with eGFP plasmid DNA. The polyplexes transfected 70.6±0.6% of the cells with 101±3% viability. The amount of DNA within the cytoplasm, nuclear envelope, and nuclei was assessed at multiple time points using fluorescent dye conjugated plasmid up to 24h post-transfection using a quantitative multi-well plate-based flow cytometry assay. Conversion to plasmid counts and degradation kinetics were accounted for via quantitative PCR (plasmid degradation rate constants were determined to be 0.62h(-1) and 0.084h(-1) for fast and slow phases respectively). Quantitative cellular uptake, nuclear association, and nuclear uptake rate constants were determined by using a four-compartment first order mass-action model. The rate limiting step for these poly(beta-amino ester)/DNA polyplex nanoparticles was determined to be cellular uptake (7.5×10(-4)h(-1)) and only 0.1% of the added dose was taken up by the human brain cancer cells, whereas 12% of internalized DNA successfully entered the nucleus (the rate of nuclear internalization of nuclear associated plasmid was 1.1h(-1)). We describe an efficient new method for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles using flow cytometry to improve understanding and design of polymeric gene delivery nanoparticles. In this work, a quantitative high throughput flow cytometry-based assay and computational modeling approach was developed for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles. This method is significant as it

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

    PubMed Central

    2009-01-01

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

  2. DNA vaccines encoding viral envelope proteins confer protective immunity against WSSV in black tiger shrimp.

    PubMed

    Rout, Namita; Kumar, Sudhir; Jaganmohan, Shanmugam; Murugan, Vadivel

    2007-04-12

    White Spot Syndrome Virus (WSSV) is a major cause of mortality in shrimp and poses a huge threat to aquaculture industry. Till now no comprehensive or individual strategy has been established to combat white spot disease. Previous efforts by other investigators have given insight of protein vaccination and its efficacy to protect shrimp against WSSV infection. In this study, we have explored the protective efficacy of DNA vaccination and tissue distribution of the immunised recombinant plasmid in black tiger shrimp (Penaeus monodon). Four recombinant constructs were generated by inserting four genes encoding the WSSV structural proteins VP15, VP28, VP35 and VP281 individually into DNA vaccine vector pVAX1. Expression of these proteins from the recombinant plasmids was confirmed in vitro in CHO cell lines. For vaccination experiments, shrimp were immunised with these DNA constructs and later challenged with WSSV. A significant level of protection was offered by the plasmids encoding VP28 or VP281 till 7 weeks whereas protein vaccination failed to protect vaccinated shrimp after 3 weeks of first immunisation. In addition, our tissue distribution study revealed the persistence of immunised DNA at least upto 2 months in the injected shrimp muscle. Thus, our results suggest that DNA vaccination strategy will have potential utility against WSSV infection in shrimp cultivation.

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

    NASA Astrophysics Data System (ADS)

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

    2000-06-01

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

  4. Three RNA cells for ribosomal lineages and three DNA viruses to replicate their genomes: a hypothesis for the origin of cellular domain.

    PubMed

    Forterre, Patrick

    2006-03-07

    The division of the living world into three cellular domains, Archaea, Bacteria, and Eukarya, is now generally accepted. However, there is no consensus about the evolutionary relationships among these domains, because all of the proposed models have a number of more or less severe pitfalls. Another drawback of current models for the universal tree of life is the exclusion of viruses, otherwise a major component of the biosphere. Recently, it was suggested that the transition from RNA to DNA genomes occurred in the viral world, and that cellular DNA and its replication machineries originated via transfers from DNA viruses to RNA cells. Here, I explore the possibility that three such independent transfers were at the origin of Archaea, Bacteria, and Eukarya, respectively. The reduction of evolutionary rates following the transition from RNA to DNA genomes would have stabilized the three canonical versions of proteins involved in translation, whereas the existence of three different founder DNA viruses explains why each domain has its specific DNA replication apparatus. In that model, plasmids can be viewed as transitional forms between DNA viruses and cellular chromosomes, and the formation of different levels of cellular organization (prokaryote or eukaryote) could be traced back to the nature of the founder DNA viruses and RNA cells.

  5. Three RNA cells for ribosomal lineages and three DNA viruses to replicate their genomes: A hypothesis for the origin of cellular domain

    PubMed Central

    Forterre, Patrick

    2006-01-01

    The division of the living world into three cellular domains, Archaea, Bacteria, and Eukarya, is now generally accepted. However, there is no consensus about the evolutionary relationships among these domains, because all of the proposed models have a number of more or less severe pitfalls. Another drawback of current models for the universal tree of life is the exclusion of viruses, otherwise a major component of the biosphere. Recently, it was suggested that the transition from RNA to DNA genomes occurred in the viral world, and that cellular DNA and its replication machineries originated via transfers from DNA viruses to RNA cells. Here, I explore the possibility that three such independent transfers were at the origin of Archaea, Bacteria, and Eukarya, respectively. The reduction of evolutionary rates following the transition from RNA to DNA genomes would have stabilized the three canonical versions of proteins involved in translation, whereas the existence of three different founder DNA viruses explains why each domain has its specific DNA replication apparatus. In that model, plasmids can be viewed as transitional forms between DNA viruses and cellular chromosomes, and the formation of different levels of cellular organization (prokaryote or eukaryote) could be traced back to the nature of the founder DNA viruses and RNA cells. PMID:16505372

  6. Programmed protection of foreign DNA from restriction allows pathogenicity island exchange during pneumococcal transformation.

    PubMed

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

    2013-02-01

    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

  7. Misregulation of DNA damage repair pathways in HPV-positive head and neck squamous cell carcinoma contributes to cellular radiosensitivity

    PubMed Central

    Nickson, Catherine M.; Moori, Parisa; Carter, Rachel J.; Rubbi, Carlos P.; Parsons, Jason L.

    2017-01-01

    Patients with human papillomavirus type 16 (HPV)-associated oropharyngeal squamous cell carcinomas (OPSCC) display increased sensitivity to radiotherapy and improved survival rates in comparison to HPV-negative forms of the disease. However the cellular mechanisms responsible for this characteristic difference are unclear. Here, we have investigated the contribution of DNA damage repair pathways to the in vitro radiosensitivity of OPSCC cell lines. We demonstrate that two HPV-positive OPSCC cells are indeed more radiosensitive than two HPV-negative OPSCC cells, which correlates with reduced efficiency for the repair of ionising radiation (IR)-induced DNA double strand breaks (DSB). Interestingly, we show that HPV-positive OPSCC cells consequently have upregulated levels of the proteins XRCC1, DNA polymerase β, PNKP and PARP-1 which are involved in base excision repair (BER) and single strand break (SSB) repair. This translates to an increased capacity and efficiency for the repair of DNA base damage and SSBs in these cells. In addition, we demonstrate that HPV-positive but interestingly more so HPV-negative OPSCC display increased radiosensitivity in combination with the PARP inhibitor olaparib. This suggests that PARP inhibition in combination with radiotherapy may be an effective treatment for both forms of OPSCC, particularly for HPV-negative OPSCC which is relatively radioresistant. PMID:28415784

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

    PubMed

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

    2005-11-01

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

  9. Cellular proteins bind to the downstream component of the lytic origin of DNA replication of Epstein-Barr virus.

    PubMed Central

    Gruffat, H; Renner, O; Pich, D; Hammerschmidt, W

    1995-01-01

    The lytic origin of DNA replication of Epstein-Barr virus, oriLyt, is a complex eukaryotic origin which is activated during the lytic phase of the viral life cycle. It consists of at least two independent cis-acting components, one of which plays a dual role in transcription and DNA replication. The binding of the viral factor BZLF1, a member of the AP1 family of transcription factors, to this upstream component is crucial for oriLyt function (A. Schepers, D. Pich, and W. Hammerschmidt, EMBO J. 12:3921-3929, 1993). The second cis-acting element, the downstream component of oriLyt, is equally indispensable; however, its function is unknown. In this study, the downstream component was found to be the binding target of several cellular proteins. One could be identified as Sp1 or as a related protein which binds twice to the downstream component of oriLyt. Mutational analysis indicated that Sp1 alone is not directly involved in mediating DNA replication; however, other factors which share the same binding sequence or bind closely to one of the Sp1 binding sites are likely candidates to contribute to a replication protein complex at the downstream component of oriLyt. The sequence requirements for the downstream component are remarkably stringent, indicating that at least one of the putative factors is a sequence-specific DNA-binding protein which is required for the activation of oriLyt. PMID:7853529

  10. 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.

  11. Protective efficacy of a DNA vaccine construct encoding the VP2 gene of infectious bursal disease and a truncated HSP70 of Mycobacterium tuberculosis in chickens.

    PubMed

    Maity, Hemanta Kumar; Dey, Sohini; Mohan, C Madhan; Khulape, Sagar A; Pathak, Dinesh C; Vakharia, Vikram N

    2015-02-18

    Infectious bursal disease (IBD) is an acute, infectious, immunosuppressive disease affecting young chicken worldwide. The etiological agent IBD virus (IBDV) is a double stranded RNA virus with outer capsid protein VP2 of IBDV is the major antigenic determinant capable of inducing neutralizing antibody. DNA vaccines encoding VP2 has been extensively studied achieving only partial protection. However, the efficacy of DNA vaccines against IBDV can be augmented by choosing a potential molecular adjuvant. The goal of the present study is to evaluate the immune response and protective efficacy of a DNA vaccine encoding the C-terminal domain of the heat shock protein 70 (cHSP70) of Mycobacterium tuberculosis gene genetically fused with the full length VP2 gene of IBDV (pCIVP2-cHSP70) in comparison to a 'DNA prime-protein boost' approach and a DNA vaccine encoding the VP2 gene (pCIVP2) alone. The results indicate that both pCIVP2-cHSP70 and 'DNA prime-protein boost' elicited humoral as well as cellular immune responses. Chickens in the pCIVP2-cHSP70 and 'DNA prime-protein boost' groups developed significantly higher levels of ELISA titer to IBDV antigen compared to the group immunized with pCIVP2 alone (p<0.01). However, significantly higher levels of lymphocyte proliferative response, IL-12 and IFN-γ production were found in the pCIVP2-cHSP70 group compared to 'DNA prime-protein boost' group. Additionally, chickens immunized with pCIVP2-cHSP70 and 'DNA prime-protein boost' vaccines were completely protected against the vvIBDV whereas pCIVP2 DNA vaccine alone was able to protect only 70%. These findings suggest that the truncated C-terminal HSP70 mediated DNA vaccine genetically fused with the VP2 gene construct stimulated both humoral and cell mediated immune responses and conferred complete protection against IBDV. This novel strategy is perhaps a seminal concept in utilizing HSP70 as an adjuvant molecule to elicit an immune response against IBD affecting chickens.

  12. Flavonoids can protect maize DNA from the induction of ultraviolet radiation damage.

    PubMed Central

    Stapleton, A E; Walbot, V

    1994-01-01

    Diverse flavonoid compounds are widely distributed in angiosperm families. Flavonoids absorb radiation in the ultraviolet (UV) region of the spectrum, and it has been proposed that these compounds function as UV filters. We demonstrate that the DNA in Zea mays plants that contain flavonoids (primarily anthocyanins) is protected from the induction of damage caused by UV radiation relative to the DNA in plants that are genetically deficient in these compounds. DNA damage was measured with a sensitive and simple assay using individual monoclonal antibodies, one specific for cyclobutane pyrimidine dimer damage and the other specific for pyrimidine(6,4)pyrimidone damage. PMID:8058838

  13. Biophysical characterization of quaternary pyridinium functionalized polynorbornenes for DNA complexation and their cellular interactions.

    PubMed

    Guler Gokce, Zeliha; Zuhal Birol, Semra; Eren, Tarık; Ercelen Ceylan, Sebnem

    2017-04-01

    Cationic polymers with hydrophobic side chains have gained great interest as DNA carriers since they form a compact complex with negatively charged DNA phosphate groups and interact with the cell membrane. Amphiphilic polyoxanorbornenes with different quaternary alkyl pyridinium side chains with ethyl-p(OPy2) and hexyl units-p(OPy6) bearing 10 kDa MWT were synthesized by living Ring-Opening Metathesis Polymerization method. The physicochemical characteristics: critical micellar concentration, size distribution, surface charge, and condensation of polymer/DNA complex were investigated. Morphology of complexes was monitored by Atomic force microscopy. Cytotoxicity and interaction of these complexes with model lipid vesicles mimicking the cell membrane were examined. These polymers were enabled to form small sized complexes of DNA, which interact with model membrane vesicles. It was found that the nature of hydrophobicity of the homopolymers significantly impacts rates of DNA complexation and the surface charge of the resulting complexes. These results highlight the prospect of the further examinations of these polymers as gene carriers. © 2016 Wiley Periodicals, Inc.

  14. DNA-binding, cytotoxicity, cellular uptake, apoptosis and photocleavage studies of Ru(II) complexes.

    PubMed

    N Deepika; C Shobha Devi; Y Praveen Kumar; K Laxma Reddy; P Venkat Reddy; D Anil Kumar; Surya S Singh; S Satyanarayana

    2016-07-01

    Two Ru(II) complexes [Ru(phen)2bppp](ClO4)2 (1) and [Ru(phen)27-Br-dppz](ClO4)2 (2) [phen=1,10 phenanthroline, 7-Br-dppz=7-fluorodipyrido[3,2-a:2',3'-c]phenazine, bppp=11-bromo-pyrido[2',3':5,6]pyrazino[2,3-f] [1,10]phenanthroline] have been synthesized and characterized by elemental analysis, ES-MS, (1)H-NMR, (13)C-NMR and IR. The in vitro cytotoxicity of the complexes examined against a panel of cancer cell lines (HeLa, Du145 and A549) by MTT method, both complexes show prominent anticancer activity against various cancer cells. Live cell imaging study and flow cytometric analysis demonstrate that both the complexes 1 and 2 could cross the cell membrane accumulating in the nucleus. Further, flow cytometry experiments showed that the cytotoxic Ru(II) complexes 1 and 2 induced apoptosis of HeLa tumor cell lines. Photo induced DNA cleavage studies have been performed and results indicate that both the complexes efficiently photo cleave pBR322 DNA. The binding properties of two complexes toward CT-DNA were investigated by various optical methods and viscosity measurements. The experimental results suggested that both Ru(II) complexes can intercalate into DNA base pairs. The complexes were docked into DNA-base pairs using the GOLD docking program.

  15. Epigenetic and genetic factors in the cellular response to radiations and DNA-damaging chemicals

    SciTech Connect

    Williams, J.R.; D'Arpa, P.

    1981-07-01

    DNA-damaging agents are widely used as therapeutic tools for a variety of disease states. Many such agents are considered to produce detrimental side effects. Thus, it is important to evaluate both therapeutic efficacy and potential risk. DNA-damaging agents can be so evaluated by comparison to agents whose therapeutic benefit and potential hazards are better known. We propose a framework for such comparison, demonstrating that a simple transformation of cytotoxicity-dose response patterns permits a facile comparison of variation between cells exposed to a single DNA-damaging agent or to different cytotoxic agents. Further, by transforming data from experiments which compare responses of 2 cell populations to an effects ratio, different patterns for the changes in cytotoxicity produced by epigenetic and genetic factors were compared. Using these transformations, we found that there is a wide variation (a factor of 4) between laboratories for a single agent (UVC) and only a slightly larger variation (factor of 6) between normal cell response for different types of DNA-damaging agents (x-ray, UVC, alkylating agents, crosslinking agents). Epigenetic factors such as repair and recovery appear to be a factor only at higher dose levels. Comparison in the cytotoxic effect of a spectrum of DNA-damaging agents in xeroderma pigmentosum, ataxia telangiectasia, and Fanconi's anemia cells indicates significantly different patterns, implying that the effect, and perhaps the nature, of these genetic conditions are quite different.

  16. Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury

    PubMed Central

    Akiba, Yasutada; Furukawa, Osamu; Guth, Paul H.; Engel, Eli; Nastaskin, Igor; Sassani, Pejvak; Dukkipatis, Ramanath; Pushkin, Alexander; Kurtz, Ira; Kaunitz, Jonathan D.

    2001-01-01

    Secretion of bicarbonate from epithelial cells is considered to be the primary mechanism by which the duodenal mucosa is protected from acid-related injury. Against this view is the finding that patients with cystic fibrosis, who have impaired duodenal bicarbonate secretion, are paradoxically protected from developing duodenal ulcers. Therefore, we hypothesized that epithelial cell intracellular pH regulation, rather than secreted extracellular bicarbonate, was the principal means by which duodenal epithelial cells are protected from acidification and injury. Using a novel in vivo microscopic method, we have measured bicarbonate secretion and epithelial cell intracellular pH (pHi), and we have followed cell injury in the presence of the anion transport inhibitor DIDS and the Cl– channel inhibitor, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). DIDS and NPPB abolished the increase of duodenal bicarbonate secretion following luminal acid perfusion. DIDS decreased basal pHi, whereas NPPB increased pHi; DIDS further decreased pHi during acid challenge and abolished the pHi overshoot over baseline observed after acid challenge, whereas NPPB attenuated the fall of pHi and exaggerated the overshoot. Finally, acid-induced epithelial injury was enhanced by DIDS and decreased by NPPB. The results support the role of intracellular bicarbonate in the protection of duodenal epithelial cells from luminal gastric acid. PMID:11748264

  17. Correlation of Cellular Immune Responses with Protection against Culture-Confirmed Influenza Virus in Young Children▿

    PubMed Central

    Forrest, Bruce D.; Pride, Michael W.; Dunning, Andrew J.; Capeding, Maria Rosario Z.; Chotpitayasunondh, Tawee; Tam, John S.; Rappaport, Ruth; Eldridge, John H.; Gruber, William C.

    2008-01-01

    The highly sensitive gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay permits the investigation of the role of cell-mediated immunity (CMI) in the protection of young children against influenza. Preliminary studies of young children confirmed that the IFN-γ ELISPOT assay was a more sensitive measure of influenza memory immune responses than serum antibody and that among seronegative children aged 6 to <36 months, an intranasal dose of 107 fluorescent focus units (FFU) of a live attenuated influenza virus vaccine (CAIV-T) elicited substantial CMI responses. A commercial inactivated influenza virus vaccine elicited CMI responses only in children with some previous exposure to related influenza viruses as determined by detectable antibody levels prevaccination. The role of CMI in actual protection against community-acquired, culture-confirmed clinical influenza by CAIV-T was investigated in a large randomized, double-blind, placebo-controlled dose-ranging efficacy trial with 2,172 children aged 6 to <36 months in the Philippines and Thailand. The estimated protection curve indicated that the majority of infants and young children with ≥100 spot-forming cells/106 peripheral blood mononuclear cells were protected against clinical influenza, establishing a possible target level of CMI for future influenza vaccine development. The ELISPOT assay for IFN-γ is a sensitive and reproducible measure of CMI and memory immune responses and contributes to establishing requirements for the future development of vaccines against influenza, especially those used for children. PMID:18448618

  18. Chemical kinetic behavior of chlorogenic acid in protecting erythrocyte and DNA against radical-induced oxidation.

    PubMed

    Tang, You-Zhi; Liu, Zai-Qun

    2008-11-26

    As an abundant ingredient in coffee, chlorogenic acid (CGA) is a well-known antioxidant. Although some works have dealt with its radical-scavenging property, the present work investigated the protective effects of CGA on the oxidation of DNA and on the hemolysis of human erythrocytes induced by 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) by means of chemical kinetics. The inhibition period (t(inh)) derived from the protective effect of CGA on erythrocyte and DNA was proportional to its concentration, t(inh) = (n/R(i))[CGA], where R(i) refers to the radical-initiation rate, and n indicates the number of radical-propagation chains terminated by CGA. It was found that the n of CGA to protect erythrocytes was 0.77, lower than that of vitamin E (2.0), but higher than that of vitamin C (0.19). Furthermore, CGA facilitated a mutual protective effect with VE and VC on AAPH-induced hemolysis by increasing n of VE and VC. CGA was also found to be a membrane-stabilizer to protect erythrocytes against hemin-induced hemolysis. Moreover, the n of CGA was only 0.41 in the process of protecting DNA. This fact revealed that CGA served as an efficient antioxidant to protect erythrocytes more than to protect DNA. Finally, the reaction between CGA and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS(+*)) or 2,2'-diphenyl-1-picrylhydrazyl (DPPH) revealed that CGA was able to trap radicals by reducing radicals more than by donating its hydrogen atoms to radicals.

  19. Antioxidant activity and protective effect on DNA strand scission of Rooibos tea (Aspalathus linearis).

    PubMed

    Lee, Eun-Jung; Jang, Hae-Dong

    2004-01-01

    Rooibos tea (Aspalathus linearis) was extracted by refluxing with water and 75% ethanol as a solvent. Antioxidant activity and protective effect on DNA strand scission were investigated by using different antioxidant assay systems and DNA strand nicking assay, respectively. 75% Ethanol extract has higher content of total soluble phenolics and flavonoid than water extract. Antioxidant activities such as hydrogen donating capacity and scavenging activity of hydrogen peroxide were higher in 75% ethanol extract than in water extract except the rate constant with hydroxyl radical. Peroxyl radical induced DNA strand scission was prevented by both 75% ethanol and water extract and hydroxyl radical induced DNA strand scission was not. This result indicates that total soluble phenolics, specially flavonoid, of Rooibos tea are responsible for several kinds of antioxidant activities and preventive activity on peroxyl radical induced DNA strand scission.

  20. Enhanced cellular radiosensitivity induced by cofilin-1 over-expression is associated with reduced DNA repair capacity

    PubMed Central

    Leu, Jyh-Der; Chiu, Yu-Wen; Lo, Chia-Chien; Chiang, Pei-Hsun; Chiu, Su-Jun; Tsai, Cheng-Han; Hwang, Jeng-Jong; Chen, Ran-Chou; Gorbunova, Vera; Lee, Yi-Jang

    2013-01-01

    Purpose A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores, the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity. Materials and Methods Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632. Results When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation induced double strand break (DSB) repair, systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1. Conclusion CFL-1 over-expression enhances radiosensitivity and this

  1. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT.

    PubMed

    Jungmann, Ralf; Avendaño, Maier S; Woehrstein, Johannes B; Dai, Mingjie; Shih, William M; Yin, Peng

    2014-03-01

    Super-resolution fluorescence microscopy is a powerful tool for biological research, but obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, a variation of point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed super-resolution imaging that achieves sub-10-nm spatial resolution in vitro on synthetic DNA structures. We also report a multiplexing approach (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-color super-resolution imaging in vitro on synthetic DNA structures as well as four-color two-dimensional (2D) imaging and three-color 3D imaging of proteins in fixed cells.

  2. Multiplexed 3D Cellular Super-Resolution Imaging with DNA-PAINT and Exchange-PAINT

    PubMed Central

    Jungmann, R.; Avendano, M.S.; Woehrstein, J.B.; Dai, M.; Shih, W.M.; Yin, P.

    2014-01-01

    While super-resolution fluorescence microscopy is a powerful tool for biological research, obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed 3D super-resolution imaging inside fixed cells and achieve sub-10 nm spatial resolution in vitro using synthetic DNA structures. We also report a novel approach for multiplexing (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-“color” super-resolution imaging in vitro on synthetic DNA structures and four-“color” imaging of proteins in a fixed cell. PMID:24487583

  3. Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

    PubMed Central

    Masuda, Yuji; Kanao, Rie; Kaji, Kentaro; Ohmori, Haruo; Hanaoka, Fumio; Masutani, Chikahide

    2015-01-01

    Translesion DNA synthesis (TLS) by the Y-family DNA polymerases Polη, Polι and Polκ, mediated via interaction with proliferating cell nuclear antigen (PCNA), is a crucial pathway that protects human cells against DNA damage. We report that Polη has three PCNA-interacting protein (PIP) boxes (PIP1, 2, 3) that contribute differentially to two distinct functions, stimulation of DNA synthesis and promotion of PCNA ubiquitination. The latter function is strongly associated with formation of nuclear Polη foci, which co-localize with PCNA. We also show that Polκ has two functionally distinct PIP boxes, like Polη, whereas Polι has a single PIP box involved in stimulation of DNA synthesis. All three polymerases were additionally stimulated by mono-ubiquitinated PCNA in vitro. The three PIP boxes and a ubiquitin-binding zinc-finger of Polη exert redundant and additive effects in vivo via distinct molecular mechanisms. These findings provide an integrated picture of the orchestration of TLS polymerases. PMID:26170230

  4. Ionizing radiation and tritium transmutation both cause formation of 5-hydroxymethyl-2'-deoxyuridine in cellular DNA.

    PubMed

    Teebor, G W; Frenkel, K; Goldstein, M S

    1984-01-01

    HeLa cells grown in the presence of [methyl-3H]thymidine contained large amounts of 5-hydroxymethyl-2'-deoxyuridine (HMdU) in their DNA. When the cells were grown in [6-3H]thymidine and their DNA was labeled to the same specific activity, no HMdU was present. When such [6-3H]thymidine-labeled cells were exposed to increasing amounts of gamma-radiation, small but increasing amounts of HMdU were formed in their DNA. This indicates that HMdU can be formed in DNA by two distinct mechanisms. The first is the result of the transmutation of 3H to 3He (beta decay) in the methyl group of thymidine, leading to formation of a carbocation. This short-lived ion reacts with hydroxide ions of water, yielding the hydroxymethyl group. HMdU that is formed by this mechanism is formed at the rate of beta decay of 3H. It appears only in [methyl-3H]thymidine residues and is present in the DNA of both nonirradiated and gamma-irradiated cells. The second mechanism is the result of the radiolysis of water caused by ionizing radiation. The resultant radical species, particularly hydroxyl radicals, may react with many sites on DNA. When the methyl group of thymine is attacked by hydroxyl radicals, the hydroxymethyl group is formed. The formation of HMdU by this mechanism was detected only when [6-3H]thymidine-labeled cells were used, since transmutation of 3H in position 6 of thymine cannot yield HMdU.

  5. Ionizing radiation and tritium transmutation both cause formation of 5-hydroxymethyl-2'-deoxyuridine in cellular DNA.

    PubMed Central

    Teebor, G W; Frenkel, K; Goldstein, M S

    1984-01-01

    HeLa cells grown in the presence of [methyl-3H]thymidine contained large amounts of 5-hydroxymethyl-2'-deoxyuridine (HMdU) in their DNA. When the cells were grown in [6-3H]thymidine and their DNA was labeled to the same specific activity, no HMdU was present. When such [6-3H]thymidine-labeled cells were exposed to increasing amounts of gamma-radiation, small but increasing amounts of HMdU were formed in their DNA. This indicates that HMdU can be formed in DNA by two distinct mechanisms. The first is the result of the transmutation of 3H to 3He (beta decay) in the methyl group of thymidine, leading to formation of a carbocation. This short-lived ion reacts with hydroxide ions of water, yielding the hydroxymethyl group. HMdU that is formed by this mechanism is formed at the rate of beta decay of 3H. It appears only in [methyl-3H]thymidine residues and is present in the DNA of both nonirradiated and gamma-irradiated cells. The second mechanism is the result of the radiolysis of water caused by ionizing radiation. The resultant radical species, particularly hydroxyl radicals, may react with many sites on DNA. When the methyl group of thymine is attacked by hydroxyl radicals, the hydroxymethyl group is formed. The formation of HMdU by this mechanism was detected only when [6-3H]thymidine-labeled cells were used, since transmutation of 3H in position 6 of thymine cannot yield HMdU. PMID:6582490

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

    NASA Astrophysics Data System (ADS)

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

    2013-07-01

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

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

    SciTech Connect

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

    2013-07-26

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

  8. Structure of the Herpes Simplex Virus 1 Genome: Manipulation of Nicks and Gaps Can Abrogate Infectivity and Alter the Cellular DNA Damage Response

    PubMed Central

    Smith, Samantha; Reuven, Nina; Mohni, Kareem N.; Schumacher, April J.

    2014-01-01

    ABSTRACT The herpes simplex virus 1 (HSV-1) virion DNA contains nicks and gaps, and in this study a novel assay for estimating the size and number of gaps in virion DNA was developed. Consistent with previous reports, we estimate that there are approximately 15 gaps per genome, and we calculate the average gap length to be approximately 30 bases. Virion DNA was isolated and treated with DNA-modifying enzymes in order to fill in the gaps and modify the ends. Interestingly, filling in gaps, blunting the ends, or adding random sequences to the 3′ ends of DNA, producing 3′ flaps, did not impair the infectivity of treated DNA following transfection of Vero cells. On the other hand, the formation of 5′ flaps in the DNA following treatment resulted in a dramatic reduction (95 to 100%) in infectivity. Virion DNA stimulated DNA-PKcs activity in transfected cells, and DNA with 5′ flaps stimulated a higher level of DNA-PKcs activity than that observed in cells transfected with untreated virion DNA. The infectivity of 5′-flapped DNA was restored in cells that do not express DNA-PKcs and in cells cotransfected with the immediate early protein ICP0, which degrades DNA-PKcs. These results are consistent with previous reports that DNA-dependent protein kinase (DNA-PK) and the nonhomologous end joining (NHEJ) repair pathway are intrinsically antiviral and that ICP0 can counteract this effect. We suggest that HSV-1 DNA with 5′ flaps may induce an antiviral state due to the induction of a DNA damage response, primarily mediated by NHEJ, that renders the HSV-1 genome less efficient for lytic infection. IMPORTANCE For productive lytic infection to occur, HSV-1 must counteract a variety of cellular intrinsic antiviral mechanisms, including the DNA damage response (DDR). DDR pathways have been associated with silencing of gene expression, cell cycle arrest, and induction of apoptosis. In addition, the fate of viral genomes is likely to play a role in whether viral genomes

  9. IFN-γ increases efficiency of DNA vaccine in protecting ducks against infection

    PubMed Central

    Long, Jian-Er; Huang, Li-Na; Qin, Zhi-Qiang; Wang, Wen-Yi; Qu, Di

    2005-01-01

    AIM: To detect the effects of DNA vaccines in combination with duck IFN-γ gene on the protection of ducks against duck hepatitis B virus (DHBV) infection. METHODS: DuIFN-γ cDNA was cloned and expressed in COS-7 cells, and the antiviral activity of DuIFN-γ was detected and neutralized by specific antibodies. Ducks were vaccinated with DHBpreS/S DNA alone or co-immunized with plasmid expressing DuIFN-γ. DuIFN-γ mRNA in peripheral blood mononuclear cells (PBMCs) from immunized ducks was detected by semi-quantitative competitive RT-PCR. Anti-DHBpreS was titrated by enzyme-linked immunosorbent assay (ELISA). DHBV DNA in sera and liver was detected by Southern blot hybridization, after ducks were challenged with high doses of DHBV. RESULTS: DuIFN-γ expressed by COS-7 was able to protect duck fibroblasts against vesicular stomatitis virus (VSV) infection in a dose-dependent fashion, and anti-DuIFN-γ antibodies neutralized the antiviral effects. DuIFN-γ in the supernatant also inhibited the release of DHBV DNA from LMH-D2 cells. When ducks were co-immunized with DNA vaccine expressing DHBpreS/S and DuIFN-γ gene as an adjuvant, the level of DuIFN-γ mRNA in PBMCs was higher than that in ducks vaccinated with DHBpreS/S DNA alone. However, the titer of anti-DHBpreS elicited by DHBpreS/S DNA alone was higher than that co-immunized with DuIFN-γ gene and DHBpreS/S DNA. After being challenged with DHBV at high doses, the load of DHBV in sera dropped faster, and the amount of total DNA and cccDNA in the liver decreased more significantly in the group of ducks co-immunized with DuIFN-γ gene and DHBpreS/S DNA than in other groups. CONCLUSION: DHBV preS/S DNA vaccine can protect ducks against DHBV infection, DuIFN-γ gene as an immune adjuvant enhances its efficacy. PMID:16124047

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

    PubMed

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

    2013-04-08

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

  11. The Influences of Glycosylation on the Antigenicity, Immunogenicity, and Protective Efficacy of Ebola Virus GP DNA Vaccines

    DTIC Science & Technology

    2006-11-22

    Microbiology. All Rights Reserved. Influences of Glycosylation on Antigenicity, Immunogenicity, and Protective Efficacy of Ebola Virus GP DNA Vaccines ...carbohydrates. We measured the influences of GP glycosylation on antigenicity, immu- nogenicity, and protection by testing DNA vaccines comprised of GP...may protect against filovirus infection (6, 39). Con- sistent with this, B-cell-deficient mice vaccinated with EBOV-like particles were not protected

  12. Protective effect of gallic acid and Syzygium cumini extract against oxidative stress-induced cellular injury in human lymphocytes.

    PubMed

    De Bona, Karine Santos; Bonfanti, Gabriela; Bitencourt, Paula Eliete Rodrigues; da Silva, Thainan Paz; Borges, Raphaela Maleski; Boligon, Aline; Pigatto, Aline; Athayde, Margareth Lynde; Moretto, Maria Beatriz

    2016-01-01

    Syzygium cumini (Myrtaceae) presents antioxidant, anti-inflammatory, hypoglycemic and antibacterial effects; however, the cellular and molecular mechanisms of action in the immune system are not yet completely elucidated. This study evaluates the in vitro effect of gallic acid and aqueous S. cumini leaf extract (ASc) on adenosine deaminase (ADA) and dipeptidyl peptidase IV (DPP-IV) activities, cell viability and oxidative stress parameters in lymphocytes exposed to 2, 2'-azobis-2-amidinopropane dihydrochloride (AAPH). Lymphocytes were incubated with ASc (100 and 500 µg/ml) and gallic acid (50 and 200 µM) at 37 °C for 30 min followed by incubation with AAPH (1 mM) at 37 °C for 2 h. After the incubation time, the lymphocytes were used for determinations of ADA, DPP-IV and lactate dehydrogenase (LDH) activities, lipid peroxidation, protein thiol (P-SH) group levels and cellular viability by colorimetric methods. (i) HPLC fingerprinting of ASc revealed the presence of catechin, epicatechin, rutin, quercitrin, isoquercitrin, quercetin, kaempferol and chlorogenic, caffeic, gallic and ellagic acids; (ii) for the first time, ASc reduced the AAPH-induced increase in ADA activity, but no effect was observed on DPP-IV activity; (iii) ASc increased P-SH groups and cellular viability and decreased LDH activity, but was not able to reduce the AAPH-induced lipid peroxidation; (iv) gallic acid showed less protective effects than ASc. ASc affects the purinergic system and may modulate adenosine levels, indicating that the extract of this plant exhibits immunomodulatory properties. ASc also may potentially prevent the cellular injury induced by oxidative stress, highlighting its cytoprotective effects.

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

    PubMed Central

    Kim, Hye Kyung

    2016-01-01

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

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

    PubMed

    Kim, Hye Kyung

    2016-07-29

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

  15. Contributions of humoral and cellular immunity to vaccine-induced protection in humans

    PubMed Central

    Amanna, Ian J.; Slifka, Mark K.

    2011-01-01

    Vaccines play a vital role in protecting the host against infectious disease. The most effective licensed vaccines elicit long-term antigen-specific antibody responses by plasma cells in addition to the development of persisting T cell and B cell memory. The relative contributions of these different immune cell subsets is context-dependent and varies depending on the attributes of the vaccine (i.e., live/attenuated, inactivated, subunit) as well as the biology of the pathogen in question. For relatively simple vaccines against bacterial antigens (e.g., tetanus toxin) or invariant viruses, the immunological correlates of protection are well-characterized. For more complex vaccines against viruses, especially ones that mutate or cause latent infections, it is more difficult to define the specific correlates of immunity. This often requires observational/natural history studies, clinical trials, or experimental evaluation in relevant animal models in order for immunological correlates to be determined or extrapolated. In this review, we will discuss the relative contributions of virus-specific T cell and B cell responses to vaccine-mediated protection against disease. PMID:21216425

  16. DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii.

    PubMed

    Lestini, Roxane; Delpech, Floriane; Myllykallio, Hannu

    2015-11-01

    Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea.

  17. Differential effects of IL-15 on the generation, maintenance and cytotoxic potential of adaptive cellular responses induced by DNA vaccination.

    PubMed

    Li, Jinyao; Valentin, Antonio; Ng, Sinnie; Beach, Rachel Kelly; Alicea, Candido; Bergamaschi, Cristina; Felber, Barbara K; Pavlakis, George N

    2015-02-25

    IL-15 is an important cytokine for the regulation of lymphocyte homeostasis. However, the role of IL-15 in the generation, maintenance and cytotoxic potential of antigen specific T cells is not fully understood. Because the route of antigenic delivery and the vaccine modality could influence the IL-15 requirement for mounting and preserving cytotoxic T cell responses, we have investigated the immunogenicity of DNA-based vaccines in IL-15 KO mice. DNA vaccination with SIV Gag induced antigen-specific CD4(+) and CD8(+) T cells in the absence of IL-15. However, the absolute number of antigen-specific CD8(+) T cells was decreased in IL-15 KO mice compared to WT animals, suggesting that IL-15 is important for the generation of maximal number of antigen-specific CD8(+) T cells. Interestingly, antigen-specific memory CD8 cells could be efficiently boosted 8 months after the final vaccination in both WT and KO strains of mice, suggesting that the maintenance of antigen-specific long-term memory T cells induced by DNA vaccination is comparable in the absence and presence of IL-15. Importantly, boosting by DNA 8-months after vaccination revealed severely reduced granzyme B content in CD8(+) T cells of IL-15 KO mice compared to WT mice. This suggests that the cytotoxic potential of the long-term memory CD8(+) T cells is impaired. These results suggest that IL-15 is not essential for the generation and maintenance of adaptive cellular responses upon DNA vaccination, but it is critical for the preservation of maximal numbers and for the activity of cytotoxic CD8(+) T cells. Published by Elsevier Ltd.

  18. Loss of cellular transformation efficiency induced by DNA irradiation with low-energy (10 eV) electrons.

    PubMed

    Kouass Sahbani, Saloua; Sanche, Leon; Cloutier, Pierre; Bass, Andrew D; Hunting, Darel J

    2014-11-20

    Low energy electrons (LEEs) of energies less than 20 eV are generated in large quantities by ionizing radiation in biological matter. While LEEs are known to induce single (SSBs) and double strand breaks (DSBs) in DNA, their ability to inactivate cells by inducing nonreparable lethal damage has not yet been demonstrated. Here we observe the effect of LEEs on the functionality of DNA, by measuring the efficiency of transforming Escherichia coli with a [pGEM-3Zf (-)] plasmid irradiated with 10 eV electrons. Highly ordered DNA films were prepared on pyrolitic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap(2+)). The uniformity of these films permits the inactivation of approximately 50% of the plasmids compared to <10% using previous methods, which is sufficient for the subsequent determination of their functionality. Upon LEE irradiation, the fraction of functional plasmids decreased exponentially with increasing electron fluence, while LEE-induced isolated base damage, frank DSB, and non DSB-cluster damage increased linearly with fluence. While DSBs can be toxic, their levels were too low to explain the loss of plasmid functionality observed upon LEE irradiation. Similarly, non-DSB cluster damage, revealed by transforming cluster damage into DSBs by digestion with repair enzymes, also occurred relatively infrequently. The exact nature of the lethal damage remains unknown, but it is probably a form of compact cluster damage in which the lesions are too close to be revealed by purified repair enzymes. In addition, this damage is either not repaired or is misrepaired by E. coli, since it results in plasmid inactivation, when they contain an average of three lesions. Comparison with previous results from a similar experiment performed with γ-irradiated plasmids indicates that the type of clustered DNA lesions, created directly on cellular DNA by LEEs, may be more difficult to repair than those produced by other species from radiolysis.

  19. A novel role of the tumor suppressor GNMT in cellular defense against DNA damage.

    PubMed

    Wang, Yi-Cheng; Lin, Wei-Li; Lin, Yan-Jun; Tang, Feng-Yao; Chen, Yi-Ming; Chiang, En-Pei Isabel

    2014-02-15

    Glycine N-methyltransferase (GNMT) is a folate binding protein commonly diminished in human hepatoma yet its role in tumor development remains to be established. GNMT binds to methylfolate but is also inhibited by it; how such interactions affect human carcinogenesis is unclear. We postulated that GNMT plays a role in folate-dependent methyl group homeostasis and helps maintain genome integrity by promoting nucleotide biosynthesis and DNA repair. To test the hypothesis, GNMT was over-expressed in GNMT-null cell lines cultured in conditions of folate abundance or restriction. The partitioning of folate dependent 1-carbon groups was investigated using stable isotopic tracers and GC/MS. DNA damage was assessed as uracil content in cell models, as well as in Gnmt wildtype (Gnmt(+/+)), heterozygote (Gnmt(+/-)) and knockout (Gnmt(-/-)) mice under folate deplete, replete, or supplementation conditions. Our study demonstrated that GMMT 1) supports methylene-folate dependent pyrimidine synthesis; 2) supports formylfolate dependent purine syntheses; 3) minimizes uracil incorporation into DNA when cells and animals were exposed to folate depletion; 4) translocates into nuclei during prolonged folate depletion. In conclusion, loss of GNMT impairs nucleotide biosynthesis. Over-expression of GNMT enhances nucleotide biosynthesis and improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. To our best knowledge, the role of GNMT in folate dependent 1-carbon transfer in nucleotide biosynthesis has never been investigated. The present study gives new insights into the underlying mechanism by which GNMT can participate in tumor prevention/suppression in humans. © 2013 UICC.

  20. 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

  1. Hispidin produced from Phellinus linteus protects against peroxynitrite-mediated DNA damage and hydroxyl radical generation.

    PubMed

    Chen, Wei; Feng, Lina; Huang, Zhaoyi; Su, Hongming

    2012-09-30

    Oxidative stress plays an important role in the progression of many chronic diseases including cardiovascular diseases, diabetes, cancer and neurodegenerative disorders. One such mediator of oxidative stress is peroxynitrite, which is highly toxic to cultured neurons and astrocytes, and has been reported to be involved in the pathogenesis of various types of neuronal diseases. Therefore, searching for natural compounds with peroxynitrite-scavenging activity might be an effective therapy for peroxynitrite-mediated cytotoxicity. Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong antioxidant, anticancer, and antidiabetic properties. However, the astrocyte protective efficacy of hispidin has been not examined. This study was undertaken to investigate whether the astrocyte protective effect of hispidin is associated with inhibition of peroxynitrite-induced DNA damage, a critical event leading to peroxynitrite-mediated cytotoxicity. Our results showed that peroxynitrite can cause DNA damage in φX-174 plasmid DNA and rat primary astrocytes. The presence of hispidin (10-20 μg/ml) was found to significantly inhibit peroxynitrite-induced DNA damage and cytotoxicity. EPR spectroscopy demonstrated that the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from peroxynitrite, and that hispidin potently diminished the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that hispidin can protect against peroxynitrite-mediated cytotoxicity, DNA damage and hydroxyl radical formation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  2. 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.

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

    PubMed

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

    2014-10-23

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

  4. 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.

  5. Rabies DNA vaccine encoding lysosome-targeted glycoprotein supplemented with Emulsigen-D confers complete protection in preexposure and postexposure studies in BALB/c mice.

    PubMed

    Kaur, Manpreet; Saxena, Ankur; Rai, Anant; Bhatnagar, Rakesh

    2010-01-01

    The worldwide incidence of rabies and the inability of currently used vaccination strategies to provide highly potent and cost-effective therapy indicate the need for an improved rabies vaccine. Thus, DNA vaccine based on lysosome-targeted glycoprotein of the rabies virus was evaluated in BALB/c mice. It imparted partial protection (60%) against challenge with 20 LD(50) of the challenge virus standard (CVS) strain of rabies virus. To improve the outcome of vaccination, to ultimately enhance the immune response, we investigated different routes for DNA vaccine delivery, varied doses of DNA, and the influence of adjuvant supplementation. The highest immune response pertaining to IgG antibody titer, with a predominantly IgG1/IgG2a subclass distribution, effective cellular immunity, and a high level of rabies virus neutralizing antibodies (RVNAs) was attained by the optimized DNA vaccine formulation comprising intramuscular administration of 100 microg of DNA vaccine supplemented with Emulsigen-D. In preexposure prophylaxis, a 3-dose regimen of this formulation generated a high RVNA titer (32 IU/ml) and conferred complete protection against challenge with 20 LD(50) of CVS. For postexposure efficacy analysis, rabies was experimentally induced with 50 LD(50) of CVS. Subsequent therapy with 5 doses of the formulation completely prevented rabies in BALB/c mice, which maintained protective RVNA titers of 4 IU/ml. The World Health Organization recommended rabies protective titer threshold is 0.5 IU/ml. Thus, this optimized DNA vaccine formulation provides an avenue for preventing and controlling rabies.

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

    PubMed

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

    2014-12-01

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

  7. Growth and origami folding of DNA on nanoparticles for high-efficiency molecular transport in cellular imaging and drug delivery.

    PubMed

    Yan, Juan; Hu, Chongya; Wang, Ping; Zhao, Bin; Ouyang, Xiangyuan; Zhou, Juan; Liu, Rui; He, Dannong; Fan, Chunhai; Song, Shiping

    2015-02-16

    A novel three-dimensional (3D) superstructure based on the growth and origami folding of DNA on gold nanoparticles (AuNPs) was developed. The 3D superstructure contains a nanoparticle core and dozens of two-dimensional DNA belts folded from long single-stranded DNAs grown in situ on the nanoparticle by rolling circle amplification (RCA). We designed two mechanisms to achieve the loading of molecules onto the 3D superstructures. In one mechanism, ligands bound to target molecules are merged into the growing DNA during the RCA process (merging mechanism). In the other mechanism, target molecules are intercalated into the double-stranded DNAs produced by origami folding (intercalating mechanism). We demonstrated that the as-fabricated 3D superstructures have a high molecule-loading capacity and that they enable the high-efficiency transport of signal reporters and drugs for cellular imaging and drug delivery, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Prostaglandin protection of human isolated gastric glands against indomethacin and ethanol injury. Evidence for direct cellular action of prostaglandin.

    PubMed Central

    Tarnawski, A; Brzozowski, T; Sarfeh, I J; Krause, W J; Ulich, T R; Gergely, H; Hollander, D

    1988-01-01

    Isolated human gastric glands from surgical specimens were preincubated in an oxygenated medium with placebo or 16,16 dimethyl prostaglandin E2 (dmPGE2) and incubated at 37 degrees C in either medium alone, medium containing 4.43 mM indomethacin or medium containing 8% ethanol. We assessed the viability of gland cells with fast green exclusion, release of lactate dehydrogenase (LDH) into the medium, and ultrastructural damage by scanning and transmission electron microscopy. Both indomethacin and ethanol significantly reduced the viability of placebo-pretreated glands, increased LDH release into the medium, and produced prominent ultrastructural damage. DmPGE2 significantly reduced both indomethacin and ethanol-induced injury, increased the number of viable cells, reduced LDH release, and diminished the extent of ultrastructural damage. These studies indicate that PG protection of gastric mucosal cells has a direct cellular action that is not limited to replacement of depleted endogenous PGs. PG protection in our experiments did not depend on PG's previously described systemic actions, such as protection of the microvessels, preservation of the mucosal blood flow, or stimulation of bicarbonate and mucus secretion. Images PMID:3350966

  9. Vitamin C in Cultured Human (HeLa) Cells: Lack of Effect on DNA Protection and Repair

    PubMed Central

    Azqueta, Amaya; Costa, Solange; Lorenzo, Yolanda; Bastani, Nasser E.; Collins, Andrew R.

    2013-01-01

    Aims: Dietary antioxidants, including vitamin C, may be in part responsible for the cancer-preventive effects of fruits and vegetables. Human intervention trials with clinical endpoints have failed to confirm their protective effects, and mechanistic studies have given inconsistent results. Our aim was to investigate antioxidant/ pro-oxidant effects of vitamin C at the cellular level. Experimental approach: We have used the comet assay to investigate effects of vitamin C on DNA damage, antioxidant status, and DNA repair, in HeLa (human tumor) cells, and HPLC to measure uptake of vitamin C into cells. Results: Even at concentrations in the medium as high as 200 μM, vitamin C did not increase the background level of strand breaks or of oxidized purines in nuclear DNA. Vitamin C is taken up by HeLa cells and accumulates to mM levels. Preincubation of cells with vitamin C did not render them resistant to strand breakage induced by H2O2 or to purine oxidation by photosensitizer plus light. Vitamin C had no effect on the rate of repair of strand breaks or oxidized bases by HeLa cells. However, vitamin C at a concentration of less than 1 μM, or extract from cells preincubated for 6 h with vitamin C, was able to induce damage (strand breaks) in lysed, histone-depleted nuclei (nucleoids). Conclusion: In these cultured human cells, vitamin C displays neither antioxidant nor pro-oxidant properties; nor does it affect DNA strand break or base excision repair. PMID:23571651

  10. Possible function of the c-myc product: promotion of cellular DNA replication.

    PubMed Central

    Iguchi-Ariga, S M; Itani, T; Kiji, Y; Ariga, H

    1987-01-01

    We have recently cloned a plasmid, pARS65, containing the sequences derived from mouse liver DNA which can autonomously replicate in mouse and human cells (Ariga et al., 1987). In this report, we show that replication of pARS65 in HL-60 cells can be inhibited by co-transfection with anti-c-myc antibody. In an in-vitro replication system using HL-60 nuclear extract, pARS65 functioned as a template. This in-vitro replication was also blocked by addition of anti-c-myc antibody. Specific binding activity of the c-myc product to pARS65 was detected by an immunobinding assay, suggesting that the c-myc protein promotes DNA replication through binding to the initiation site of replication. This has been substantiated using the antibody to help isolate a human DNA segment that can autonomously replicate in the cells. Images Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:3665880

  11. DNA Demethylation Upregulated Nrf2 Expression in Alzheimer’s Disease Cellular Model

    PubMed Central

    Cao, Huimin; Wang, Li; Chen, Beibei; Zheng, Peng; He, Yi; Ding, Yubin; Deng, Yushuang; Lu, Xi; Guo, Xiuming; Zhang, Yuping; Li, Yu; Yu, Gang

    2016-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important transcription factor in the defense against oxidative stress. Cumulative evidence has shown that oxidative stress plays a key role in the pathogenesis of Alzheimer’s disease (AD). Previous animal and clinical studies had observed decreased expression of Nrf2 in AD. However, the underlying regulation mechanisms of Nrf2 in AD remain unclear. Here, we used the DNA methyltransferases (Dnmts) inhibitor 5-aza-2′-deoxycytidine (5-Aza) to test whether Nrf2 expression was regulated by methylation in N2a cells characterizing by expressing human Swedish mutant amyloid precursor protein (N2a/APPswe). We found 5-Aza treatment increased Nrf2 at both messenger RNA and protein levels via downregulating the expression of Dnmts and DNA demethylation. In addition, 5-Aza-mediated upregulation of Nrf2 expression was concomitant with increased nuclear translocation of Nrf2 and higher expression of Nrf2 downstream target gene NAD(P)H:quinone oxidoreductas (NQO1). Our study showed that DNA demethylation promoted the Nrf2 cell signaling pathway, which may enhance the antioxidant system against AD development. PMID:26779013

  12. Protection from heat-induced protein migration and DNA repair inhibition by cycloheximide.

    PubMed

    Armour, E P; Lee, Y J; Corry, P M; Borrelli, M J

    1988-12-15

    The mechanism by which Cycloheximide (CHM) protects cells from heat induced killing has been investigated. Cycloheximide (10 micrograms/ml) added for 2 hr before and during a 3 hour heating at 43 degrees C prevented a 40% increase of heat-induced protein accumulation in the nucleus and protected cells (0.0001 vs. 0.15 surviving fraction) from heat-induced killing. Heat-induced DNA repair inhibition was also suppressed when cells were treated with CHM in the above manner. This combination of results suggests that protein accumulation in the nucleus and inhibition of DNA repair are related and these events are associated with CHM protection from heat induced cell killing.

  13. Cellular FLICE-inhibitory protein protects against cardiac remodeling induced by angiotensin II in mice.

    PubMed

    Li, Hongliang; Tang, Qi-Zhu; Liu, Chen; Moon, Mark; Chen, Manyin; Yan, Ling; Bian, Zhou-Yan; Zhang, Yan; Wang, Ai-Bing; Nghiem, Mai P; Liu, Peter P

    2010-12-01

    The development of cardiac hypertrophy in response to increased hemodynamic load and neurohormonal stress is initially a compensatory response that may eventually lead to ventricular dilatation and heart failure. Cellular FLICE-inhibitory protein (cFLIP) is a homologue of caspase 8 without caspase activity that inhibits apoptosis initiated by death receptor signaling. Previous studies showed that cFLIP expression was markedly decreased in the ventricular myocardium of patients with end-stage heart failure. However, the critical role of cFLIP on cardiac remodeling remains unclear. To specifically determine the role of cFLIP in pathological cardiac remodeling, we used heterozygote cFLIP(+/-) mice and transgenic mice with cardiac-specific overexpression of the human cFLIP(L) gene. Our results demonstrated that the cFLIP(+/-) mice were susceptible to cardiac hypertrophy and fibrosis through inhibition of mitogen-activated protein kinase kinase-extracellular signal-regulated kinase 1/2 signaling, whereas the transgenic mice displayed the opposite phenotype in response to angiotensin II stimulation. These studies indicate that cFLIP protein is a crucial component of the signaling pathway involved in cardiac remodeling and heart failure.

  14. DNA Vaccination Affords Significant Protection against Feline Immunodeficiency Virus Infection without Inducing Detectable Antiviral Antibodies

    PubMed Central

    Hosie, Margaret J.; Flynn, J. Norman; Rigby, Mark A.; Cannon, Celia; Dunsford, Thomas; Mackay, Nancy A.; Argyle, David; Willett, Brian J.; Miyazawa, Takayuki; Onions, David E.; Jarrett, Oswald; Neil, James C.

    1998-01-01

    To test the potential of a multigene DNA vaccine against lentivirus infection, we generated a defective mutant provirus of feline immunodeficiency virus (FIV) with an in-frame deletion in pol (FIVΔRT). In a first experiment, FIVΔRT DNA was administered intramuscularly to 10 animals, half of which also received feline gamma interferon (IFN-γ) DNA. The DNA was administered in four 100-μg doses at 0, 10, and 23 weeks. Immunization with FIVΔRT elicited cytotoxic T-cell (CTL) responses to FIV Gag and Env in the absence of a serological response. After challenge with homologous virus at week 26, all 10 of the control animals became seropositive and viremic but 4 of the 10 vaccinates remained seronegative and virus free. Furthermore, quantitative virus isolation and quantitative PCR analysis of viral DNA in peripheral blood mononuclear cells revealed significantly lower virus loads in the FIVΔRT vaccinates than in the controls. Immunization with FIVΔRT in conjunction with IFN-γ gave the highest proportion of protected cats, with only two of five vaccinates showing evidence of infection following challenge. In a second experiment involving two groups (FIVΔRT plus IFN-γ and IFN-γ alone), the immunization schedule was reduced to 0, 4, and 8 weeks. Once again, CTL responses were seen prior to challenge in the absence of detectable antibodies. Two of five cats receiving the proviral DNA vaccine were protected against infection, with an overall reduction in virus load compared to the five infected controls. These findings demonstrate that DNA vaccination can elicit protection against lentivirus infection in the absence of a serological response and suggest the need to reconsider efficacy criteria for lentivirus vaccines. PMID:9696827

  15. Roadmap to cellular reprogramming--manipulating transcriptional networks with DNA, RNA, proteins and small molecules.

    PubMed

    Wörsdörfer, P; Thier, M; Kadari, A; Edenhofer, F

    2013-06-01

    Recent reports demonstrate that the plasticity of mammalian somatic cells is much higher than previously assumed and that ectopic expression of transcription factors may have the potential to induce the conversion of any cell type into another. Fibroblast cells can be converted into embryonic stem cell-like cells, neural cells, cardiomyocytes, macrophage-like cells as well as blood progenitors. Additionally, the conversion of astrocytes into neurons or neural stem cells into monocytes has been demonstrated. Nowadays, in the era of systems biology, continuously growing holistic data sets are providing increasing insights into core transcriptional networks and cellular signaling pathways. This knowledge enables cell biologists to understand how cellular fate is determined and how it could be manipulated. As a consequence for biomedical applications, it might be soon possible to convert patient specific somatic cells directly into desired transplantable other cell types. The clinical value, however, of such reprogrammed cells is currently limited due to the invasiveness of methods applied to induce reprogramming factor activity. This review will focus on experimental strategies to ectopically induce cell fate modulators. We will emphasize those strategies that enable efficient and robust overexpression of transcription factors by minimal genetic alterations of the host genome. Furthermore, we will discuss procedures devoid of any genomic manipulation, such as the direct delivery of mRNA, proteins, or the use of small molecules. By this, we aim to give a comprehensive overview on state of the art techniques that harbor the potential to generate safe reprogrammed cells for clinical applications.

  16. A broad spectrum high-SPF photostable sunscreen with a high UVA-PF can protect against cellular damage at high UV exposure doses.

    PubMed

    Cole, Curtis; Appa, Yohini; Ou-Yang, Hao

    2014-08-01

    Adva