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Sample records for cellular dna synthesis

  1. Cellular factors required for papillomavirus DNA replication.

    OpenAIRE

    Melendy, T; Sedman, J; Stenlund, A

    1995-01-01

    In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells. DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication. The E2 protein stimulates DNA synthesis in a binding site-independent manner. Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a ...

  2. Exploration of cellular DNA lesion, DNA-binding and biocidal ordeal of novel curcumin based Knoevenagel Schiff base complexes incorporating tryptophan: Synthesis and structural validation

    Science.gov (United States)

    Chandrasekar, Thiravidamani; Raman, Natarajan

    2016-07-01

    A few novel Schiff base transition metal complexes of general formula [MLCl] (where, L = Schiff base, obtained by the condensation reaction of Knoevenagel condensate of curcumin, L-tryptophan and M = Cu(II), Ni(II), Co(II), and Zn(II)), were prepared by stencil synthesis. They were typified using UV-vis, IR, EPR spectral techniques, micro analytical techniques, magnetic susceptibility and molar conductivity. Geometry of the metal complexes was examined and recognized as square planar. DNA binding and viscosity studies revealed that the metal(II) complexes powerfully bound via an intercalation mechanism with the calf thymus DNA. Gel-electrophoresis technique was used to investigate the DNA cleavage competence of the complexes and they establish to approve the cleavage of pBR322 DNA in presence of oxidant H2O2. This outcome inferred that the synthesized complexes showed better nuclease activity. Moreover, the complexes were monitored for antimicrobial activities. The results exposed that the synthesized compounds were forceful against all the microbes under exploration.

  3. Cellular responses to environmental DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Viral DNA synthesis-dependent titration of a cellular repressor activates transcription of the human adenovirus type 2 IVa2 gene

    OpenAIRE

    C. Iftode; Flint, S J

    2004-01-01

    Synthesis of progeny DNA genomes in cells infected by human subgroup C adenoviruses leads to several changes in viral gene expression. These changes include transcription from previously silent, late promoters, such as the IVa2 promoter, and a large increase in the efficiency of major-late (ML) transcription. Some of these changes appear to take place sequentially, because the product of the IVa2 gene has been implicated in stimulation of ML transcription. Our previous biochemical studies sug...

  5. Synthesis, DNA binding, cellular DNA lesion and cytotoxicity of a series of new benzimidazole-based Schiff base copper(II) complexes.

    Science.gov (United States)

    Paul, Anup; Anbu, Sellamuthu; Sharma, Gunjan; Kuznetsov, Maxim L; Koch, Biplob; Guedes da Silva, M Fátima C; Pombeiro, Armando J L

    2015-12-14

    A series of new benzimidazole containing compounds 2-((1-R-1-H-benzimidazol-2-yl)phenyl-imino)naphthol HL(1-3) (R = methyl, ethyl or propyl, respectively) have been synthesized by Schiff base condensation of 2-(1-R-1-H-benzo[d]imidazol-2-yl)aniline and 2-hydroxy-1-naphthaldehyde. The reactions of HL(1-3) with Cu(NO3)2·2.5H2O led to the corresponding copper(II) complexes [Cu(L)(NO3)] 1-3. All the compounds were characterized by conventional analytical techniques and, for 1 and 3, also by single-crystal X-ray analysis. The interactions of complexes 1-3 with calf thymus DNA were studied by absorption and fluorescence spectroscopic techniques and the calculated binding constants (K(b)) are in the range of 3.5 × 10(5) M(-1)-3.2 × 10(5) M(-1). Complexes 1-3 effectively bind DNA through an intercalative mode, as proved by molecular docking studies. The binding affinity of the complexes decreases with the size increase of the N-alkyl substituent, in the order of 1 > 2 > 3, which is also in accord with the calculated LUMO(complex) energies. They show substantial in vitro cytotoxic effect against human lung (A-549), breast (MDA-MB-231) and cervical (HeLa) cancer cell lines. Complex 1 exhibits a significant inhibitory effect on the proliferation of the A-549 cancer cells. The antiproliferative efficacy of 1 has also been analysed by a DNA fragmentation assay, fluorescence activated cell sorting (FACS) and nuclear morphology using a fluorescence microscope. The possible mode for the apoptosis pathway of 1 has also been evaluated by a reactive oxygen species (ROS) generation study.

  6. Depletion of Cellular Pre-Replication Complex Factors Results in Increased Human Cytomegalovirus DNA Replication

    OpenAIRE

    Tamara Evans Braun; Emma Poole; John Sinclair

    2012-01-01

    Although HCMV encodes many genes required for the replication of its DNA genome, no HCMV-encoded orthologue of the origin binding protein, which has been identified in other herpesviruses, has been identified. This has led to speculation that HCMV may use other viral proteins or possibly cellular factors for the initiation of DNA synthesis. It is also unclear whether cellular replication factors are required for efficient replication of viral DNA during or after viral replication origin recog...

  7. Synthesis of DNA

    Science.gov (United States)

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  8. Synthesis of chemically modified DNA.

    Science.gov (United States)

    Shivalingam, Arun; Brown, Tom

    2016-06-15

    Naturally occurring DNA is encoded by the four nucleobases adenine, cytosine, guanine and thymine. Yet minor chemical modifications to these bases, such as methylation, can significantly alter DNA function, and more drastic changes, such as replacement with unnatural base pairs, could expand its function. In order to realize the full potential of DNA in therapeutic and synthetic biology applications, our ability to 'write' long modified DNA in a controlled manner must be improved. This review highlights methods currently used for the synthesis of moderately long chemically modified nucleic acids (up to 1000 bp), their limitations and areas for future expansion. PMID:27284032

  9. Celecoxib transiently inhibits cellular protein synthesis.

    Science.gov (United States)

    Pyrko, Peter; Kardosh, Adel; Schönthal, Axel H

    2008-01-15

    To uncover the full spectrum of its pharmacological activities, the selective COX-2 inhibitor celecoxib is routinely being used at concentrations of up to 100 microM in cell culture. At these elevated concentrations, several COX-2-independent effects were identified, although many details of these events have remained unclear. Here, we report a COX-2-independent effect of celecoxib that might have profound consequences for the interpretation of previous results obtained at elevated concentrations of this drug in vitro. We found that celecoxib rapidly inhibits general protein translation at concentrations as low as 30 microM. This appears to be a consequence of endoplasmic reticulum (ER) stress and entails the phosphorylation and inactivation of eukaryotic translation initiation factor 2 alpha (eIF2alpha). These effects were not achieved by other coxibs (rofecoxib, valdecoxib) or traditional NSAIDs (indomethacin, flurbiprofen), but were mimicked by the COX-2-inactive celecoxib analog, 2,5-dimethyl-celecoxib (DMC), indicating COX-2 independence. Considering the obvious impact of blocked translation on cellular function, we provide evidence that this severe inhibition of protein synthesis might suffice to explain some of the previously reported COX-2-independent effects of celecoxib, such as the down-regulation of the essential cell cycle regulatory protein cyclin D, which is a short-lived protein that rapidly disappears in response to the inhibition of protein synthesis. Taken together, our findings establish ER stress-induced inhibition of general translation as a critical outcome of celecoxib treatment in vitro, and suggest that this effect needs to be considered when interpreting observations from the use of this drug in cell culture. PMID:17920040

  10. Effects of aluminum on DNA synthesis, cellular polyamines, polyamine biosynthetic enzymes and inorganic ions in cell suspension cultures of a woody plant, Catharanthus roseus

    Energy Technology Data Exchange (ETDEWEB)

    Minocha, R.; Shortle, W.C. (USDA Forest Service, Durham (US)); Minocha, S.C.; Long, S.L. (Dept. of Plant Biology, Univ. of New Hamshire, Durham (US))

    1992-01-01

    Increased aluminium (Al) solubility in soil waters due to acid precipitation has aroused considerable interest in the problem of Al toxicity in plants. In the present study, an in vitro suspension culture system of Catharanthus roseus (L.) G. Don was used to analyze the effects of aluminum on several biochemical processes in these cells. The aliphatic polyamines, spermine and spermidine, and their precusor, putrescine, have been implicated in a number of stress responses of plants. Addition of 0.2, 0.5 or 1.0 mM AlCl{sub 3} to cells cultured for 3 days caused a small but significant increase in cellular levels of putrescine at 4 h followed by a sharp decline by 16 h. There was no further decline in levels of putrescine during the next 32 h. Spermidine levels did not change appreciably compared to those in the control cultures. However, spermine levels increased by 2-3-fold at 24 and 48 h. Cellular activities of arginine decarboxylase (ADC; EC 4.1.1.19) and S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) were both inhibited by 20-25% at 4 and 7 h. Ornithine decarboxylase (ODC; EC 4.1.1.17) was less than 10% of ADC activity at all times. Whereas all concentrations of Al caused a slight decrease in total cell number, cell viability was affected only by 1.0 mM Al. There was a decrease in the cellular levels of Ca, Mg, Na, K, Mn, P and Fe in the cells treated with Al at 4 h, but a significant increase by 16 and 24 h. The results presented here suggest that both the absolute amounts of Al and the length of exposure to it are important for cell toxicity. (au).

  11. DNA origami: Nanorobots grab cellular control

    Science.gov (United States)

    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.

  12. Inhibition of DNA replication, DNA repair synthesis, and DNA polymerases α and δ by butylphenyl deoxyguanosine triphosphate

    International Nuclear Information System (INIS)

    Semiconservative DNA replication in growing mammalian cells and ultraviolet (UV)-induced DNA repair synthesis in nongrowing mammalian cells are mediated by one or both of the aphidicolin-sensitive DNA polymerases, α and/or δ. They have studied the inhibition of replication and repair synthesis in permeable human cells by N2 (p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh dGTP), an agent which inhibits polymerase α strongly and polymerase δ weakly. Both processes are inhibited by BuPh-dGTP in competition with dGTP. The K/sub i/'s are, for replication, 2-3 μM and, for repair synthesis, 3-4 μM, consistent with the involvement of the same DNA polymerase in both processes. Inhibition of isolated human polymerase α by BuPh-dGTP is also competitive with dGTP, but the K/sub i/ is approximately 10 nM, several hundred-fold lower than the K/sub i/'s of replication and repair synthesis. Isolated polymerase δ is inhibited by BuPh-dGTP at doses similar to those which inhibit replication and repair synthesis, however, attempts to determine the K/sub i/ of polymerase δ were hampered by the finding that the dependence of δ activity on deoxyribunucleotide concentration is parabolic at low doses. This behavior differs from the behavior of polymerase α and of cellular DNA replication and repair synthesis, all of which show a simple, hyperbolic relationship between activity and deoxyribonucleotide concentration. Thus, inhibition of DNA replication and UV induced DNA repair synthesis by BuPh dGTP is quantitatively similar to DNA polymerase δ, but some other characteristics of the cellular processes are more similar to those of polymerase α

  13. Anticancer agent CHS-828 inhibits cellular synthesis of NAD

    DEFF Research Database (Denmark)

    Olesen, U.H.; Christensen, M.K.; Bjorkling, F.;

    2008-01-01

    Malignant cells display increased demands for energy production and DNA repair. Nicotinamide adenine dinucleotide (NAD) is required for both processes and is also continuously degraded by cellular enzymes. Nicotinamide phosphoribosyltransferase (Nampt) is a crucial factor in the resynthesis of NA...

  14. Analysis of cellular and extracellular DNA in fingerprints

    Energy Technology Data Exchange (ETDEWEB)

    Button, Julie M. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    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.

  15. DNA polymerase δ and DNA repair: DNA repair synthesis in human fibroblasts requires DNA polymerase δ

    International Nuclear Information System (INIS)

    When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernate of similarly treated HeLa cells. Monoclonal antibody to KB cell DNA polymerase α, while binding to HeLa DNA polymerase α, did not bind to the HeLa DNA polymerase δ. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGT) and 2(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase α, but did not inhibit the DNA polymerase δ. Neither purified DNA polymerase α nor β could promote repair DNA synthesis in the permeabilized cells. Furthermore, if monoclonal antibodies to DNA polymerase α BuPdGTP, or BuAdATP was added to the reconstituted system, there was no significant inhibition

  16. DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination.

    OpenAIRE

    Islas, L; Fairley, C F; Morgan, W. F.

    1998-01-01

    DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well a...

  17. Cellular Responses to Cisplatin-Induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Alakananda Basu

    2010-01-01

    Full Text Available Cisplatin is one of the most effective anticancer agents widely used in the treatment of solid tumors. It is generally considered as a cytotoxic drug which kills cancer cells by damaging DNA and inhibiting DNA synthesis. How cells respond to cisplatin-induced DNA damage plays a critical role in deciding cisplatin sensitivity. Cisplatin-induced DNA damage activates various signaling pathways to prevent or promote cell death. This paper summarizes our current understandings regarding the mechanisms by which cisplatin induces cell death and the bases of cisplatin resistance. We have discussed various steps, including the entry of cisplatin inside cells, DNA repair, drug detoxification, DNA damage response, and regulation of cisplatin-induced apoptosis by protein kinases. An understanding of how various signaling pathways regulate cisplatin-induced cell death should aid in the development of more effective therapeutic strategies for the treatment of cancer.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

    Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision re-pair (BER). SSBs generally have blocked termini lacking the conventional 5'-phosphate and 3'-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 (polβ) 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 MMS-treated XRCC1-/-, and to a lesser extent in polβ-/- 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 andpolβ-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induccd cytotoxicity. XRCC1-/- cellsare also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role inmodulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.

  19. HSV-I and the cellular DNA damage response

    OpenAIRE

    Smith, Samantha; Weller, Sandra K.

    2015-01-01

    Peter Wildy first observed genetic recombination between strains of HSV in 1955. At the time, knowledge of DNA repair mechanisms was limited, and it has only been in the last decade that particular DNA damage response (DDR) pathways have been examined in the context of viral infections. One of the first reports addressing the interaction between a cellular DDR protein and HSV-1 was the observation by Lees-Miller et al. that DNA-dependent protein kinase catalytic subunit levels were depleted i...

  20. Spontaneous unscheduled DNA synthesis in human lymphocytes

    International Nuclear Information System (INIS)

    The rate of spontaneous unscheduled DNA synthesis in human lymphocytes was estimated from measurements of tritiated thymidine incorporation into double-stranded DNA (ds-DNA) during incubation of cells in vitro. The contribution of scheduled DNA synthesis to the observed incorporation was reduced by inhibiting replication with hydroxyurea and by separating freshly replicated single-stranded DNA (ss-DNA) from repaired ds-DNA by column chromatography. The residual contribution of scheduled DNA synthesis was estimated by observing effects on thymidine incorporation of: (a) increasing the rate of production of apurinic sites, and alternatively, (b) increasing the number of cells in S-phase. Corrections based on estimates of endogenous pool size were also made. The rate of spontaneous unscheduled DNA synthesis is estimated to be 490 +- 120 thymidine molecules incorporated per cell per hour. These results compare favorably with estimates made from rates of depurination and depyrimidination of DNA, measured in molecular systems if we assume thymidine is incorporated by a short patch mechanism which incorporates an average of four bases per lesion

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

    International Nuclear Information System (INIS)

    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

  2. Continuous induction of unscheduled DNA synthesis by gamma irradiation

    International Nuclear Information System (INIS)

    The induction of DNA-synthesis in non-S-phase cells is a very sensitive measure of a preceding damage of the DNA. Usually, in an in vivo -in vitro test (treatment of an animal, incorporation of H3-thymidine in a cell suspension) the damaging of DNA takes place hours to days before the evaluation. In this case, the time course of the UDS-induction after a single dose of 1 Gy gamma irradiation should be observed for a long time (21 months). C57 black mice served as test animals. In an age of about 80 days they were irradiated and the induction of unscheduled DNA synthesis was measured at ten points of time during the whole life-span of the animals. Although the repair in this gamma radiation damage in DNA is a very quick process - with centrifugation in alkaline sucrose you find a half time of some minutes - an induction of unscheduled DNA synthesis could be seen at the irradiated animals until the end of their life (640 days). The reason for this could be permanent disorders in cellular regulation caused by the gamma irradiation. 4 figs. (Author)

  3. Cellular Uptake of Tile-Assembled DNA Nanotubes

    Directory of Open Access Journals (Sweden)

    Samet Kocabey

    2014-12-01

    Full Text Available 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.

  4. Cellular processing and destinies of artificial DNA nanostructures.

    Science.gov (United States)

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

    2016-08-01

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

  5. Ultraviolet radiation-mediated damage to cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Cadet, Jean [Laboratoire Lesions des Acides Nucleiques, Service de Chimie Inorganique et Biologique, CEA/DSM/Departement de Recherche Fondamentale sur la Matiere Condensee, CEA-Grenoble, 17, Av. des Martyrs, Grenoble Cedex 9 F-38054 (France)]. E-mail: jcadet@cea.fr; Sage, Evelyne [Institut Curie, CNRS/IC UMR 2027, Centre Universitaire, Orsay (France); Douki, Thierry [Laboratoire Lesions des Acides Nucleiques, Service de Chimie Inorganique et Biologique, CEA/DSM/Departement de Recherche Fondamentale sur la Matiere Condensee, CEA-Grenoble, 17, Av. des Martyrs, Grenoble Cedex 9 F-38054 (France)

    2005-04-01

    Emphasis is placed in this review article on recent aspects of the photochemistry of cellular DNA in which both the UVB and UVA components of solar radiation are implicated individually or synergistically. Interestingly, further mechanistic insights into the UV-induced formation of DNA photoproducts were gained from the application of new accurate and sensitive chromatographic and enzymic assays aimed at measuring base damage. Thus, each of the twelve possible dimeric photoproducts that are produced at the four main bipyrimidine sites can now be singled out as dinucleoside monophosphates that are enzymatically released from UV-irradiated DNA. This was achieved using a recently developed high-performance liquid chromatography-tandem mass spectrometry assay (HPLC-MS/MS) assay after DNA extraction and appropriate enzymic digestion. Interestingly, a similar photoproduct distribution pattern is observed in both isolated and cellular DNA upon exposure to low doses of either UVC or UVB radiation. This applies more specifically to the DNA of rodent and human cells, the cis-syn cyclobutadithymine being predominant over the two other main photolesions, namely thymine-cytosine pyrimidine (6-4) pyrimidone adduct and the related cyclobutyl dimer. UVA-irradiation was found to generate cyclobutane dimers at TT and to a lower extent at TC sites as a likely result of energy transfer mechanism involving still unknown photoexcited chromophore(s). Oxidative damage to DNA is also induced although less efficiently by UVA-mediated photosensitization processes that mostly involved {sup 1}O{sub 2} together with a smaller contribution of hydroxyl radical-mediated reactions through initially generated superoxide radicals.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Ishai, R.; Scharf, R.; Sharon, R.; Kapten, I. (Technion-Israel Institute of Technology, Haifa (Israel))

    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.

  7. Measurement of oxidatively generated base damage in cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Cadet, Jean, E-mail: jean.cadet@cea.fr [Laboratoire ' Lesions des Acides Nucleiques' , SCIB-UMR-E no3 (CEA/UJF), FRE CNRS 3200, Departement de Recherche Fondamentale sur la Matiere Condensee, CEA/Grenoble, F-38054 Grenoble Cedex 9 (France); Douki, Thierry; Ravanat, Jean-Luc [Laboratoire ' Lesions des Acides Nucleiques' , SCIB-UMR-E no3 (CEA/UJF), FRE CNRS 3200, Departement de Recherche Fondamentale sur la Matiere Condensee, CEA/Grenoble, F-38054 Grenoble Cedex 9 (France)

    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 {sup 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.

  8. Biological evaluation of new nickel(II) metallates: Synthesis, DNA/protein binding and mitochondrial mediated apoptosis in human lung cancer cells (A549) via ROS hypergeneration and depletion of cellular antioxidant pool.

    Science.gov (United States)

    Kalaivani, P; Saranya, S; Poornima, P; Prabhakaran, R; Dallemer, F; Vijaya Padma, V; Natarajan, K

    2014-07-23

    A series of novel nickel(II) thiosemicarbazone complexes(1-4) have been prepared and characterized by various spectral, analytical techniques and X-ray crystallography. Further, their efficacy to interact with CT-DNA/BSA has been explored. From the binding studies, it is inferred that complex 4 found to be more active than other complexes. The complexes bound with CT-DNA by intercalation mode. Moreover, static quenching was observed for their interaction with BSA. The new complexes were tested for their in vitro cytotoxicity against human lung adenocarcinoma (A549) cell line. The results showed that the new complexes exhibited significant degree of cytotoxicity at given experimental condition. Further, the results of LDH and NO release supported the cytotoxic nature of the complexes. The observed cytotoxicity of the complexes may be routed through ROS-hypergeneration and lipid-peroxidation with subsequent depletion of cellular antioxidant pool (GSH, SOD, CAT, GPx and GST) resulted in the reduction of mitochondrial-membrane potential, caspase-3 activation and DNA fragmentation. Thus, the data from the present study disclose that the complexes could induce apoptosis in A549 cells through mitochondrial mediated fashion and inhibited the migration of lung cancer cells and by metastasis. PMID:24946146

  9. Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs

    OpenAIRE

    Adams, Drew J.; Dai, Mingji; Pellegrino, Giovanni; Wagner, Bridget K.; Stern, Andrew M.; Shamji, Alykhan F.; Schreiber, Stuart L.

    2012-01-01

    Piperlongumine is a naturally occurring small molecule recently identified to be toxic selectively to cancer cells in vitro and in vivo. This compound was found to elevate cellular levels of reactive oxygen species (ROS) selectively in cancer cell lines. The synthesis of 80 piperlongumine analogs has revealed structural modifications that retain, enhance, and ablate key piperlongumine-associated effects on cells, including elevation of ROS, cancer cell death, and selectivity for cancer cells ...

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

    Science.gov (United States)

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

    2016-04-12

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

  11. DNA synthesis in ataxia telangiectasia

    OpenAIRE

    Jaspers, Nicolaas

    1985-01-01

    textabstractAfter the discovery that cultured cells from AT patients are hypersensitive to ionizing radiation the suggestion was made that AT-could be the 1 X-ray-analogue 1 of xeroderma pigmentosum. The latter syndrome (XP) is characterized by hypersensitivity to short-wave UV-radiation, caused by a reduced ability to properly remove UV-induced DNA damage. The evidence for a DNA repair defect in AT cells is not as strong as in the case of XP (see section 2.2.5 of this thesis). Different XP p...

  12. DNA precursor compartmentation in mammalian cells: metabolic and antimetabolic studies of nuclear and mitochondrial DNA synthesis

    International Nuclear Information System (INIS)

    HeLa cells were used for the quantitation of cellular and mitochondrial deoxyribonucleoside triphosphate (dNTP) and ribonucleoside triphosphate (rNTP) pools and of changes in pools in response to treatment with the antimetabolites methotrexate (mtx) and 5-fluorodeoxyuridine (FUdR). Use of an enzymatic assay of dNTPs and of improved nucleotide extraction methods allowed quantitation of mitochondrial dNTP pools. All four mitochondrial dNTP pools expand following treatment with mtx or FUdR whereas cellular dTTP and dGTP pools are depleted. Mitochrondrial rNTP pools were also found to expand in response to these antimetabolites. Mouse L-cells were used to determine the relative contributions of an exogenously supplied precursor to nuclear and mitochrondrial DNA replication. Cells were labeled to near steady state specific activities with 32P-orthophosphate and subsequently labeled with [3H]uridine, a general pyrimidine precursor, in the continuing presence of 32P. Deoxyribonucleoside monophosphates derived from these DNAs were separated by HPLC and the 3H/32P ratio in each pyrimidine determined. The dCMP residues in mitochondrial DNA (mtDNA) were found to be derived exclusively from the exogenous supplied uridine. The dTMP residues from nuclear and mtDNA and the dCMP residues from nuclear DNA were seen to be synthesized partly from exogenous sources and partly from other sources, presumably de novo pyrimidine synthesis

  13. DNA-Destabilizing Agents as an Alternative Approach for Targeting DNA: Mechanisms of Action and Cellular Consequences

    Directory of Open Access Journals (Sweden)

    Gaëlle Lenglet

    2010-01-01

    Full Text Available DNA targeting drugs represent a large proportion of the actual anticancer drug pharmacopeia, both in terms of drug brands and prescription volumes. Small DNA-interacting molecules share the ability of certain proteins to change the DNA helix's overall organization and geometrical orientation via tilt, roll, twist, slip, and flip effects. In this ocean of DNA-interacting compounds, most stabilize both DNA strands and very few display helix-destabilizing properties. These types of DNA-destabilizing effect are observed with certain mono- or bis-intercalators and DNA alkylating agents (some of which have been or are being developed as cancer drugs. The formation of locally destabilized DNA portions could interfere with protein/DNA recognition and potentially affect several crucial cellular processes, such as DNA repair, replication, and transcription. The present paper describes the molecular basis of DNA destabilization, the cellular impact on protein recognition, and DNA repair processes and the latter's relationships with antitumour efficacy.

  14. Stimulation of mouse DNA primase-catalyzed oligoribonucleotide synthesis by mouse DNA helicase B.

    OpenAIRE

    Saitoh, A; S. Tada; Katada, T; Enomoto, T.

    1995-01-01

    Many prokaryotic and viral DNA helicases involved in DNA replication stimulate their cognate DNA primase activity. To assess the stimulation of DNA primase activity by mammalian DNA helicases, we analyzed the synthesis of oligoribonucleotides by mouse DNA polymerase alpha-primase complex on single-stranded circular M13 DNA in the presence of mouse DNA helicase B. DNA helicase B was purified by sequential chromatography through eight columns. When the purified DNA helicase B was applied to a M...

  15. Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function

    International Nuclear Information System (INIS)

    Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and α-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at high concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent

  16. Thymidine Analogues for Tracking DNA Synthesis

    Directory of Open Access Journals (Sweden)

    Brenton L. Cavanagh

    2011-09-01

    Full Text Available Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in “tagging DNA synthesis” is the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU. The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using ‘Huisgen’s reaction’ (1,3-dipolar cycloaddition or ‘click chemistry’. This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other “unnatural bases”. These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.

  17. Detection, characterization and measure of a new radiation-induced damage in isolated and cellular DNA

    International Nuclear Information System (INIS)

    Deoxyribonucleic acid (DNA) contains the genetic information and chemical injury to this macromolecule may have severe biological consequences. We report here the detection of 4 new radiation-induced DNA lesions by using a high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) approach. For that purpose, the characteristic fragmentation of most 2'-deoxy-ribo nucleosides, the loss of 116 Da corresponding to the loss of the 2-deoxyribose moiety, was used in the so-called neutral loss mode of the HPLC-MS/MS. One of the newly detected lesions, named dCyd341 because it is a 2'-deoxycytidine modification exhibiting a molecular weight of 341 Da, was also detected in cellular DNA. Characterization of this modified nucleoside was performed using NMR and exact mass determination of the product obtained by chemical synthesis. A mechanism of formation was then proposed, in which the first event is the H-abstraction at the C4 position of a 2-deoxyribose moiety. Then, the sugar modification produced exhibits a reactive aldehyde that, through reaction with a vicinal cytosine base, gives rise to dCyd341. dCyd341 could be considered as a complex damage since its formation involves a DNA strand break and a cross-link between a damaged sugar residue and a vicinal cytosine base located most probably on the complementary DNA strand. In addition to its characterization, preliminary biological studies revealed that cells are able to remove the lesion from DNA. Repair studies have revealed the ability of cells to excise the lesion. Identification of the repair systems involved could represent an interesting challenge. (author)

  18. Phospholipase C-gamma 1 can induce DNA synthesis by a mechanism independent of its lipase activity.

    OpenAIRE

    Smith, M. R.; Liu, Y.L.; Matthews, N T; Rhee, S G; Sung, W K; Kung, H F

    1994-01-01

    Inositol phospholipid-specific phospholipase C (PLC) is involved in several signaling pathways leading to cellular growth and differentiation. Our previous studies reported the induction of DNA synthesis in quiescent NIH 3T3 cells after microinjection of PLC and the inhibition of serum- or Ras-stimulated DNA synthesis by a mixture of monoclonal antibodies to PLC-gamma 1. In the course of our investigation of anti-PLC-gamma 1 monoclonal antibodies, we found that each antibody exerts different ...

  19. DNA Nanoparticles for Improved Protein Synthesis In Vitro.

    Science.gov (United States)

    Galinis, Robertas; Stonyte, Greta; Kiseliovas, Vaidotas; Zilionis, Rapolas; Studer, Sabine; Hilvert, Donald; Janulaitis, Arvydas; Mazutis, Linas

    2016-02-24

    The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 10(4)  clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription-translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.

  20. Unscheduled DNA synthesis in frog lens at 50C

    International Nuclear Information System (INIS)

    Unscheduled DNA labeling occurs in the frog even at low temperatures. It is concluded tentatively that UV-induced labeling observed in cold incubated lenses represents repair synthesis of DNA. (author)

  1. Inhibitory effect of benzene metabolites on nuclear DNA synthesis in bone marrow cells

    International Nuclear Information System (INIS)

    Effects of endogenously produced and exogenously added benzene metabolites on the nuclear DNA synthetic activity were investigated using a culture system of mouse bone marrow cells. Effects of the metabolites were evaluated by a 30-min incorporation of [3H]thymidine into DNA following a 30-min interaction with the cells in McCoy's 5a medium with 10% fetal calf serum. Phenol and muconic acid did not inhibit nuclear DNA synthesis. However, catechol, 1,2,4-benzenetriol, hydroquinone, and p-benzoquinone were able to inhibit 52, 64, 79, and 98% of the nuclear DNA synthetic activity, respectively, at 24 μM. In a cell-free DNA synthetic system, catechol and hydroquinone did not inhibit the incorporation of [3H]thymidine triphosphate into DNA up to 24 μM but 1,2,4-benzenetriol and p-benzoquinone did. The effect of the latter two benzene metabolites was completely blocked in the presence of 1,4-dithiothreitol (1 mM) in the cell-free assay system. Furthermore, when DNA polymerase α, which requires a sulfhydryl (SH) group as an active site, was replaced by DNA polymerase 1, which does not require an SH group for its catalytic activity, p-benzoquinone and 1,2,4-benzenetriol were unable to inhibit DNA synthesis. Thus, the data imply the p-benzoquinone and 1,2,4-benzenetriol inhibited DNA polymerase α, consequently resulting in inhibition of DNA synthesis in both cellular and cell-free DNA synthetic systems. The present study identifies catechol, hydroquinone, p-benzoquinone, and 1,2,4-benzenetriol as toxic benzene metabolites in bone marrow cells and also suggests that their inhibitory action on DNA synthesis is mediated by mechanism(s) other than that involving DNA damage as a primary cause

  2. DNA sequencing by synthesis with degenerate primers

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The degenerate primer-based sequencing Was developed by a synthesis method(DP-SBS)for high-throughput DNA sequencing,in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays.In this method,adifferent set of degenerate primers containing a give nnumber(n)of degenerate nucleotides at the 3'-ends were annealed to the sequenced templates that were immobilized on the solid surface.The nucleotides(n+1)on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides.The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately.The main advanmge of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension.From the present study,it is found that the DP-SBS method is reliable,simple,and cost-effective for laboratory-sequencing a large amount of short DNA fragments.

  3. Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents

    International Nuclear Information System (INIS)

    The induction of unscheduled DNA synthesis has been considered as a suitable endpoint for the screening of genotoxic agents. Experimentally, unscheduled DNA synthesis is most frequently measured by autoradiography. The purpose of this report was to examine the usefulness of the liquid scintillation counting technique in measuring unscheduled DNA synthesis response in isolated rat hepatocytes. The various liquid scintillation counting-based unscheduled DNA synthesis assay procedures were examined according to the following groupings: (1) procedures based on the acid precipitation of cellular macromolecules, (2) procedures based on isopycnic gradient centrifugation of solubilized cells, (3) procedures based on nuclei isolation in conjunction with other DNA purification methods, and (4) procedures based on the selective retention of hepatocellular DNA. Limited cases in which test chemicals gave positive unscheduled DNA synthesis response in liquid scintillation counting-based assays and negative unscheduled DNA synthesis response in autoradiography-based assays are presented. It is concluded that liquid scintillation counting-based unscheduled DNA synthesis assays represent an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs

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

    Science.gov (United States)

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

    2016-01-01

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

  5. Synthesis of marmycin A and investigation into its cellular activity

    Science.gov (United States)

    Cañeque, Tatiana; Gomes, Filipe; Mai, Trang Thi; Maestri, Giovanni; Malacria, Max; Rodriguez, Raphaël

    2015-09-01

    Anthracyclines such as doxorubicin are used extensively in the treatment of cancers. Anthraquinone-related angucyclines also exhibit antiproliferative properties and have been proposed to operate via similar mechanisms, including direct genome targeting. Here, we report the chemical synthesis of marmycin A and the study of its cellular activity. The aromatic core was constructed by means of a one-pot multistep reaction comprising a regioselective Diels-Alder cycloaddition, and the complex sugar backbone was introduced through a copper-catalysed Ullmann cross-coupling, followed by a challenging Friedel-Crafts cyclization. Remarkably, fluorescence microscopy revealed that marmycin A does not target the nucleus but instead accumulates in lysosomes, thereby promoting cell death independently of genome targeting. Furthermore, a synthetic dimer of marmycin A and the lysosome-targeting agent artesunate exhibited a synergistic activity against the invasive MDA-MB-231 cancer cell line. These findings shed light on the elusive pathways through which anthraquinone derivatives act in cells, pointing towards unanticipated biological and therapeutic applications.

  6. An autoradiographic demonstration of nuclear DNA replication by DNA polymerase alpha and of mitochondrial DNA synthesis by DNA polymerase gamma.

    OpenAIRE

    Geuskens, M.; Hardt, N; Pedrali-Noy, G; Spadari, S

    1981-01-01

    The incorporation of thymidine into the DNA of eukaryotic cells is markedly depressed, but not completely inhibited, by aphidicolin, a highly specific inhibitor of DNA polymerase alpha. An electron microscope autoradiographic analysis of the synthesis of nuclear and mitochondrial DNA in vivo in Concanavalin A stimulated rabbit spleen lymphocytes and in Hamster cell cultures, in the absence and in the presence of aphidicolin, revealed that aphidicolin inhibits the nuclear but not the mitochond...

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

    Science.gov (United States)

    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. PMID:25208528

  8. Differential sensitivity to aphidicolin of replicative DNA synthesis and ultraviolet-induced unscheduled DNA synthesis in vivo in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Seki,Shuji

    1984-06-01

    Full Text Available In vivo in mammalian cells, ultraviolet-induced unscheduled DNA synthesis was less sensitive to aphidicolin than was replicative DNA synthesis. Replicative DNA synthesis in HeLa, HEp-2, WI-38 VA-13 and CV-1 cells was inhibited more than 97% by aphidicolin at 10 micrograms/ml, whereas aphidicolin inhibition of DNA synthesis in ultraviolet-irradiated cells varied between 30% and 90% depending on cell types and assay conditions. Aphidicolin inhibition of unscheduled DNA synthesis (UDS in HeLa cells increased gradually with increasing aphidicolin concentration and reached approximately 90% at 100 micrograms/ml aphidicolin. A significant fraction of UDS in ultraviolet-irradiated HEp-2 cells was resistant to aphidicolin even at 300 micrograms/ml. Considered along with related information reported previously, the present results suggest that both aphidicolin-sensitive and insensitive DNA polymerases, DNA polymerase alpha and a non-alpha DNA polymerase (possibly DNA polymerase beta, are involved in in situ UDS in these ultraviolet-irradiated cells. Comparison of staphylococcal nuclease sensitivity between DNAs repaired in the presence and in the absence of aphidicolin in HEp-2 cells suggested that the involvement of DNA polymerase alpha in UDS favored DNA synthesis in the intranucleosomal region.

  9. DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair.

    OpenAIRE

    Mossi, R; Ferrari, E.; Hübscher, U

    1998-01-01

    The joining of single-stranded breaks in double-stranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination. Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA polymerases delta and epsilon show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by th...

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

    Science.gov (United States)

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

    1999-01-01

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

  11. Quantitation by flow microfluorometry of total cellular DNA in Acanthamoeba

    Energy Technology Data Exchange (ETDEWEB)

    Coulson, P.B.; Tyndall, R.

    1978-01-01

    The DNA content of five speciea of Acanthamoeba was determined by flow microfluorometry. Acanthamoeba castellanii (AC-30), acanthamoeba polyphaga (APG and P-23), acanthamoeba rhysodes, acanthamoeba culbertsoni (A-1), and acanthamoeba royreba were grown in a casitone based medium 24 to 48 hr. The trophozoites were harvested, fixed in 70% ethanol (acidified), pretreated with RNase, stained with propidium diiodide, and evaluated for DNA-bound fluorescence. All species tested had DNA values between 2.0 to 5.0 pg/cell. These results placed DNA/cell values of Acanthamoeba slightly lower than DNA/cell values of other eucaryotic cells and much lower than Amoeba proteus values. These results indicate that FMF may be a useful adjunct in distinguishing Acanthamoeba cells from either eucaryotic cells or some other amoeba. However, differences in DNA/cell between species of Acanthamoeba are small and would not be useful in identification of species.

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

    OpenAIRE

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

    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. ...

  13. Potential role of DNA-dependent protein kinase in cellular resistance to ionizing radiation

    Institute of Scientific and Technical Information of China (English)

    LI Ning; ZHANG Hong; WANG Yanling; WANG Xiaohu; HAO Jifang

    2009-01-01

    In this paper, we study the ability of DNA-PK-deficient (M059J) and -proficient (M059K) cells to undergo the rate of cellular proliferation, cell cycle distribution and apoptosis after 10 Gy X-ray irradiation, and the role of DNA-PK in radiosensitivity. The results showed that M059J cells exhibited hyper-radiosensitivity compared with M059K cells. A strong G2 phase arrest was observed in M059J cells post irradiation. Significant accumulation in the G2 phase in M059J cells was accompanied by apoptosis at 12 h. Altogether, the data suggested that DNA-PK may have two roles in mammalian cells after DNA damage, a role in DNA DSB repair and a second role in DNA-damaged cells to traverse a G2 checkpoint, by which DNA-PK may affect cellular sensitivity to ionizing radiation.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Xurui Zhang

    Full Text Available 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.

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

    Science.gov (United States)

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

    2015-04-01

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

  17. Caveolar vesicles generate DNA damage and perpetuate cellular aging

    Institute of Scientific and Technical Information of China (English)

    Keith Wheaton

    2011-01-01

    @@ The replicative limit of human fibroblasts has long provided a model to assess the molecular mechanisms underlying cellular aging [1].In culture, fibroblasts which reach the end of their proliferative lifespan acquire profound molecular changes that limit their response to growth factors, and cause permanent exit from the cell cycle [2].

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

    Science.gov (United States)

    Taylor, Duncan

    2016-09-01

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

  19. Analytical Devices Based on Direct Synthesis of DNA on Paper.

    Science.gov (United States)

    Glavan, Ana C; Niu, Jia; Chen, Zhen; Güder, Firat; Cheng, Chao-Min; Liu, David; Whitesides, George M

    2016-01-01

    This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

  20. Photoenzyme probes of photodamage to cells and cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, B. M.

    1979-01-01

    Development of photoenzyme probes for detection of ultraviolet damage to cells and DNA is reviewed with special emphasis on a process using polyethylene glycol to induce cell fusion. Polyethylene glycol is easy to obtain and handle, is gentle to the cells and does not induce latent or productive virus infection; therefore, it may be a general method for insertion of exogenous enzymes into mammalian cells. (PCS)

  1. Replication stress activates DNA repair synthesis in mitosis

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A;

    2015-01-01

    mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest...

  2. Inhibition of DNA-dependent RNA synthesis by 8-methoxypsoralen.

    Science.gov (United States)

    Gniazdowski, M; Czyz, M; Wilmańska, D; Studzian, K; Frasunek, M; Płucienniczak, A; Szmigiero, L

    1988-09-01

    The effect of the photobinding of 8-methoxypsoralen to phage T7 DNA on different steps of RNA synthesis in vitro was assayed. Total RNA synthesis is reduced to a few percent and the transcript size is decreased, as shown by means of gel filtration on a Sepharose 4B column when DNA of the adduct content of six drug molecules per 10(3) nucleotides is used. The initiation of RNA chains seems to be less affected, as inferred from an abortive initiation assay. Synthesis of pppApU on DNA of the same adduct content is inhibited to 34% of the corresponding controls, while the overall RNA synthesis is inhibited to 6%. The amount of the enzyme needed for maximal retention of DNA, the kinetics of its binding and the decay of the polymerase-DNA complex at high ionic strength (or on decrease of the temperature) are similar with DNA either irradiated in the absence of the drug or DNA bearing six 8-methoxypsoralen molecules per 10(3) nucleotides. It is concluded from this study that 8-methoxypsoralen partially inhibits initiation and blocks movement of RNA polymerase along the template, inducing premature termination. It does not appear to influence the binding of the enzyme to DNA. PMID:3048406

  3. The Yin-Yang of DNA Damage Response: Roles in Tumorigenesis and Cellular Senescence

    Directory of Open Access Journals (Sweden)

    Sang Soo Kim

    2013-01-01

    Full Text Available Senescent cells are relatively stable, lacking proliferation capacity yet retaining metabolic activity. In contrast, cancer cells are rather invasive and devastating, with uncontrolled proliferative capacity and resistance to cell death signals. Although tumorigenesis and cellular senescence are seemingly opposite pathological events, they are actually driven by a unified mechanism: DNA damage. Integrity of the DNA damage response (DDR network can impose a tumorigenesis barrier by navigating abnormal cells to cellular senescence. Compromise of DDR, possibly due to the inactivation of DDR components, may prevent cellular senescence but at the expense of tumor formation. Here we provide an overview of the fundamental role of DDR in tumorigenesis and cellular senescence, under the light of the Yin-Yang concept of Chinese philosophy. Emphasis is placed on discussing DDR outcome in the light of in vivo models. This information is critical as it can help make better decisions for clinical treatments of cancer patients.

  4. Cellular aging of mitochondrial DNA-depleted cells

    International Nuclear Information System (INIS)

    We have reported that mitochondrial DNA-depleted ρ0 cells are resistant to cell death. Because aged cells have frequent mitochondrial DNA mutations, the resistance of ρ0 cells against cell death might be related to the apoptosis resistance of aged cells and frequent development of cancers in aged individuals. We studied if ρ0 cells have features simulating aged cells. SK-Hep1 hepatoma ρ0 cells showed typical morphology associated with aging such as increased size and elongated appearance. They had increased senescence-associated β-Gal activity, lipofuscin pigment, and plasminogen activator inhibitor-1 expression. Consistent with their decreased proliferation, the expression of mitotic cyclins was decreased and that of cdk inhibitors was increased. Rb hypophosphorylation and decreased telomerase activity were also noted. Features simulating aged cells were also observed in MDA-MB-435 ρ0 cells. These results support the mitochondrial theory of aging, and suggest that ρ0 cells could serve as an in vitro model for aged cells

  5. Divergent synthesis and identification of the cellular targets of deoxyelephantopins

    Science.gov (United States)

    Lagoutte, Roman; Serba, Christelle; Abegg, Daniel; Hoch, Dominic G.; Adibekian, Alexander; Winssinger, Nicolas

    2016-08-01

    Herbal extracts containing sesquiterpene lactones have been extensively used in traditional medicine and are known to be rich in α,β-unsaturated functionalities that can covalently engage target proteins. Here we report synthetic methodologies to access analogues of deoxyelephantopin, a sesquiterpene lactone with anticancer properties. Using alkyne-tagged cellular probes and quantitative proteomics analysis, we identified several cellular targets of deoxyelephantopin. We further demonstrate that deoxyelephantopin antagonizes PPARγ activity in situ via covalent engagement of a cysteine residue in the zinc-finger motif of this nuclear receptor.

  6. DNA display III. Solid-phase organic synthesis on unprotected DNA.

    Directory of Open Access Journals (Sweden)

    David R Halpin

    2004-07-01

    Full Text Available DNA-directed synthesis represents a powerful new tool for molecular discovery. Its ultimate utility, however, hinges upon the diversity of chemical reactions that can be executed in the presence of unprotected DNA. We present a solid-phase reaction format that makes possible the use of standard organic reaction conditions and common reagents to facilitate chemical transformations on unprotected DNA supports. We demonstrate the feasibility of this strategy by comprehensively adapting solid-phase 9-fluorenylmethyoxycarbonyl-based peptide synthesis to be DNA-compatible, and we describe a set of tools for the adaptation of other chemistries. Efficient peptide coupling to DNA was observed for all 33 amino acids tested, and polypeptides as long as 12 amino acids were synthesized on DNA supports. Beyond the direct implications for synthesis of peptide-DNA conjugates, the methods described offer a general strategy for organic synthesis on unprotected DNA. Their employment can facilitate the generation of chemically diverse DNA-encoded molecular populations amenable to in vitro evolution and genetic manipulation.

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

    Directory of Open Access Journals (Sweden)

    Juan Cristóbal Conde-Pérezprina

    2012-01-01

    Full Text Available 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.

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

    OpenAIRE

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

    2015-01-01

    Background 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. Results We perform whole genome bisulfite sequencing on a set of unmatched sampl...

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

    Directory of Open Access Journals (Sweden)

    Jitesh Neupane

    2015-11-01

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

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

    Science.gov (United States)

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

    2015-11-17

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

  11. Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ.

    Science.gov (United States)

    Copeland, William C; Kasiviswanathan, Rajesh; Longley, Matthew J

    2016-01-01

    Mitochondrial DNA is replicated by the nuclear-encoded DNA polymerase γ (pol γ) which is composed of a single 140 kDa catalytic subunit and a dimeric 55 kDa accessory subunit. Mitochondrial DNA is vulnerable to various forms of damage, including several types of oxidative lesions, UV-induced photoproducts, chemical adducts from environmental sources, as well as alkylation and inter-strand cross-links from chemotherapy agents. Although many of these lesions block DNA replication, pol γ can bypass some lesions by nucleotide incorporation opposite a template lesion and further extension of the DNA primer past the lesion. This process of translesion synthesis (TLS) by pol γ can occur in either an error-free or an error-prone manner. Assessment of TLS requires extensive analysis of oligonucleotide substrates and replication products by denaturing polyacrylamide sequencing gels. This chapter presents protocols for the analysis of translesion DNA synthesis.

  12. Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ

    Science.gov (United States)

    Copeland, William C.; Kasiviswanathan, Rajesh; Longley, Matthew J.

    2016-01-01

    Summary Mitochondrial DNA is replicated by the nuclear encoded DNA polymerase γ (pol γ) which is composed of a single 140 kDa catalytic subunit and a dimeric 55 kDa accessory subunit. Mitochondrial DNA is vulnerable to various forms of damage, including several types of oxidative lesions, UV-induced photoproducts, chemical adducts from environmental sources, as well as alkylation and inter-strand crosslinks from chemotherapy agents. Although many of these lesions block DNA replication, Pol γ can bypass some lesions by nucleotide incorporation opposite a template lesion and further extension of the DNA primer past the lesion. This process of translesion synthesis (TLS) by Pol γ can occur in either an error-free or an error-prone manner. Assessment of TLS requires extensive analysis of oligonucleotide substrates and replication products by denaturing polyacrylamide sequencing gels. This chapter presents protocols for the analysis of translesion DNA synthesis. PMID:26530671

  13. The relationship between cellular radiosensitivity and radiation-induced DNA damage measured by the comet assay

    International Nuclear Information System (INIS)

    The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by γ-irradiation was examined in three kinds of cells, Chinese hamster ovary fibroblast CHO-K1, human melanoma HMV-II and mouse leukemia L5178Y. Cell survival was determined by a clonogenic assay. The induction and rejoining of DNA strand breaks induced by radiation were measured by the alkaline and neutral comet assay. L5178Y cells were the most radiosensitive, while CHO-K1 cells and HMV-II cells were radioresistant. There was an inverse relationship between the survival fraction at 2 Gy (SF2) and the yield of initial DNA strand breaks per unit dose under the alkaline condition of the comet assay, and also a relationship between SF2 and the residual DNA strand breaks (for 4 hr after irradiation) under the neutral condition for the comet assay, the latter being generally considered to be relative to cellular radiosensitivity. In the present analysis, it was considered that the alkaline condition for the comet assay was optimal for evaluating the initial DNA strand breaks, while the neutral condition was optimal for evaluating the residual DNA strand breaks. Since the comet assay is simpler and more rapid than other methods for detecting radiation-induced DNA damage, this assay appears to be a useful predictive assay for evaluating cellular clonogenic radiosensitivity of tumor cells. (author)

  14. Experimental genomics: The application of DNA microarrays in cellular and molecular biology studies

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The genome sequence information in combination with DNA microarrays promises to revolutionize the way of cellular and molecular biological research by allowing complex mixtures of RNA and DNA to interrogated in a parallel and quant itative fashion. DNA microarrays can be used to measure levels of gene expressio n for tens of thousands of gene simultaneously and take advantage of all availab le sequence information for experimental design and data interpretation in pursu it of biological understanding. Recent progress in experimental genomics allows DNA microarrays not simply to provide a catalogue of all the genes and informati on about their function, but to understand how the components work together to comprise functioning cells and organisms. This brief review gives a survey of DNA microarrays technology and its applications in genome and gene function analysis, gene expression studies, biological signal and defense system, cell cyclereg ulation, mechanism of transcriptional regulation, proteomics, and the functional ity of food component.

  15. Cellular DNA repair cofactors affecting hepatitis B virus infection and replication

    Institute of Scientific and Technical Information of China (English)

    Fan Zhao; Ning-Bo Hou; Ting Song; Xiang He; Zi-Rui Zheng; Qing-Jun Ma; Li Li; Yan-Hong Zhang; Hui Zhong

    2008-01-01

    AIM: To investigate whether hepatitis B virus (HBV)infection activates DNA damage response and DNA repair cofactors inhibit HBV infection and replication.METHODS: Human hepatocyte cell line HL7702 was studied. Immunoblotting was performed to test the expression of ataxia telangiectasia-mutated (ATM)-Rad3-related protein (ATR), p21 and the level of phosphorylation of Chk1, p53, H2AX, ATM in HBV-infected or non-infected-cells. Special short RNAi oligos was transfected to induce transient ATR knockdown in HL7702. ATR-ATM chemical inhibitors caffeine (CF) and theophylline (TP), or Chk1 inhibitor 7-hydroxystaurosporine (UCN01) was studied to determine whether they suppress cellular DNA damage response and MG132 inhibits proteasome.RESULTS: The ATR checkpoint pathway, responding to single-strand breaks in DNA, was activated in response to HBV infection. ATR knockdown cells decreased the HBV DNA yields, implying that HBV infection and replication could activate and exploit the activated DNA damage response. CF/TP or UCN01 reduced the HBV DNA yield by 70% and 80%, respectively. HBV abrogated the ATR-dependent DNA damage signaling pathway by degrading p21, and introduction of the p21 protein before HBV infection reduced the HBV DNA yield. Consistent with this result, p21 accumulation after MG132 treatment also sharply decreased the HBV DNA yield.CONCLUSION: HBV infection can be treated with therapeutic approaches targeting host cell proteins by inhibiting a cellular gene required for HBV replication or by restoring a response abrogated by HBV, thus providing a potential approach to the prevention and treatrnent of HBV infection.

  16. The involvement of XPC protein in the cisplatin DNA damaging treatment-mediated cellular response

    Institute of Scientific and Technical Information of China (English)

    Gan WANG; Alan DOMBKOWSKI; Lynn CHUANG; Xiao Xin S XU

    2004-01-01

    Recognition of DNA damage is a critical step for DNA damage-mediated cellular response. XPC is an important DNA damage recognition protein involved in nucleotide excision repair (NER). We have studied the XPC protein in cisplatin DNA damaging treatment-mediated cellular response. Comparison of the microarray data from both normal and XPCdefective human fibroblasts identified 861 XPC-responsive genes in the cisplatin treatment (with minimum fold change≥1.5).The cell cycle and cell proliferation-related genes are the most affected genes by the XPC defect in the treatment. Many other cellular function genes, especially the DNA repair and signal transduction-related genes, were also affected by the XPC defect in the treatment. To validate the microarray data, the transcription levels of some microarray-identified genes were also determined by an RT-PCR based real time PCR assay. The real time PCR results are consistent with the microarray data for most of the tested genes, indicating the reliability of the microarray data. To further validate the microarray data, the cisplatin treatment-mediated caspase-3 activation was also determined. The Western blot hybridization results indicate that the XPC defect greatly attenuates the cisplatin treatment-mediated Caspase-3 activation. We elucidated the role of p53 protein in the XPC protein DNA damage recognition-mediated signaling process. The XPC defect reduces the cisplatin treatment-mediated p53 response. These results suggest that the XPC protein plays an important role in the cisplatin treatment-mediated cellular response. It may also suggest a possible mechanism of cancer cell drug resistance.

  17. D-ribose inhibits DNA repair synthesis in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Zunica, G.; Marini, M.; Brunelli, M.A.; Chiricolo, M.; Franceschi, C.

    1986-07-31

    D-ribose is cytotoxic for quiescent human lymphocytes and severely inhibits their PHA-induced proliferation at concentrations (25-50 mM) at which other simple sugars are ineffective. In order to explain these effects, DNA repair synthesis was evaluated in PHA-stimulated human lymphocytes treated with hydroxyurea and irradiated. D-ribose, in contrast to other reducing sugars, did not induce repair synthesis and therefore did not apparently damage DNA in a direct way, although it markedly inhibited gamma ray-induced repair. Taking into account that lymphocytes must rejoin physiologically-formed DNA strand breaks in order to enter the cell cycle, we suggest that D-ribose exerts its cytotoxic activity by interfering with metabolic pathways critical for the repair of DNA breaks.

  18. DNA-synthesis inhibition and repair DNA-synthesis in CHO Ade- C cells: An alternative approach to genotoxicity testing

    International Nuclear Information System (INIS)

    We describe an alternative assay to determine genotoxicity. Its main feature is that it combines two measures in a single experiment; the inhibition of replicative DNA synthesis together with the stimulation of DNA repair. We show that, in tests of four different genotoxic agents, the assay gives results that are entirely consistent with what is known about the mode of action of these agents. In addition, we have demonstrated that chemical carcinogens requiring metabolic activation can be examined using a standard procedure of incubation with a microsomal activating fraction. We consider the combined assay for DNA synthesis inhibition and repair synthesis to be a useful way for the rapid pre-screening of chemicals suspected of genotoxic activity on the level of mammalian cells. (author)

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

    Science.gov (United States)

    Wolfe, Annie; Phipps, Kara; Weitao, Tao

    2014-01-01

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

  20. New approaches to detect 8-hydroxyguanine in {gamma}-irradiated cellular DNA

    Energy Technology Data Exchange (ETDEWEB)

    Mei, Nan; Tamae, Kazuyoshi; Hirano, Takeshi; Kasai, Hiroshi [University of Occupational and Environmental Health, Institute of Industrial Ecological Sciences, Kitakyushu, Fukuoka (Japan); Kunugita, Naoki [University of Occupational and Environmental Health, School of Health Sciences, Kitakyushu, Fukuoka (Japan)

    2003-07-01

    This report describes an assay to detect 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) in cellular DNA by modification of enzyme treatment after DNA extraction, using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). This modification greatly reduces the measured background level of 8-hydroxyguanine (8-OH-Gua) in DNA, and improves the HPLC-ECD sensitivity to measure oxidative DNA damage. The 8-OH-Gua value in the DNA was expressed by the ratio of 8-OH-dGMP to deoxycytidine 5'-monophosphate (dCMP). Background level of 8-OH-Gua in DNA under our conditions was several times lower than that by a previous method. The human lung carcinoma cells (A549) were exposed to {gamma}-rays of 20-100 Gy. A dose-dependent increase in oxidative DNA damage of 8-OH-Gua was observed. Furthermore, using commercial FITC-kit of an immunohistochemical type procedure, 8-OH-Gua was clearly detected in A549 cells and the fluorescence intensity of cells with oxidative DNA damage increased with the doses of {gamma}-irradiation. Using an 8-OH-Gua repair activity assay, we also found that {gamma}-rays decreased the repair enzyme activity. We conclude that the 8-OH-Gua level in human cellular DNA increases partly by the generation of reactive oxygen species (ROS) and partly by the inhibition of repair activity for 8-OH-Gua. (author)

  1. CONSECUTIVE IMMUNIZATION WITH RECOMBINANT FOWLPOX VIRUS AND PLASMID DNA FOR ENHANCING CELLULAR AND HUMORAL IMMUNITY

    Institute of Scientific and Technical Information of China (English)

    罗坤; 金宁一; 郭志儒; 秦云龙; 郭炎; 方厚华; 安汝国; 殷震

    2001-01-01

    To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD4+ T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.

  2. DNA and RNA induced enantioselectivity in chemical synthesis

    NARCIS (Netherlands)

    Roelfes, Gerard

    2007-01-01

    One of the hallmarks of DNA and RNA structures is their elegant chirality. Using these chiral structures to induce enantioselectivity in chemical synthesis is as enticing as it is challenging. In recent years, three general approaches have been developed to achieve this, including chirality transfer

  3. CELLULAR RESPONSES TO DNA DAMAGE AND ONCOGENESIS BY THE p53 AND pRb/E2F PATHWAYS

    OpenAIRE

    Elza Ibrahim Auerkari; Ismu Suharsono Suwelo; Achmad Tjarta; Santoso Cornain; T. W. Rahardjo; Eto, K; Ikeda, M.A

    2015-01-01

    Cellular responses to stress including DNA damage, show multiple options involving the mechanisms of growth arrest. DNA repair and programmed cell death or apoptosis. Failures in these mechanisms can result in oncogenesis or accelerated senescence. Much of the response is coordinated by p53, a nuclear phosphoprotein with a central role in the defences against physical, chemical and pathogenic agents which challenge the DNA integrity. The p53 pathways for mobilising the cellular defences are l...

  4. Novel pattern of post-γ ray de novo DNA synthesis in a radioresistant human strain

    International Nuclear Information System (INIS)

    Enhanced resistance to radiation cytotoxicity in a fibroblast strain from an afflicted member of a Li-Fraumeni syndrome family may be largely ascribable to a change in the pattern of DNA replicative synthesis following γ ray exposure. That is, the extent of the initial radiogenic inhibition of replicative synthesis and the time interval before its subsequent recovery were both found to be greater in radioresistant (RR) compared to normal cells. In addition, the post-recovery replication rates in the RR cells were both higher and longer lasting than those in the control cells. A similar differential pattern was also seen following treatment with 4NQO, another DNA-damaging agent to which this RR strain displays enhanced resistance. Moreover, several conventional DNA repair assays indicated that the RR cells repair radiogenic damage at normal rates. The authors therefore suggest that the increased inhibition and prolonged lag in resumption of replicative synthesis seen in the RR strain upon exposure to certain genotoxic agents may enhance cellular recovery by ''buying additional time'' for processing of potentially lethal lesions

  5. Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers

    Directory of Open Access Journals (Sweden)

    Jana eSlyskova

    2014-05-01

    Full Text Available Thousands of DNA lesions are estimated to occur in each cell every day and almost all are recognized and repaired. DNA repair is an essential system that prevents accumulation of mutations which can lead to serious cellular malfunctions. Phenotypic evaluation of DNA repair activity of individuals is a relatively new approach. Methods to assess base and nucleotide excision repair pathways (BER and NER in peripheral blood cells based on modified comet assay protocols have been widely applied in human epidemiological studies. These provided some interesting observations of individual DNA repair activity being suppressed among cancer patients. However, extension of these results to cancer target tissues requires a different approach. Here we describe the evaluation of BER and NER activities in extracts from deep-frozen colon biopsies using an upgraded version of the in vitro comet-based DNA repair assay in which twelve reactions on one microscope slide can be performed. The aim of this report is to provide a detailed, easy-to-follow protocol together with results of optimization experiments. Additionally, results obtained by functional assays were analysed in the context of other cellular biomarkers, namely single nucleotide polymorphisms and gene expressions. We have shown that measuring DNA repair activity is not easily replaceable by genomic or transcriptomic approaches, but should be applied with the latter techniques in a complementary manner. The ability to measure DNA repair directly in cancer target tissues might finally answer questions about the tissue-specificity of DNA repair processes and their real involvement in the process of carcinogenesis.

  6. Using a cDNA microarray to study cellular gene expression altered by Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    徐永忠; 谢建平; 李瑶; 乐军; 陈建平; 淳于利娟; 王洪海

    2003-01-01

    Objective To examine the global effects of Mycobacterium tuberculosis (M.tuberculosis) infection on macrophages. Methods The gene expression profiling of macrophage U937, in response to infection with M.tuberculosis H37Ra, was monitored using a high-density cDNA microarray. Results M.tuberculosis infection caused 463 differentially expressed genes, of which 366 genes are known genes registered in the Gene Bank. These genes function in various cellular processes including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity as well as a variety of cellular metabolic pathways, and may play key roles in M.tuberculosis infection and intracellular survival. Conclusions M.tuberculosis infection alters the expression of host-cell genes, and these genes will provide a foundation for understanding the infection process of M.tuberculosis. The cDNA microarray is a powerful tool for studying pathogen-host cell interaction.

  7. Charged particle-induced modification of cellular genomic DNA and gene expression level

    International Nuclear Information System (INIS)

    Aim of this study is to understand cellular and molecular nature of cancer cells survived for long term after charged particle therapy. During the period of 1st year, clonogenic sensitivity of various cancer cell lines against charged particles was investigated by two experimental strategies. Firstly, human glioblastoma cell line, Becker, was investigated for the phenotypic changes after long term survival period (3 weeks). Especially, the cells were revealed to be sensitized toward secondary exposure of charged particles in a way of primary dose-dependence. However, this tendency was clearly eliminated when cells were treated by 5-azacytidine, a DNA methylation inhibitor, before the primary exposure. Thus, epigenetic regulations of cellular genomic DNA were supposed to play important roles in the radiation sensitivity changes of the long-term survived cells. In the second approach, mouse cancer cell line analysis in the presence of 5-azacytidine revealed epigenetic heterogeneity of charged particle sensitivity within the cell population. (author)

  8. At the intersection of non-coding transcription, DNA repair, chromatin structure, and cellular senescence

    Directory of Open Access Journals (Sweden)

    Ryosuke eOhsawa

    2013-07-01

    Full Text Available It is well accepted that non-coding RNAs play a critical role in regulating gene expression. Recent paradigm-setting studies are now revealing that non-coding RNAs, other than microRNAs, also play intriguing roles in the maintenance of chromatin structure, in the DNA damage response, and in adult human stem cell aging. In this review, we will discuss the complex inter-dependent relationships among non-coding RNA transcription, maintenance of genomic stability, chromatin structure and adult stem cell senescence. DNA damage-induced non-coding RNAs transcribed in the vicinity of the DNA break regulate recruitment of the DNA damage machinery and DNA repair efficiency. We will discuss the correlation between non-coding RNAs and DNA damage repair efficiency and the potential role of changing chromatin structures around double-strand break sites. On the other hand, induction of non-coding RNA transcription from the repetitive Alu elements occurs during human stem cell aging and hinders efficient DNA repair causing entry into senescence. We will discuss how this fine balance between transcription and genomic instability may be regulated by the dramatic changes to chromatin structure that accompany cellular senescence.

  9. DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis

    Science.gov (United States)

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J.; Xing, Chao; Wang, Richard C.; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K.; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R.; Burstein, Ezra

    2016-01-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  10. Synthesis of furan-based DNA binders and their interaction with DNA

    International Nuclear Information System (INIS)

    In recent years, many substances, based on naturally occurring DNA-binding molecules have been developed for the use in cancer therapy and as virostatica. Most of these substances are binding specifically to A-T rich sequences in the DNA minor groove. Neutral and positively charged DNA-binders are known. BNCT is most effective, which the boron is directly located in the cellular nucleus, so that the intercation with thermal neutrons can directly damage the DNA. To reach this aim, we have connected ammonioundecahydrododecaborate(1-) to DNA-binding structures such as 2,5-bis(4-formylphenyl)furan via a Schiff-Base reaction followed by a reduction of the imine to a secondary amine. In a following step the amine can be alkylated to insert positive charges to prevent repulsion between the compounds and the negatively charged sugar-phosphate-backbone of the DNA. (author)

  11. Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

    DEFF Research Database (Denmark)

    Skov, Joan; Bryld, Torsten; Lindegaard, Dorthe;

    2011-01-01

    We report the synthesis of two C4'-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4'-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization...... multiple sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both...... modifications are tolerated in DNA:RNA hybrids but leave their melting temperatures virtually unaffected. Fluorescence data indicate that the pyrene moiety is residing outside the helix. The available data suggest that the DNA discrimination is due to (i) the positive charge of the piperazino ring having a...

  12. Involvement of DNA polymerase δ in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

    International Nuclear Information System (INIS)

    DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase δ as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, the authors describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, α or δ. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase α several hundred times more strongly than it inhibits DNA polymerase δ. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase δ. It appears that repair synthesis at late time after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase δ

  13. Simultaneous disruption of two DNA polymerases, Polη and Polζ, in Avian DT40 cells unmasks the role of Polη in cellular response to various DNA lesions.

    Directory of Open Access Journals (Sweden)

    Kouji Hirota

    2010-10-01

    Full Text Available Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR and translesion DNA synthesis (TLS. TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V, and DNA polymerase ζ by generating POLη(-/-/POLζ(-/- cells from the chicken DT40 cell line. POLζ(-/- cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Polη plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLη(-/-/POLζ(-/- cells shows that, unexpectedly, the loss of Polη significantly rescued all mutant phenotypes of POLζ(-/- cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Polη contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells.

  14. Effect of X irradiation on a heterotransplanted human colonic carcinoma before and after a change in the cellular DNA content

    DEFF Research Database (Denmark)

    Spang-Thomsen, M; Vindeløv, L L; Nielsen, A

    1983-01-01

    analysis (FCM), were compared to results obtained with the tumor prior to the evolutionary event. The results showed that the radiation effects on growth rate and on cell kinetics had changed after the change in cellular DNA content. In the hyperpentaploid tumor the irradiation had no effect......A spontaneous change of cellular DNA content occurred in a hyperdiploid human colonic carcinoma grown in nude mice. After the change to hyperpentaploidy the tumor was exposed to single-dose X irradiation, and the effects on growth curves and on the cell cycle, determined by flow cytometric DNA...... to provide rapid information on cellular synchronization and on redistribution time....

  15. Sequencing of chloroplast genome using whole cellular DNA and Solexa sequencing technology

    Directory of Open Access Journals (Sweden)

    Jian eWu

    2012-11-01

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

  16. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells.

    Science.gov (United States)

    Maddocks, Oliver D K; Labuschagne, Christiaan F; Adams, Peter D; Vousden, Karen H

    2016-01-21

    Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids.

  17. Enhancing repair of radiation-induced strand breaks in cellular DNA as a radiotherapeutic potential

    International Nuclear Information System (INIS)

    Protection of mammalian organisms including man from deleterious effects of ionizing radiation is of paramount importance and development of effective approaches to combat radiation damages using non-toxic radioprotectors is of considerable interest for defence, nuclear industries, radiation accidents, space travels, etc., besides the protection of normal tissues during radiotherapy of tumours. Many synthetic as well as natural compounds have been investigated in the recent past for their efficacy to protect the biological systems from radiation induced damages. They include sulfhydryl compounds, antioxidants, plant extracts, immune-modulators, and other agents. However, the inherent toxicity of many of the synthetic agents at the effective radio-protective concentration warranted further search for safer and more effective radio-protectors. In this context, therapeutic radioprotectors which are effective on post irradiation administration are of special relevance. One of the property that can be applied while screening for such radiation protective therapeutics is their ability to enhance repair of radiation-induced lesions in cellular DNA in terms of cellular repair index based on the parameters of the DNA following comet assay. Post irradiation administration of some natural and synthetic agents have shown their potential to enhance repair of radiation-induced strand breaks in cellular DNA in mice. These include phytoceuticals such as gallic acid, sesamol etc., extracts of medicinal plants such as Andrographis panniculata, and a few synthetic compounds such as tocopherol-mono-glucoside. The talk will give an overview of the work on DNA repair enhancement by a few natural and synthetic agents. (author)

  18. Synthesis of cellular organelles containing nano-magnets stunts growth of magnetotactic bacteria.

    Science.gov (United States)

    Naresh, Mohit; Hasija, Vivek; Sharma, Megha; Mittal, Aditya

    2010-07-01

    Magnetotactic bacteria are unique prokaryotes possessing the feature of cellular organelles called magnetosomes (membrane bound 40-50 nm vesicles entrapping a magnetic nano-crystal of magnetite or greigite). The obvious energetic impact of sophisticated eukaryotic-like membrane-bound organelle assembly on a presumably simpler prokaryotic system is not addressed in literature. In this work, while presenting evidence of direct coupling of carbon source consumption to synthesis of magnetosomes, we provide the first experimentally derived estimate of energy for organelle synthesis by Magnetospirillum gryphiswaldense as approximately 5 nJoules per magnetosome. Considering our estimate of approximately 0.2 microJoules per bacterial cell as the energy required for growth, we show that the energetic load of organelle synthesis results in stunting of cell growth. We also show that removal of soluble iron or sequestration by exogenous compounds in the bacterial cell cultures reverses the impact of the excess metabolic load exerted during magnetosomal synthesis. Thus, by taking advantage of the magnetotactic bacterial system we present the first experimental evidence for the presumed energy consumption during assembly of naturally occurring sub-100 nm intra-cellular organelles. PMID:21128392

  19. Tunable Hydrophobicity in DNA Micelles : Design, Synthesis, and Characterization of a New Family of DNA Amphiphiles

    NARCIS (Netherlands)

    Anaya, Milena; Kwak, Minseok; Musser, Andrew J.; Muellen, Klaus; Herrmann, Andreas; Müllen, Klaus

    2010-01-01

    This work describes the synthesis and characterization of a new family of DNA amphiphiles containing modified nucleobases. The hydrophobicity was imparted by the introduction of a dodec-1-yne chain at the 5-position of the uracil base, which allowed precise and simple tuning of the hydrophobic prope

  20. Design and synthesis of threading intercalators to target DNA.

    Science.gov (United States)

    Howell, Lesley A; Gulam, Rosul; Mueller, Anja; O'Connell, Maria A; Searcey, Mark

    2010-12-01

    Threading intercalators are high affinity DNA binding agents that bind by inserting a chromophore into the duplex and locating one group in each groove. The first threading intercalators that can be conjugated to acids, sulfonic acids and peptides to target them to duplex DNA are described, based upon the well studied acridine-3- or 4-carboxamides. Cellular uptake of the parent acridine is rapid and it can be visualized in the nucleus of cells. Both the parent compounds and their conjugates maintain antitumor activity.

  1. The inhibition of DNA synthesis in vitamin-E-depleted lymphosarcoma cells by X-rays and cytostatics

    International Nuclear Information System (INIS)

    Since there is evidence that the lipid-soluble anti-oxidant vitamin E may protect the polyunsaturated fatty acids of cellular membranes from free-radical attack, a shortage of vitamin E should increase the radiosensitivity of the membranes. An investigation has been carried out into the in vivo incorporation of 3H-thymidine in spleen lymphosarcomas growing in X-irradiated (500 rad) normal and vitamin-E-deficient C57BL mice. The results showed that DNA synthesis was significantly more radiosensitive in the vitamin-E-depleted lymphosarcoma cells, and that the effect was most pronounced 3 to 5 hours post irradiation. Studied of the effects of intraperitoneal injections of the cancer therapeutic agents 1-β-D-Arabinofuranosylcytosine (ARA-C) and Adriamycin on the inhibition of thymidine incorporation into DNA showed no significant differences between normal and vitamin-E deficient lymphosarcoma cells. The inhibition of DNA synthesis by these drugs does not involve free radicals. The vitamin E deficient tumour cells had a higher lipid peroxidation rate at 370C (0.5 +- 0.1 nmoles/mg protein per hour) than the normal cells (0.2 +- 0.1 nmoles/mg protein per hour). The higher lipid peroxidation capacity corresponded with the enhanced radiosensitivity. The results provide indirect evidence for the involvement of cellular membranes in the mechanism of radiation-induced inhibition of DNA synthesis. (U.K.)

  2. Synthesis, DNA binding and cytotoxic evaluation of aminoquinoline scaffolds

    Indian Academy of Sciences (India)

    Gopal Senthil Kumar; Mohamed Ashraf Ali; Tan Soo Choon; Rajendra Prasad Karnam Jayarampillai

    2016-03-01

    An effortless synthetic route has been developed for the synthesis of a new class of aminoquinoline substituted isoindolin-1,3-diones from regio-isomerical hydrazinylquinolines with phthalic anhydride in presence of Eaton’s reagent. DNA binding studies of selected isomeric compounds showed interaction withDNA via intercalation mode with higher binding affinity of 4-substituted quinolines rather than 2-substituted counterparts. Further, all compounds were screened for cytotoxic activity against three human cancer cell lines,among them compound 2c outranged standard doxorubicin against CCRF-CEM cell line.

  3. The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

    Science.gov (United States)

    Ramachandran, Aparna; Nandakumar, Divya; Deshpande, Aishwarya P; Lucas, Thomas P; R-Bhojappa, Ramanagouda; Tang, Guo-Qing; Raney, Kevin; Yin, Y Whitney; Patel, Smita S

    2016-08-01

    Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA. PMID:27311715

  4. Evaluation of cellular responses for a chimeric HBsAg-HCV core DNA vaccine in BALB/c mice

    Directory of Open Access Journals (Sweden)

    Maryam Yazdanian

    2015-01-01

    Conclusion: Fusion of HBsAg to HCVcp in the context of a DNA vaccine modality could augment Th1-oriented cellular and CTL responses toward a protective epitope, comparable to that of HCVcp (subunit HCV vaccine immunization.

  5. Toxicity DNA damage and inhibition of DNA repair synthesis in human melanoma cells by concentrated sunlight

    International Nuclear Information System (INIS)

    A water lens was used to focus solar radiation, giving an 8-fold concentration of the total spectrum and a cytocidal flux similar to that of laboratory UV sources. Survival curves for human melanoma cells were similar for sunlight and 254 nm UV. An xeroderma pigmentosum lymphoblastoid line was equally sensitive to both agents and human cell lines sensitive to ionizing radiation (lymphoblastoid lines), crosslinking agents or monofunctional alkylating agents (melanoma lines) had the same 254 nm UV and solar survival responses as appropriate control lines. Two melanoma sublines derived separately by 16 cycles of treatment with sunlight or 254 nm UV were crossresistant to both agents. In one melanoma cell line, DNA strand breaks and DNA protein crosslinking were induced in melanoma cells by sunlight but pyrimidine dimers and DNA interstrand crosslinking could not be detected. The solar fluence response of DNA repair synthesis was much less than that from equitoxic 254 nm UV, reaching a maximum near the D0 value and then declining; but semiconservative DNA synthesis remained high. These effects were not due to changes in thymidine pool sizes. Solar exposure did not have a major effect on 254 nm UV-induced repair synthesis. (author)

  6. Different patterns of bacterial DNA synthesis during postantibiotic effect.

    OpenAIRE

    Gottfredsson, M; Erlendsdóttir, H; Gudmundsson, A.; Gudmundsson, S.

    1995-01-01

    Studies on bacterial metabolism during the postantibiotic effect (PAE) period are limited but might provide insight into the nature of the PAE. We evaluated the rate of DNA synthesis in bacteria during the PAE period after a 1-h exposure of organisms in the logarithmic growth phase to various antibiotics. Staphylococcus aureus ATCC 25923 was exposed to vancomycin, dicloxacillin, rifampin, and ciprofloxacin; Escherichia coli ATCC 25922 was exposed to gentamicin, tobramycin, rifampin, imipenem,...

  7. A germline polymorphism of DNA polymerase beta induces genomic instability and cellular transformation.

    Directory of Open Access Journals (Sweden)

    Jennifer Yamtich

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

  8. Dimethylarsenic acid damages cellular DNA and inhibits gap junctional intercellular communication between human skin fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    GuoXB; DengFR

    2002-01-01

    Although arsenic is identified as a human carcinogen,there is currently no accepted mechanism for its action or an established animal model for evaluating the carcinogenic activity of arsenic.To elucidate the mechanism of arsenic arcinogenesis,we investigated the effect of dimethylarsenic acid(DMAA),the main metabolite of inorganic arsenic in humans,on the cellular DNA and gap junctional intercellular communication (GJIC) between human skin fibroblast cells.Single-cell gel electrophoresis (SCGE) assay was used to detect the DNA damage in human skin fibroblast cells exposed to DMAA,and the GJIC between cells was detected by the scrape loading/dye transfer assay.DMAA at concentrations of 0.01-1.0 mmol·L-1 induced DNA damage in a dose-dependent manner,and GJIC between human skin fibroblast cells was significantly inhibited by DMAA at 1.0 mmol·L-1.Our results suggest that both genotoxic and nongenotoxic mechanism are involved in the mechanism of DMAA-induced cellular toxicity.

  9. Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis

    International Nuclear Information System (INIS)

    A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with γ rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited

  10. Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis

    International Nuclear Information System (INIS)

    A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with gamma rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited

  11. Pyrazinoic Acid Decreases the Proton Motive Force, Respiratory ATP Synthesis Activity, and Cellular ATP Levels▿†

    OpenAIRE

    Lu, P.; Haagsma, A.C.; Pham, H.; Maaskant, J. J.; Mol, S; Lill, H.; Bald, D

    2011-01-01

    Pyrazinoic acid, the active form of the first-line antituberculosis drug pyrazinamide, decreased the proton motive force and respiratory ATP synthesis rates in subcellular mycobacterial membrane assays. Pyrazinoic acid also significantly lowered cellular ATP levels in Mycobacterium bovis BCG. These results indicate that the predominant mechanism of killing by this drug may operate by depletion of cellular ATP reserves.

  12. Replication stress activates DNA repair synthesis in mitosis.

    Science.gov (United States)

    Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A; Bursomanno, Sara; Aleliunaite, Aiste; Wu, Wei; Mankouri, Hocine W; Shen, Huahao; Liu, Ying; Hickson, Ian D

    2015-12-10

    Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS 'expression'), particularly when cells have been exposed to replicative stress. The MUS81-EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

  13. In situ amplification of DNA fragments specific for human Y chromosome in cellular nuclei by PCR

    Institute of Scientific and Technical Information of China (English)

    张锡元; 姜海波; 李立家; 马琦; 杨建琪; 刘汀

    1996-01-01

    Using single primer pairs Y3 and Y4, in siru polymerase chain reaction (in situ PCR) was successfully performed on the specimen slides of peripheral leukocytes. By both of the direct digpxiginin-11-dUTP incorporation into PCR products with in situ PCR (direct in situ PCR) and in situ PCR followed by detection of in situ hybridization (indirect in siru PCR), DNA fragments specific for human Y chromosome were obviously amplified in cellular nuclei of specimens on the slides. The results were verified by Southern analysis. The methodology of in situ PCR and its application were discussed.

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Science.gov (United States)

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

    2015-12-25

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

  16. Computational method and system for modeling, analyzing, and optimizing DNA amplification and synthesis

    Science.gov (United States)

    Vandersall, Jennifer A.; Gardner, Shea N.; Clague, David S.

    2010-05-04

    A computational method and computer-based system of modeling DNA synthesis for the design and interpretation of PCR amplification, parallel DNA synthesis, and microarray chip analysis. The method and system include modules that address the bioinformatics, kinetics, and thermodynamics of DNA amplification and synthesis. Specifically, the steps of DNA selection, as well as the kinetics and thermodynamics of DNA hybridization and extensions, are addressed, which enable the optimization of the processing and the prediction of the products as a function of DNA sequence, mixing protocol, time, temperature and concentration of species.

  17. Studies on enzymes involved in DNA synthesis and thymine nucleotide formation in potato tuber slices

    International Nuclear Information System (INIS)

    Activity changes of several enzymes involved in DNA synthesis were investigated in potato tuber tissue in which DNA synthesis was induced by slicing. Nucleoside phosphotransferase activity increased only slightly during aging of the tissue discs. Thymidine monophosphate (TMP) kinase activity increased about 36% after aging for 24 hr. Protein synthesis in an early stage of aging was necessary for the activity increase. A 2.7-fold increase was observed in DNA polymerase activity after aging for 36 hr. The activity increase was due to continuous synthesis of enzyme protein. In vivo examination of TMP synthetase suggests that its activity does not necessarily increase before full development of DNA synthesis. It was concluded that among the enzymes examined, TMP kinase activity may increase shortly after slicing to support a massive supply of thymidine triphosphate and the increased activity of DNA polymerane may contribute to the active synthesis of DNA in aged discs. (auth.)

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

    Directory of Open Access Journals (Sweden)

    Gunjan Guha

    2011-01-01

    Full Text Available 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.

  19. Relative ultraviolet radiation sensitivity of certain functions of polyoma virus. Stimulation of cell DNA synthesis

    International Nuclear Information System (INIS)

    Peritoneal Mouse macrophages were used to study the stimulation of cell DNA synthesis by polyoma virus. Using ultraviolet-irradiated polyoma virus, it was possible to show a difference between the inactivation of infectivity and of induction of DNA synthesis. By statistical analysis of these two phenomena it was found that 39% of the viral genome is necessary for the induction of cell DNA synthesis

  20. Facile Synthesis of Biocompatible Fluorescent Nanoparticles for Cellular Imaging and Targeted Detection of Cancer Cells.

    Science.gov (United States)

    Tang, Fu; Wang, Chun; Wang, Xiaoyu; Li, Lidong

    2015-11-18

    In this work, we report the facile synthesis of functional core-shell structured nanoparticles with fluorescence enhancement, which show specific targeting of cancer cells. Biopolymer poly-l-lysine was used to coat the silver core with various shell thicknesses. Then, the nanoparticles were functionalized with folic acid as a targeting agent for folic acid receptor. The metal-enhanced fluorescence effect was observed when the fluorophore (5-(and-6)-carboxyfluorescein-succinimidyl ester) was conjugated to the modified nanoparticle surface. Cellular imaging assay of the nanoparticles in folic acid receptor-positive cancer cells showed their excellent biocompatibility and selectivity. The as-prepared functional nanoparticles demonstrate the efficiency of the metal-enhanced fluorescence effect and provide an alternative approach for the cellular imaging and targeting of cancer cells.

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

    Institute of Scientific and Technical Information of China (English)

    SCHMITT Estelle; PAQUET Claudie; BEAUCHEMIN Myriam; BERTRAND Richard

    2007-01-01

    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. A DNA-dependent stress response involving DNA-PK occurs in hypoxic cells and contributes to cellular adaptation to hypoxia.

    Science.gov (United States)

    Bouquet, Fanny; Ousset, Marielle; Biard, Denis; Fallone, Frédérique; Dauvillier, Stéphanie; Frit, Philippe; Salles, Bernard; Muller, Catherine

    2011-06-01

    DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand break (DSB) signalling and repair. We report that DNA-PK is activated by mild hypoxia conditions (0.1-1% O₂) as shown by (1) its autophosphorylation on Ser2056, and (2) its mobilisation from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. The recruitment of DNA-PK was not followed by activation and recruitment of the XRCC4-DNA-ligase-IV complex, suggesting that DSBs are not responsible for activation of DNA-PK. To unravel the mechanism of DNA-PK activation, we show that exposure of cells to trichostatin A, a histone deacetylase inhibitor, leads to DNA-PK autophosphorylation and relocalisation to DNA. Histone acetylation (mainly H3K14) is increased in hypoxic cells and treatment with anacardic acid, an inhibitor of histone acetyl transferase, prevented both histone modifications and DNA-PK activation in hypoxic conditions. Importantly, in using either silenced DNA-PK cells or cells exposed to a specific DNA-PK inhibitor (NU7026), we demonstrated that hypoxic DNA-PK activation positively regulates the key transcription factor HIF-1 and one subsequent target gene, GLUT1. Our results show that hypoxia initiates chromatin modification and consequently DNA-PK activation, which positively regulate cellular oxygen-sensing and oxygen-signalling pathways. PMID:21576354

  3. Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants of Ustilago maydis

    Energy Technology Data Exchange (ETDEWEB)

    Unrau, P.

    1977-01-01

    The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungus Ustilago maydis after incubation at the restrictive temperature (32/sup 0/C) for eight hours. Mutants ts-220, ts-207, ts-432 and ts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutants ts-20, tsd 1-1, ts-84 and pol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutant pol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutant ts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32/sup 0/C. tsd 1-1 and ts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis which correlates to increasing UV sensitivity of these strains on incubation at 32/sup 0/C. A pol 1-1 ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.

  4. Radiation hypersensitivity and radioresistant DNA synthesis in ataxia-telangiectasia

    International Nuclear Information System (INIS)

    Patients with the autosomal recessive genetic disease, ataxia-telangiectasia (A-T), are cancer-prone and hypersensitive to the killing effects of ionizing radiation. In an attempt to isolate the gene(s) responsible for the hypersensitivity of A-T cells, they were transfected with normal human DNA in cosmid vectors containing a rescuable marker (G-418 resistance), and revertants to normal sensitivity were isolated and characterized. The failure of radioresistant revertants to demonstrate a reversion of the phenotype, radioresistant DNA synthesis, shows that this feature is dependent on a gene separate from the one conferring resistance to cell killing. Cells from every A-T patient thus far examined demonstrate both hypersensitivity, in terms of radiation-induced cell killing, and radioresistant DNA synthesis. The results reported here, however, show that the former is not a result of the latter, as previously proposed. Moreover, the fact that these two characteristics can be uncoupled obscures the role(s) that either of them plays in the etiology of the disease, or in the development in its other features, including cancer-proneness

  5. Studies on bleomycin-induced repair DNA synthesis in permeable mouse ascites sarcoma cells.

    Directory of Open Access Journals (Sweden)

    Mori,Shigeru

    1989-04-01

    Full Text Available To study the mechanism of DNA excision repair, a DNA repair system employing permeable mouse sarcoma (SR-C3H/He cells was established and characterized. SR-C3H/He cells were permeabilized with a 0.0175% Triton X-100 solution. The permeable cells were treated with 1 mM ATP and 0.11 mM bleomycin, and then washed thoroughly to remove ATP and bleomycin. Repair DNA synthesis occurred in the bleomycin-damaged, permeable SR-C3H/He cells when incubated with ATP and four deoxyribonucleoside triphosphates. The repair nature of the DNA synthesis was confirmed by the BrdUMP density shift technique, and by the reduced sensitivity of the newly synthesized DNA to Escherichia coli exonuclease III. The DNA synthesis was optimally enhanced by addition of 0.08 M NaCl. Studies using selective inhibitors of DNA synthesis showed that aphidicolin-sensitive DNA polymerase (DNA polymerase alpha and/or delta and DNA polymerase beta were involved in the repair process. The present DNA repair system is thought to be useful to study nuclear DNA damage by bleomycin, removal of the damaged ends by an exonuclease, repair DNA synthesis by DNA polymerases and repair patch ligation by DNA ligase(s.

  6. Gymnemagenin-a triterpene saponin prevents γ-radiation induced cellular DNA damage

    International Nuclear Information System (INIS)

    Gymnema sylvestre an ethno-medicinally important plant was investigated for its protecting activity against radiation induced DNA damage. The major bioactive component present in Gymnema sylvestre such as gymnemic acid and gymnemagenin a triterpene saponin, were tested for its radioprotective effects against 60Co irradiation induced DNA damage in fish model using fresh water fish Pangasius sutchi. Fishes subjected to a dose of 133 Gy of gamma radiation and observed for eight days. The genotoxic assessment by micronucleus assay showed us that that the plant extract helped in reducing the frequency of micronucleated and binucleated erythrocytes compared to the irradiated control group. The genotoxic assessment by alkaline comet assay by single gel electrophoresis shows that pretreatment with the plant extract appreciably decreased the percentage of tail DNA towards the levels close to those of normal control group. The gradual increase in the level of the antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT) during the course of the experiment indicates that the antioxidant enzyme activities play an important role in protecting organisms against gamma radiation-induced cellular oxidative stress. In conclusion the leaf extracts of Gymnema sylvstre exerts its radio protective potential by suppressing the toxic assault of ROS generated by the ionizing radiation through its ability to boost the levels of antioxidant enzymes (CAT and SOD) due to the presence of its phytochemicals like gymnemgenenin- a Triterpene Saponin. (author)

  7. A germline polymorphism of thymine DNA glycosylase induces genomic instability and cellular transformation.

    Directory of Open Access Journals (Sweden)

    Ashley Sjolund

    2014-11-01

    Full Text Available Thymine DNA glycosylase (TDG functions in base excision repair, a DNA repair pathway that acts in a lesion-specific manner to correct individual damaged or altered bases. TDG preferentially catalyzes the removal of thymine and uracil paired with guanine, and is also active on 5-fluorouracil (5-FU paired with adenine or guanine. The rs4135113 single nucleotide polymorphism (SNP of TDG is found in 10% of the global population. This coding SNP results in the alteration of Gly199 to Ser. Gly199 is part of a loop responsible for stabilizing the flipped abasic nucleotide in the active site pocket. Biochemical analyses indicate that G199S exhibits tighter binding to both its substrate and abasic product. The persistent accumulation of abasic sites in cells expressing G199S leads to the induction of double-strand breaks (DSBs. Cells expressing the G199S variant also activate a DNA damage response. When expressed in cells, G199S induces genomic instability and cellular transformation. Together, these results suggest that individuals harboring the G199S variant may have increased risk for developing cancer.

  8. Reduced cellular DNA repair capacity after environmentally relevant arsenic exposure. Influence of Ogg1 deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Bach, Jordi; Peremartí, Jana; Annangi, Balasubramnayam [Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona (Spain); Marcos, Ricard, E-mail: ricard.marcos@uab.es [Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona (Spain); CIBER Epidemiología y Salud Pública, ISCIII, Madrid (Spain); Hernández, Alba, E-mail: alba.hernandez@uab.es [Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona (Spain); CIBER Epidemiología y Salud Pública, ISCIII, Madrid (Spain)

    2015-09-15

    Highlights: • Repair ability under long-term exposure to arsenic was tested using the comet assay. • Effects were measured under Ogg1 wild-type and deficient backgrounds. • Exposed cells repair less efficiency the DNA damage induced by SA, KBrO{sub 3}, MMA{sup III} or UVC radiation. • Oxidative damage and Ogg1 deficient background exacerbate repair deficiencies. • Overexpression of the arsenic metabolizing enzyme As3mt acts as adaptive mechanism. - Abstract: Inorganic arsenic (i-As) is a genotoxic and carcinogenic environmental contaminant known to affect millions of people worldwide. Our previous work demonstrated that chronic sub-toxic i-As concentrations were able to induce biologically significant levels of genotoxic and oxidative DNA damage that were strongly influenced by the Ogg1 genotype. In order to study the nature of the observed levels of damage and the observed differences between MEF Ogg1{sup +/+} and Ogg1{sup −/−} genetic backgrounds, the genotoxic and oxidative DNA repair kinetics of 18-weeks exposed MEF cells were evaluated by the comet assay. Results indicate that MEF Ogg1{sup +/+} and Ogg1{sup −/−} cells chronically exposed to i-As repair the DNA damage induced by arsenite, potassium bromide and UVC radiation less efficiently than control cells, being that observation clearly more pronounced in MEF Ogg1{sup −/−} cells. Consequently, exposed cells accumulate a higher percentage of unrepaired DNA damage at the end of the repair period. As an attempt to eliminate i-As associated toxicity, chronically exposed MEF Ogg1{sup −/−} cells overexpress the arsenic metabolizing enzyme As3mt. This adaptive response confers cells a significant resistance to i-As-induced cell death, but at expenses of accumulating high levels of DNA damage due to their repair impairment. Overall, the work presented here evidences that i-As chronic exposure disrupts the normal cellular repair function, and that oxidative DNA damage—and Ogg1 deficiency

  9. Reduced cellular DNA repair capacity after environmentally relevant arsenic exposure. Influence of Ogg1 deficiency

    International Nuclear Information System (INIS)

    Highlights: • Repair ability under long-term exposure to arsenic was tested using the comet assay. • Effects were measured under Ogg1 wild-type and deficient backgrounds. • Exposed cells repair less efficiency the DNA damage induced by SA, KBrO3, MMAIII or UVC radiation. • Oxidative damage and Ogg1 deficient background exacerbate repair deficiencies. • Overexpression of the arsenic metabolizing enzyme As3mt acts as adaptive mechanism. - Abstract: Inorganic arsenic (i-As) is a genotoxic and carcinogenic environmental contaminant known to affect millions of people worldwide. Our previous work demonstrated that chronic sub-toxic i-As concentrations were able to induce biologically significant levels of genotoxic and oxidative DNA damage that were strongly influenced by the Ogg1 genotype. In order to study the nature of the observed levels of damage and the observed differences between MEF Ogg1+/+ and Ogg1−/− genetic backgrounds, the genotoxic and oxidative DNA repair kinetics of 18-weeks exposed MEF cells were evaluated by the comet assay. Results indicate that MEF Ogg1+/+ and Ogg1−/− cells chronically exposed to i-As repair the DNA damage induced by arsenite, potassium bromide and UVC radiation less efficiently than control cells, being that observation clearly more pronounced in MEF Ogg1−/− cells. Consequently, exposed cells accumulate a higher percentage of unrepaired DNA damage at the end of the repair period. As an attempt to eliminate i-As associated toxicity, chronically exposed MEF Ogg1−/− cells overexpress the arsenic metabolizing enzyme As3mt. This adaptive response confers cells a significant resistance to i-As-induced cell death, but at expenses of accumulating high levels of DNA damage due to their repair impairment. Overall, the work presented here evidences that i-As chronic exposure disrupts the normal cellular repair function, and that oxidative DNA damage—and Ogg1 deficiency—exacerbates this phenomenon. The observed cell

  10. Effects of Pulsed Electric Fields on DNA Synthesis in an Osteoblast-Like Cell Line (UMR-106)

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The study of the bioeffects of electromagnetic fields (EMFs) is an important national task in biological physics. Using EMFs to treat bone diseases involves electrical technology, biology, and medicine. But the effects of EMFs are still controversial and the mechanisms are not yet clear. Therefore, more effect is needed to detect the effects at the cellular and molecular levels. This paper investigates the effects of low-energy, low-frequency pulsed capacitively coupled electric fields (PCCEFs) on DNA synthesis in UMR-106 osteoblast-like cells. The equipment can generate 25250Hz frequency, 0300V amplitude and 0.2ms pulse width signal. DNA synthesis is judged by the uptake of 3H-thymidine (3H-TdR). The results showed that the response of UMR-106 cells to electric field exposure are characterized by: (a) a frequency window for increased DNA synthesis, with a peak near 125Hz; (b) decreased synthesis with increasing electric intensity with repression at 100V/cm and 25Hz.

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

    Energy Technology Data Exchange (ETDEWEB)

    Roper, Katherine; Coverley, Dawn, E-mail: dc17@york.ac.uk

    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

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

    International Nuclear Information System (INIS)

    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 naïve 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 naïve nuclei. At the same time, H2AX is phosphorylated in naïve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naïve 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: ► A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. ► Damage-activated extracts impose the cellular response to DNA damage on naïve nuclei. ► PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. ► Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. ► LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening approach.

  13. Peptide Synthesis on a Next-Generation DNA Sequencing Platform.

    Science.gov (United States)

    Svensen, Nina; Peersen, Olve B; Jaffrey, Samie R

    2016-09-01

    Methods for displaying large numbers of peptides on solid surfaces are essential for high-throughput characterization of peptide function and binding properties. Here we describe a method for converting the >10(7) flow cell-bound clusters of identical DNA strands generated by the Illumina DNA sequencing technology into clusters of complementary RNA, and subsequently peptide clusters. We modified the flow-cell-bound primers with ribonucleotides thus enabling them to be used by poliovirus polymerase 3D(pol) . The primers hybridize to the clustered DNA thus leading to RNA clusters. The RNAs fold into functional protein- or small molecule-binding aptamers. We used the mRNA-display approach to synthesize flow-cell-tethered peptides from these RNA clusters. The peptides showed selective binding to cognate antibodies. The methods described here provide an approach for using DNA clusters to template peptide synthesis on an Illumina flow cell, thus providing new opportunities for massively parallel peptide-based assays.

  14. 酶促DNA合成研究的进展%Advance in Enzymatic DNA Synthesis

    Institute of Scientific and Technical Information of China (English)

    向义和

    2011-01-01

    The advance in enzymatic DNA synthesis is introduced. Kornberg and his colleagues went through deoxyribonucleotide.de-oxynucleoside try phosphates and DNA synthesis. The immediate precursor of DNA synthesis was known. DNA polymerase was separated and purified. The chemical mechanism of DNA synthesis was revealed and infectious phage φX174DNA was synthesized.%笔者介绍了酶促DNA合成研究的进展.科恩伯格和他的同事经历了从合成核苷酸、核苷三磷酸到合成DNA的历程.他们分离并提纯了DNA聚合酶,弄清了合成DNA的最直接的前体,揭示了DNA合成的化学机理,合成了具有感染性的噬菌体φX174DNA.

  15. CELLULAR RESPONSES TO DNA DAMAGE AND ONCOGENESIS BY THE p53 AND pRb/E2F PATHWAYS

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-07-01

    Full Text Available Cellular responses to stress including DNA damage, show multiple options involving the mechanisms of growth arrest. DNA repair and programmed cell death or apoptosis. Failures in these mechanisms can result in oncogenesis or accelerated senescence. Much of the response is coordinated by p53, a nuclear phosphoprotein with a central role in the defences against physical, chemical and pathogenic agents which challenge the DNA integrity. The p53 pathways for mobilising the cellular defences are linked to the pRB/E2D pathways regulating the cell cycle progression. This paper aims to review the current understanding on the networks and main molecular machinery of these processes. In addition, the implications on cellular decision making for the defences as well as revolutionary aspects of these mechanisms are discussed in brief.

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

    Energy Technology Data Exchange (ETDEWEB)

    Du, Fengxia [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Zhang, Minjie [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Xiaohua; Yang, Caiyun [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Meng, Hao; Wang, Dong; Chang, Shuang [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xu, Ye [Department of Radiation Oncology, Division of Genomic Stability, Dana Farber Cancer Institute, Harvard Medical School, MA 02134 (United States); Price, Brendan, E-mail: Brendan_Price@dfci.harvard.edu [Department of Radiation Oncology, Division of Genomic Stability, Dana Farber Cancer Institute, Harvard Medical School, MA 02134 (United States); Sun, Yingli, E-mail: sunyl@big.ac.cn [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China)

    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.

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

    International Nuclear Information System (INIS)

    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

  18. A candidate DNA vaccine elicits HCV specific humoral and cellular immune responses

    Institute of Scientific and Technical Information of China (English)

    Li-Xin Zhu; Jing Liu; Ye Ye; You-Hua Xie; Yu-Ying Kong; Guang-Di Li; Yuan Wang

    2004-01-01

    AIM: To investigate the immunogenicity of candidate DNA vaccine against hepatitis C virus (HCV) delivered by two plasmids expressing HCV envelope protein 1 (E1) and envelope protein 2 (E2) antigens respectively and to study the effect of CpG adjuvant on this candidate vaccine.METHODS: Recombinant plasmids expressing HCV E1 and E2 antigens respectively were used to simultaneously inoculate mice with or without CpG adjuvant. Antisera were then collected and titers of anti-HCV antibodies were analyzed by ELISA. One month after the last injection, animals were sacrificed to prepare single-cell suspension of splenocytes.These cells were subjected to HCVantigen specific proliferation assays and cytokine secretion assays to evaluate the cellular immune responses of the vaccinated animals.RESULTS: Antibody responses to HCV E1 and E2 antigens were detected in vaccinated animals. Animals receiving CpG adjuvant had slightly lower titers of anti-HCV antibodies in the sera, while the splenocytes from these animals showed higher HCV-antigen specific proliferation. Analysis of cytokine secretion from the splenocytes was consistent with the above results. While no antigen-specific IL-4 secretion was detected for all vaccinated animals, HCV antigen-specific INF-γ secretion was detected for the splenocytes of vaccinated animals. CpG adjuvant enhanced the secretion of INF-γ but did not change the profile of IL-4 secretion.CONCLUSION: Vaccination of mice with plasmids encoding HCV E1 and E2 antigens induces humoral and cellular immune responses. CpG adjuvant significantly enhances the cellular immune response.

  19. Antiproliferative activity of bicyclic benzimidazole nucleosides: synthesis, DNA-binding and cell cycle analysis.

    Science.gov (United States)

    Sontakke, Vyankat A; Lawande, Pravin P; Kate, Anup N; Khan, Ayesha; Joshi, Rakesh; Kumbhar, Anupa A; Shinde, Vaishali S

    2016-04-26

    An efficient route was developed for synthesis of bicyclic benzimidazole nucleosides from readily available d-glucose. The key reactions were Vörbruggen glycosylation and ring closing metathesis (RCM). Primarily, to understand the mode of DNA binding, we performed a molecular docking study and the binding was found to be in the minor groove region. Based on the proposed binding model, UV-visible and fluorescence spectroscopic techniques using calf thymus DNA (CT-DNA) demonstrated a non-intercalative mode of binding. Antiproliferative activity of nucleosides was tested against MCF-7 and MDA-MB-231 breast cancer cell lines and found to be active at low micromolar concentrations. Compounds and displayed significant antiproliferative activity as compared to and with the reference anticancer drug, doxorubicin. Cell cycle analysis showed that nucleoside induced cell cycle arrest at the S-phase. Confocal microscopy has been performed to validate the induction of cellular apoptosis. Based on these findings, such modified bicyclic benzimidazole nucleosides will make a significant contribution to the development of anticancer drugs. PMID:27074628

  20. Synthesis of full length and truncated microcin B17 analogues as DNA gyrase poisons.

    Science.gov (United States)

    Thompson, Robert E; Collin, Frédéric; Maxwell, Anthony; Jolliffe, Katrina A; Payne, Richard J

    2014-03-14

    Microcin B17 (MccB17) is a post-translationally modified peptide containing thiazole and oxazole heterocycles that interrupt the peptide backbone. MccB17 is capable of poisoning DNA gyrase through stabilization of the gyrase-DNA cleavage complex and has therefore attracted significant attention. Using a combination of Fmoc-strategy solid-phase peptide synthesis and solution-phase fragment assembly we have prepared a library of full-length and truncated MccB17 analogues to investigate key structural requirements for gyrase-poisoning activity. Synthetic peptides lacking the glycine-rich N-terminal portion of the full-length sequence showed strong stabilization of the gyrase-DNA cleavage complex with increased potency relative to the full-length sequences. This truncation, however, led to a decrease in antibacterial activity of these analogues relative to their full-length counterparts indicating a potential role of the N-terminal region of the natural product for cellular uptake.

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

    Science.gov (United States)

    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.

  2. Colon Cancer-associated DNA Polymerase β Variant Induces Genomic Instability and Cellular Transformation*

    Science.gov (United States)

    Nemec, Antonia A.; Donigan, Katherine A.; Murphy, Drew L.; Jaeger, Joachim; Sweasy, Joann B.

    2012-01-01

    Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. We have identified several mutations of the DNA polymerase β (pol β) gene in human colorectal cancer. We have demonstrated that the expression of the pol β G231D variant increased chromosomal aberrations and induced cellular transformation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished, suggesting that it resulted as a consequence of genomic instability. Biochemical analysis revealed that its catalytic rate was 140-fold slower than WT pol β, and this was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of pol β lies in close proximity to the template strand of the DNA. Molecular modeling demonstrated that the change from a small and nonpolar glycine to a negatively charged aspartate resulted in a repulsion between the template and residue 231 leading to the distortion of the dNTP binding pocket. In addition, expression of G231D was insufficient to rescue pol β-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. More importantly, this suggests that the G231D variant has impaired base excision repair. Together, these data indicate that the G231D variant plays a role in driving cancer. PMID:22573322

  3. DNA synthesis in the imaginal wing discs of the American bollworm Helicoverpa armigera (Hübner)

    Indian Academy of Sciences (India)

    A Josephrajkumar; B Subrahmanyam

    2002-03-01

    The effect of two insect growth regulators of plant origin viz. plumbagin and azadirachtin and the ecdysteroids 20-hydroxyecdysone, makisterone A and a phytoecdysteroid on DNA synthesis in imaginal wing discs of day 4 final instar Helicoverpa armigera larvae was studied. DNA synthesis increased with increase in time of incubation up to 8 h and decreased later without the addition of moulting hormone. Addition of 20-hydroxyecdysone supported long term acquisition of competence for DNA synthesis in the wing discs. Both DNA synthesis and protein content were drastically reduced in plumbagin and azadirachtin-treated insects. Under in vitro conditions, plumbagin had a more pronounced inhibitory effect than azadirachtin. All the ecdysteroids tested, viz. makisterone A, 20-hydroxyecdysone and the ecdysteroidal fraction from the silver fern Cheilanthes farinosa enhanced DNA synthesis.

  4. Multi-line split DNA synthesis: a novel combinatorial method to make high quality peptide libraries

    Directory of Open Access Journals (Sweden)

    Ueno Shingo

    2004-09-01

    Full Text Available Abstract Background We developed a method to make a various high quality random peptide libraries for evolutionary protein engineering based on a combinatorial DNA synthesis. Results A split synthesis in codon units was performed with mixtures of bases optimally designed by using a Genetic Algorithm program. It required only standard DNA synthetic reagents and standard DNA synthesizers in three lines. This multi-line split DNA synthesis (MLSDS is simply realized by adding a mix-and-split process to normal DNA synthesis protocol. Superiority of MLSDS method over other methods was shown. We demonstrated the synthesis of oligonucleotide libraries with 1016 diversity, and the construction of a library with random sequence coding 120 amino acids containing few stop codons. Conclusions Owing to the flexibility of the MLSDS method, it will be able to design various "rational" libraries by using bioinformatics databases.

  5. ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.

    Science.gov (United States)

    Lewis, Samantha C; Uchiyama, Lauren F; Nunnari, Jodi

    2016-07-15

    Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. In human cells, hundreds to thousands of mtDNA copies are replicated asynchronously, packaged into protein-DNA nucleoids, and distributed within a dynamic mitochondrial network. The mechanisms that govern how nucleoids are chosen for replication and distribution are not understood. Mitochondrial distribution depends on division, which occurs at endoplasmic reticulum (ER)-mitochondria contact sites. These sites were spatially linked to a subset of nucleoids selectively marked by mtDNA polymerase and engaged in mtDNA synthesis--events that occurred upstream of mitochondrial constriction and division machine assembly. Our data suggest that ER tubules proximal to nucleoids are necessary but not sufficient for mtDNA synthesis. Thus, ER-mitochondria contacts coordinate licensing of mtDNA synthesis with division to distribute newly replicated nucleoids to daughter mitochondria.

  6. Cross Talk between Nucleotide Synthesis Pathways with Cellular Immunity in Constraining Hepatitis E Virus Replication.

    Science.gov (United States)

    Wang, Yijin; Wang, Wenshi; Xu, Lei; Zhou, Xinying; Shokrollahi, Ehsan; Felczak, Krzysztof; van der Laan, Luc J W; Pankiewicz, Krzysztof W; Sprengers, Dave; Raat, Nicolaas J H; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

    2016-05-01

    Viruses are solely dependent on host cells to propagate; therefore, understanding virus-host interaction is important for antiviral drug development. Since de novo nucleotide biosynthesis is essentially required for both host cell metabolism and viral replication, specific catalytic enzymes of these pathways have been explored as potential antiviral targets. In this study, we investigated the role of different enzymatic cascades of nucleotide biosynthesis in hepatitis E virus (HEV) replication. By profiling various pharmacological inhibitors of nucleotide biosynthesis, we found that targeting the early steps of the purine biosynthesis pathway led to the enhancement of HEV replication, whereas targeting the later step resulted in potent antiviral activity via the depletion of purine nucleotide. Furthermore, the inhibition of the pyrimidine pathway resulted in potent anti-HEV activity. Interestingly, all of these inhibitors with anti-HEV activity concurrently triggered the induction of antiviral interferon-stimulated genes (ISGs). Although ISGs are commonly induced by interferons via the JAK-STAT pathway, their induction by nucleotide synthesis inhibitors is completely independent of this classical mechanism. In conclusion, this study revealed an unconventional novel mechanism of cross talk between nucleotide biosynthesis pathways and cellular antiviral immunity in constraining HEV infection. Targeting particular enzymes in nucleotide biosynthesis represents a viable option for antiviral drug development against HEV. HEV is the most common cause of acute viral hepatitis worldwide and is also associated with chronic hepatitis, especially in immunocompromised patients. Although often an acute and self-limiting infection in the general population, HEV can cause severe morbidity and mortality in certain patients, a problem compounded by the lack of FDA-approved anti-HEV medication available. In this study, we have investigated the role of the nucleotide synthesis pathway

  7. Activation of WIP1 phosphatase by HTLV-1 Tax mitigates the cellular response to DNA damage.

    Directory of Open Access Journals (Sweden)

    Tajhal Dayaram

    Full Text Available 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 G(1 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 G(1/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G(1 exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome.

  8. Effects of uniconazole waterless-dressing seed on DNA synthesis in seed germination of wheat

    International Nuclear Information System (INIS)

    Effects of uniconazole waterless-dressing seed on the synthesis of DNA in seed germination of wheat, c.v. Mianyang 26, were studied with the method of 3H-TdR. The results showed that uniconazole treatments could promote DNA synthesis, when the concentration of uniconazole was 20 mg/kg, the speed of DNA synthesis was the quickest. At the same time, the incorporating 3H-TdR indicated that uniconazole treatment were beneficial to the DNA repairing in the early period of wheat seed germination. (authors)

  9. Bleomycin-induced DNA synthesis in a cell-free system using a permeable mouse sarcoma cell Extract.

    Directory of Open Access Journals (Sweden)

    Seki,Shuji

    1987-10-01

    Full Text Available To investigate factors involved in excision repair DNA synthesis, a soluble extract was prepared from permeable mouse sarcoma (SR-C3H/He cells by homogenization and ultracentrifugation. DNA synthesis measured by using native calf thymus DNA as the template-primer and the extract as the polymerase source showed low activity. The DNA synthesis was enhanced more than ten-fold by the addition of an appropriate concentration of bleomycin, a radiomimetic DNA-damaging drug. Using selective inhibitors of DNA polymerases, it was shown that the DNA polymerase involved in the bleomycin-induced DNA synthesis was DNA polymerase beta. In addition to DNA polymerase beta, an exonuclease which converts bleomycin-damaged DNA into suitable template-primers for repair DNA synthesis appeared to be present in the permeable cell extract.

  10. Human herpesvirus-6 and cytomegalovirus DNA in liver donor biopsies and their correlation with HLA matches and acute cellular rejection

    Directory of Open Access Journals (Sweden)

    Ana Carolina Guardia

    2014-04-01

    Full Text Available Herpesvirus reactivation is common after liver transplantation. OBJECTIVE: Analyze the presence of cytomegalovirus (HCMV and human herpesvirus-6 (HHV-6 DNA in liver donor biopsies, seeking to better understand issues involving human donor leukocyte antigens (HLA-A, B and DR, as well as correlations with acute cellular rejection. METHODS: Fifty-nine liver transplantation patients were investigated for the presence of HCMV and HHV-6 DNA in liver donor biopsies, using the Nested-PCR technique. The clinical donor information and HLA matches were obtained from the São Paulo State Transplant System. The recipients' records regarding acute cellular rejection were studied. RESULTS: Seven (11.8% biopsies were positive for HCMV DNA and 29 (49% were positive for HHV-6 DNA. In 14 donors with HLA-DR 15 nine had HHV-6 DNA positive liver biopsy with a tendency for significant association (p=0.09, 22 recipients developed acute cellular rejection and 9/22 were positive for HLA-DR 15 (p=0.03; χ 2=4.51, which was statistically significant in univariate analysis and showed a tendency after multivariate analysis (p=0.08. CONCLUSION: HHV-6 DNA was prevalent in liver donors studied as well as HLA-DR 15. These findings suggest that patients with HLA-DR 15 in liver donor biopsies develop more rejection after liver transplantation.

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

    International Nuclear Information System (INIS)

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

  12. EFFECT OF GLYCYRRHETINIC ACID ON DNA DAMAGE AND UNSCHEDULED DNA SYNTHESIS INDUCED BY BENZO (α) PYRENE

    Institute of Scientific and Technical Information of China (English)

    陈晓光; 韩锐

    1995-01-01

    Glycyrrhetinic acid (GA) is an active component of Glycyrrhiza uraleusis fisch. In this study, GA was found to inhibit ear edema and ornithine decarboxykase (ODC)activity induced by croton oil in mice. GA could also protect rapid DNA damage and decrease the unscheduled DNA synthesis induced by benzo(α)pyrene, The results demonstrate that GA has a potential cancer chemopreventive activity.

  13. The initiation mechanism of translesion DNA synthesis in response to UV irradiation

    International Nuclear Information System (INIS)

    Ultraviolet (UV) light causes DNA damage and increases a person's risk for both melanoma and non-melanoma skin cancer. If the DNA damage is unrepaired, cells can often tolerate it by using specialized DNA polymerases during DNA replication to insert a base opposite a lesion and bypass the damage, in a process called translesion DNA synthesis (TLS). This review addresses recent advances in our understanding of TLS. (author)

  14. Synergistic bombesin and insulin stimulation of DNA synthesis in human fetal kidney in serum-free culture.

    Science.gov (United States)

    Brière, N; Chailler, P

    1993-05-01

    The respective influences of growth factors during kidney development can be directly evaluated using the chemically-defined serum-free culture system perfected in our laboratory. Since, in this culture model, conditions are minimal for growth and differentiation, DNA synthesis sharply decreases during the first 48 h. The addition of epidermal growth factor (EGF, 100 ng/ml), insulin (5 micrograms/ml) and transferrin (5 micrograms/ml) significantly restores this important cellular function. The objective of the present study was to determine the influence of bombesin, a potent mitogen, supplemented alone or in combination with insulin, transferrin and/or EGF. Cortical explants of human fetal kidneys (17-20 weeks) were maintained during 5 days in culture. When compared with 5 day controls (L-15 medium only), bombesin generated a maximal though weak effect on DNA synthesis at a concentration of 0.3 nM, corresponding to a stimulation index (SI) of 22%. When combined with either transferrin or EGF, or with transferrin plus EGF, bombesin did not alter the SI of individual factors. Insulin, in turn, greatly increased DNA synthesis (SI = 169%), while bombesin strongly potentiated this effect (SI = 275%). Transferrin also enhanced insulin SI from 169 to 240%. When added as a third factor, bombesin further potentiated the effectiveness (SI = 338%) of the combination insulin plus transferrin. These results indicate that bombesin controls cell proliferation in synergism with other regulators and hence may act as a competence growth factor during nephrogenesis.

  15. DNA-content and synthesis rate in human melanoma cells in vitro after hyperthermia and radiation

    International Nuclear Information System (INIS)

    Human melanoma cells were cultured over 168 hours. DNA-content and 3H-thymidine incorporation were measured after 800 R and hyperthermia of 400C and 440C for 3 and 6 hours. An early and a late effect could be distinguished. 3 and 6 hours after irradiation alone no alteration of DNA-synthesis was observed. After heat treatment at 400C for 6 hours the DNA-synthesis was increased immediately. 420C and 440C deminished the rate of DNA-synthesis. The combined treatment (heat and irradiation) suppressed the overshooting rate of DNA-synthesis. Accordingly after heat treatment at 400C for 6 hours the DNA-content was higher than the controls and the 3 hours-400C treated cultures measured over a period of 48 hours. Thereafter the DNA-content showed little or no alterations compared with the controls. The heat treatment at 440C reduced the DNA-content heavily, followed by a relative increase at 120 hours. The DNA-synthesis rate showed the same effect. The combined treatment suppressed this late increase. However, 24 hours after combined treatment the incorporation of thymidine into the DNA was higher at 440C-6 than 440C-3 hours, 40C-3 hours, than 400C-6 hours, although the DNA-content was very low. The results show synergistic effects of hyperthermia and radiation on the DNA-synthesis and content if one considers the effects at the later periods (120-168 hours). However, a stimulating effect is found on the DNA-synthesis if the melanoma cells are incubated at 400C for 6 hours. (orig.)

  16. Synthesis and cellular uptake of folic acid-conjugated cellulose nanocrystals for cancer targeting.

    Science.gov (United States)

    Dong, Shuping; Cho, Hyung Joon; Lee, Yong Woo; Roman, Maren

    2014-05-12

    Elongated nanoparticles have recently been shown to have distinct advantages over spherical ones in targeted drug delivery applications. In addition to their oblong geometry, their lack of cytotoxicity and numerous surface hydroxyl groups make cellulose nanocrystals (CNCs) promising drug delivery vectors. Herein we report the synthesis of folic acid-conjugated CNCs for the targeted delivery of chemotherapeutic agents to folate receptor-positive cancer cells. Folate receptor-mediated cellular binding/uptake of the conjugate was demonstrated on human (DBTRG-05MG, H4) and rat (C6) brain tumor cells. Folate receptor expression of the cells was verified by immunofluorescence staining. Cellular binding/uptake of the conjugate by DBTRG-05MG, H4, and C6 cells was 1452, 975, and 46 times higher, respectively, than that of nontargeted CNCs. The uptake mechanism was determined by preincubation of the cells with the uptake inhibitors chlorpromazine or genistein. DBTRG-05MG and C6 cells internalized the conjugate primarily via caveolae-mediated endocytosis, whereas H4 cells internalized the conjugate primarily via clathrin-mediated endocytosis. PMID:24716601

  17. Reconstructing the emergence of cellular life through the synthesis of model protocells.

    Science.gov (United States)

    Mansy, S S; Szostak, J W

    2009-01-01

    The complexity of modern biological life has long made it difficult to understand how life could emerge spontaneously from the chemistry of the early earth. The key to resolving this mystery lies in the simplicity of the earliest living cells, together with the ability of the appropriate molecular building blocks to spontaneously self-assemble into larger structures. In our view, the two key components of a primitive cell are not only self-assembling, but also self-replicating, structures: the nucleic acid genome and the cell membrane. Here, we summarize recent experimental progress toward the synthesis of efficient self-replicating nucleic acid and membrane vesicle systems and discuss some of the issues that arise during efforts to integrate these two subsystems into a coherent whole. We have shown that spontaneous nucleic-acid-copying chemistry can take place within membrane vesicles, using externally supplied activated nucleotides as substrates. Thus, membranes need not be a barrier to the uptake of environmentally supplied nutrients. We examine some of the remaining obstacles that must be overcome to enable the synthesis of a complete self-replicating protocell, and we discuss the implications of these experiments for our understanding of the emergence of Darwinian evolution and the origin and early evolution of cellular life. PMID:19734203

  18. Nerve growth factor enhances DNA synthesis in cultured cerebellar neuroblasts.

    Science.gov (United States)

    Confort, C; Charrasse, S; Clos, J

    1991-10-01

    The cerebellar neuroblasts in primary cultures from five-day-old rats bore NGF receptor immunoreactivity, suggesting a potential responsive to NGF. At low plating density, NGF was found to enhance DNA synthesis in these cells in a dose-dependent manner. As these cells synthesize NGF, one possibility to account for the lack of response of neuroblasts plated at high density is that the amount of endogenous trophic agent produced in this culture condition is sufficient to ensure an optimal effect. The results demonstrate that premitotic neuroblasts in the CNS, as well postmitotic neurons, are responsive to NGF. At the early stage of its development, the cerebellum therefore appears to be a very good autocrine model of NGF action. PMID:1661619

  19. Nerve growth factor enhances DNA synthesis in cultured cerebellar neuroblasts.

    Science.gov (United States)

    Confort, C; Charrasse, S; Clos, J

    1991-10-01

    The cerebellar neuroblasts in primary cultures from five-day-old rats bore NGF receptor immunoreactivity, suggesting a potential responsive to NGF. At low plating density, NGF was found to enhance DNA synthesis in these cells in a dose-dependent manner. As these cells synthesize NGF, one possibility to account for the lack of response of neuroblasts plated at high density is that the amount of endogenous trophic agent produced in this culture condition is sufficient to ensure an optimal effect. The results demonstrate that premitotic neuroblasts in the CNS, as well postmitotic neurons, are responsive to NGF. At the early stage of its development, the cerebellum therefore appears to be a very good autocrine model of NGF action.

  20. Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Horwitz, M.S.; Friefeld, B.R.; Keiser, H.D.

    1982-12-01

    Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60/sup 0/C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases ..cap alpha.., BETA, ..gamma.. and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.

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

    Directory of Open Access Journals (Sweden)

    Tuomas Rönnberg

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

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

    Science.gov (United States)

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

    2012-01-01

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

  3. DNA polymerase I-mediated ultraviolet repair synthesis in toluene-treated Escherichia coli

    International Nuclear Information System (INIS)

    DNA synthesis after ultraviolet irradiation is low in wild type toluene-treated cells. The level of repair incorporation is greater in strains deficient in DNA polymerase I. The low level of repair synthesis is attributable to the concerted action of DNA polymerase I and polynucleotide ligase. Repair synthesis is stimulated by blocking ligase activity with the addition of nicotinamide mononucleotide (NMN) or the use of a ligase temperature-sensitive mutant. NMN stimulation is specific for DNA polymerase I-mediated repair synthesis, as it is absent in isogenic strains deficient in the polymerase function or the 5' yields 3' exonuclease function associated with DNA polymerase I. DNA synthesis that is stimulated by NMN is proportional to the ultraviolet exposure at low doses, nonconservative in nature, and is dependent on the uvrA gene product but is independent of the recA gene product. These criteria place this synthesis in the excision repair pathway. The NMN-stimulated repair synthesis requires ATP and is N-ethylmaleimide-resistant. The use of NMN provides a direct means for evaluating the involvement of DNA polymerase I in excision repair

  4. Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

    Directory of Open Access Journals (Sweden)

    Olsen Birgitte B

    2012-03-01

    Full Text Available Abstract Background The DNA-dependent protein kinase (DNA-PK is a nuclear complex composed of a large catalytic subunit (DNA-PKcs and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks. Results In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks. Conclusions Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  7. Synthesis, characterization, DNA binding, DNA cleavage, protein binding and cytotoxic activities of Ru(II) complexes.

    Science.gov (United States)

    Thota, Sreekanth; Vallala, Srujana; Yerra, Rajeshwar; Rodrigues, Daniel Alencar; Raghavendra, Nulgumnalli Manjunathaiah; Barreiro, Eliezer J

    2016-01-01

    We report on the synthesis of novel Ru(II) compounds (Ru-1 to Ru-8) bearing R-pdc, 4-Cl-pbinh ligands (where R=4-CF3, 4-F, 4-OH pdc=3-phenyl-5-(1H-pyrrol-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide, pbinh=phenoxybenzylidene isonicotinyl hydrazides) and their in vitro antitumor activity toward the cell lines murine leukemia L1210, human lymphocyte CEM, human epithelial cervical carcinoma HeLa, BEL-7402 and Molt4/C8. Some of the complexes exhibited more potent antiproliferative activity against cell lines than the standard drug cisplatin. Ruthenium complex Ru-2 displayed potent cytotoxicity with than that of cisplatin. DNA-binding, DNA cleavage and protein binding properties of ruthenium complexes with these ligands are reported. Interactions of these ruthenium complexes with DNA revealed an intercalative mode of binding between them. Synchronous fluorescence spectra proved that the interaction of ruthenium complexes with bovine serum albumin (BSA) resulted in a conformational change of the latter.

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

    Science.gov (United States)

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

    2016-01-01

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

  9. Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

    OpenAIRE

    Han, Chunhua; Wani, Gulzar; Zhao, Ran; Qian, Jiang; Sharma, Nidhi; He, Jinshan; Zhu, Qianzheng; Wang, Qi-En; Wani, Altaf A.

    2014-01-01

    Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV ir...

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kiran, Shashi; Oddi, Vineesha [Laboratory of Cancer Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, 500001 (India); Ramakrishna, Gayatri, E-mail: gayatrirama1@gmail.com [Laboratory of Cancer Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, 500001 (India); Laboratory of Cancer Cell Biology, Department of Research, Institute of Liver and Biliary Sciences, Delhi 110070 (India)

    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

  12. Nucleotide excision repair DNA synthesis by excess DNA polymerase beta: a potential source of genetic instability in cancer cells.

    Science.gov (United States)

    Canitrot, Y; Hoffmann, J S; Calsou, P; Hayakawa, H; Salles, B; Cazaux, C

    2000-09-01

    The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error-free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases delta and/or epsilon participate to the repair DNA synthesis step. Using UV and cisplatin-modified DNA templates, we measured in vitro that extracts from cells overexpressing the error-prone DNA polymerase beta exhibited a five- to sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol beta in this step. By using a 28 nt gapped, double-stranded DNA substrate mimicking the product of the incision step, we showed that Pol beta is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error-prone Pol beta in cancer cells could perturb the well-defined specific functions of DNA polymerases during error-free DNA transactions. PMID:10973926

  13. Modulation of cellular and humoral immune responses to anHIV-1 DNA vaccine by interleukin-12 and interleukin-18 DNA immunization

    Institute of Scientific and Technical Information of China (English)

    孙永涛; 王福祥; 孙永年; 徐哲; 王临旭; 刘娟; 白雪帆; 黄长形

    2004-01-01

    Objective: To investigate the effect of interleukin-12 (IL-12) and interleukin-18 (IL-18)DNA immunization on immune response induced by HIV-1 DNA vaccine and to explore new strategies for therapeutic HIV DNA vaccine.Methods: The recombinant expression vector pCI-neoGAG was constructed by inserting HIV Gag gene into the eukaryotic expression vector pCI-neo. Balb/c mice were immunized with pCI-neoGAG alone or co-immunized with the DNA encoding for IL-12 or IL-18. Anti-HIV antibody and IFN-γ were tested by ELISA, and splenocytes were isolated for detecting antigen-specific lymphoproliferative responses and specific CTL response by MTT assay and LDH assay respectively. Results: The antiHIV antibody titers of mice co-immunized with pCI-neoGAG and the DNA encoding for IL-12 or IL-18 were lower than that of mice immunized with pCI-neoGAG alone( P < 0.01). In contrast, the IFN-γ level of mice co-immunized with pCI-neoGAG and the DNA encoding for IL-12 or IL-18 was higher than that of mice immunized with pCI-neoGAG alone ( P <0.01). Furthermore, compared with mice injected with pCI-neoGAG alone, the specific CTL cytotoxity activity and antigenspecific lymphoproliferative responses of mice immunized with pCI-neoGAG and the DNA encoding for IL-12 or IL-18 were significantly enhanced respectively ( P < 0.01). Conclusion: The DNA encoding for IL-12 or IL-18 together with HIV DNA vaccine may enhance specific Th-1 responses and cellular immune response elicited in mice. Hence, the DNA encoding for IL-12 or IL-18 are promising immune adjuvants for HIV-1 DNA vaccine.

  14. DNA synthesis as an index of the cell reaction to irradiation and other damaging exposures

    International Nuclear Information System (INIS)

    Recent investigation results, showing the outlook of DNA synthesis suppresion determination method as a test for estimating and predicting cell sensitivity to irradiation and other damageing exposures are presented. Advantages of such a method are noted

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

    Science.gov (United States)

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

    2012-08-21

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

  16. Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases. delta. and. beta. are involved in DNA repair synthesis induced by N-methyl-N prime -nitro-N-nitrosoguanidine

    Energy Technology Data Exchange (ETDEWEB)

    Hammond, R.A.; Miller, M.R. (West Virginia Univ. Health Sciences Center, Morgantown (USA)); McClung, J.K. (Samuel Roberts Noble Foundation, Inc., East Ardmore, OK (USA))

    1990-01-09

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

  17. In vitro synthesis of ribosomal proteins directed by Escherichia coli DNA.

    Science.gov (United States)

    Kaltschmidt, E; Kahan, L; Nomura, M

    1974-02-01

    In vitro synthesis of a number of E. coli 30S ribosomal proteins has been demonstrated in a cell-free system consisting of ribosomes, initiation factors, RNA polymerase, a fraction containing soluble enzymes and factors, and E. coli DNA. DNA-dependent synthesis of the following 30S proteins has been demonstrated: S4, S5, S7, S8, S9, S10, S13, S14, S16, S19, and S20.

  18. Stimulation of rat bladder epithelial DNA synthesis intravesical instillation of distilled water

    International Nuclear Information System (INIS)

    Two commonly used cystoscopic infusion fluids were examined to determine whether their infusion stimulates DNA synthesis of the bladder epithelium. Following a single intravesical dose of 0.5 ml of distilled water or 1.5% L-glycine solution, rats were killed periodically up to 1 week. A transient but significant increase in epithelial cell [3H]thymidine labeling was observed at 48 hr after distilled water instillation. Glycine solution did not stimulate DNA synthesis

  19. DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis.

    Science.gov (United States)

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J; Xing, Chao; Wang, Richard C; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R; Burstein, Ezra

    2016-05-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.

  20. Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips.

    Science.gov (United States)

    Kosuri, Sriram; Eroshenko, Nikolai; Leproust, Emily M; Super, Michael; Way, Jeffrey; Li, Jin Billy; Church, George M

    2010-12-01

    Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

  1. DNA mismatch repair efficiency and fidelity are elevated during DNA synthesis in human cells

    International Nuclear Information System (INIS)

    DNA mismatch repair (MMR) within human cells is hypothesized to occur primarily at the replication fork. However, experimental models measuring MMR activity at specific phases of the cell cycle and during genomic DNA synthesis are lacking. We have investigated MMR activity within the nuclear environment of HeLa cells after enriching for G1, S and G2/M phase of the cell cycle by centrifugal elutriation. This approach preserves physiologically normal MMR activity in cell populations subdivided into different phases of the cell cycle. Here we have shown that nuclear protein concentration of hMutSα and hMutLα increases as cells progress into S phase during routine cell culture. MMR activity, as measured by both in vitro and in vivo approaches, increases during S phase to the highest extent within normally growing cells. Both fidelity and activity of MMR are highest on actively replicating templates within intact cells during S phase. The MMR pathway however, is also active at lower levels at other phases of the cell cycle, and on nonreplicating templates

  2. A proposed role played by benzene itself in the induction of acute cytopenia: inhibition of DNA synthesis.

    Science.gov (United States)

    Lee, E W; Garner, C D; Johnson, J T

    1988-04-01

    A single intraperitoneal dose of benzene (880 mg/kg) in mice inhibited DNA synthesis of bone marrow cells within one hour postinjection. However, there was no inhibitory effect on the synthesis of heme and protein at that dosage. Dose-dependent inhibition of DNA synthesis by benzene was observed over the range of 440 to 1760 mg/kg, supporting the idea that cytopenia which was observed by others following multiple doses of benzene (e.g., 440 or 880 mg/kg) might be due to the inhibitory effect of benzene on DNA synthesis. In our studies, benzene concentrations above 81 micrograms/g wet bone marrow resulted in inhibition of DNA synthesis, regardless of whether it was given ip or by inhalation. The effect of benzene itself, rather than its toxic metabolites, on DNA synthesis was further seen in experiments using a bone marrow cell culture system and cell-free DNA synthetic system. Experimental results demonstrated that benzene alone was capable of inhibiting the DNA synthesis of bone marrow cells and that the reduced DNA synthesis resulted from the inhibitory effect of benzene on DNA polymerase alpha, the enzyme that catalyzes the last step of the DNA synthetic pathway. Thus, benzene itself could play a significant role in inducing myelotoxicity in the case of acute or subacute toxicity by exerting its inhibitory effect on DNA synthesis.

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

    Science.gov (United States)

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

    2010-06-23

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

  4. Quantification of cellular uptake of DNA nanostructures by qPCR

    DEFF Research Database (Denmark)

    Okholm, Anders Hauge; Nielsen, Jesper Sejrup; Vinther, Mathias;

    2014-01-01

    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 peptid...

  5. Macro-cellular silica foams: synthesis during the natural creaming process of an oil-in-water emulsion.

    Science.gov (United States)

    Sen, T; Tiddy, G J T; Casci, J L; Anderson, M W

    2003-09-01

    The room-temperature synthesis of a macro-mesoporous silica material during the natural creaming process of an oil-in-water emulsion is reported. The material has 3-dimensional interconnected macropores with a strut-like structure similar to meso-cellular silica foams with mesoporous walls of worm-hole structure. The material has very high surface area (approximately 800 m2 g(-1)) with narrow mesopore size distribution.

  6. Mutations for Worse or Better: Low-Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly Regulated Double-Edged Sword.

    Science.gov (United States)

    Jaszczur, Malgorzata; Bertram, Jeffrey G; Robinson, Andrew; van Oijen, Antoine M; Woodgate, Roger; Cox, Michael M; Goodman, Myron F

    2016-04-26

    1953, the year of Watson and Crick, bore witness to a less acclaimed yet highly influential discovery. Jean Weigle demonstrated that upon infection of Escherichia coli, λ phage deactivated by UV radiation, and thus unable to form progeny, could be reactivated by irradiation of the bacterial host. Evelyn Witkin and Miroslav Radman later revealed the presence of the SOS regulon. The more than 40 regulon genes are repressed by LexA protein and induced by the coproteolytic cleavage of LexA, catalyzed by RecA protein bound to single-stranded DNA, the RecA* nucleoprotein filament. Several SOS-induced proteins are engaged in repairing both cellular and extracellular damaged DNA. There's no "free lunch", however, because error-free repair is accompanied by error-prone translesion DNA synthesis (TLS), involving E. coli DNA polymerase V (UmuD'2C) and RecA*. This review describes the biochemical mechanisms of pol V-mediated TLS. pol V is active only as a mutasomal complex, pol V Mut = UmuD'2C-RecA-ATP. RecA* donates a single RecA subunit to pol V. We highlight three recent insights. (1) pol V Mut has an intrinsic DNA-dependent ATPase activity that governs polymerase binding and dissociation from DNA. (2) Active and inactive states of pol V Mut are determined at least in part by the distinct interactions between RecA and UmuC. (3) pol V is activated by RecA*, not at a blocked replisome, but at the inner cell membrane.

  7. Does trans-lesion synthesis explain the UV-radiation resistance of DNA synthesis in C. elegans embryos

    Energy Technology Data Exchange (ETDEWEB)

    Hartman, Phil; Reddy, Jennifer; Svendsen, Betty-Ann (Texas Christian Univ., Fort Worth, TX (United States). Dept. of Biology)

    1991-09-01

    Over 10-fold larger fluences were required to inhibit both DNA synthesis and cell division in wild-type C.elegans embryos as compared with other model systems or C.elegans rad mutants. In addition, unlike in other organisms, the molecular weight of daughter DNA strands was reduced only after large, superlethal fluences. The molecular weight of nascent DNA fragments exceeded the interdimer distance by up to 19-fold, indicating that C.elegans embryos can replicate through non-instructional lesions. This putative trans-lesion synthetic capability may explain the refractory nature of UV-radiation on embryonic DNA synthesis and nuclear division in C.elegans. (author). 42 refs.; 7 figs.

  8. Impact and mechanism of TiO2 nanoparticles on DNA synthesis in vitro

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The impact of TiO2 nanoparticles on DNA synthesis in vitro in the dark and the molecular mechanism of such impact were studied. The impact of TiO2 nanoparticles on DNA synthesis was investigated by adding TiO2 nanoparticles in different sizes and at various concentrations into the polymerase chain reaction (PCR) system. TiO2 nanoparticles were premixed with the DNA polymerase, the primer or the template, respectively and then the supernatant and the precipitation of each mixture were added into the PCR system separately to observe the impact on DNA synthesis. Sequentially the interaction be- tween TiO2 nanoparticles and the DNA polymerase, the primer or the template was further analyzed by using UV-visible spectroscopy and polyacrylamide gel electrophoresis (PAGE). The results suggest that TiO2 nanoparticles inhibit DNA synthesis in the PCR system in the dark more severely than mi- croscale TiO2 particles at the equivalent concentration and the inhibition effect of TiO2 nanoparticles is concentration dependent. The molecular mechanism of such inhibition is that in the dark, TiO2 nanoparticles interact with the DNA polymerase through physical adsorption while TiO2 nanoparticles do with the primer or the template in a chemical adsorption manner. The disfunction levels of the bio-molecules under the impact of TiO2 nanoparticles are in the following order: the primer > the tem- plate > the DNA polymerase.

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

    Science.gov (United States)

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

    2012-02-01

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

  10. Involvement of sulfoquinovosyl diacylglycerol in DNA synthesis in Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Aoki Motohide

    2012-02-01

    Full Text Available Abstract Background Sulfoquinovosyl diacylglycerol (SQDG is present in the membranes of cyanobacteria and their postulated progeny, plastids, in plants. A cyanobacterium, Synechocystis sp. PCC 6803, requires SQDG for growth: its mutant (SD1 with the sqdB gene for SQDG synthesis disrupted can grow with external supplementation of SQDG. However, upon removal of SQDG from the medium, its growth is retarded, with a decrease in the cellular content of SQDG throughout cell division, and finally ceases. Concomitantly with the decrease in SQDG, the maximal activity of photosynthesis at high-light intensity is repressed by 40%. Findings We investigated effects of SQDG-defect on physiological aspects in Synechocystis with the use of SD1. SD1 cells defective in SQDG exhibited normal photosynthesis at low-light intensity as on culturing. Meanwhile, SD1 cells defective in SQDG were impaired in light-activated heterotrophic growth as well as in photoautotrophic growth. Flow cytometric analysis of the photoautotrophically growing cells gave similar cell size histograms for the wild type and SD1 supplemented with SQDG. However, the profile of SD1 defective in SQDG changed such that large part of the cell population was increased in size. Of particular interest was the microscopic observation that the mitotic index, i.e., population of dumbbell-like cells with a septum, increased from 14 to 29% in the SD1 culture without SQDG. Flow cytometric analysis also showed that the enlarged cells of SD1 defective in SQDG contained high levels of Chl, however, the DNA content was low. Conclusions Our experiments strongly support the idea that photosynthesis is not the limiting factor for the growth of SD1 defective in SQDG, and that SQDG is responsible for some physiologically fundamental process common to both photoautotrophic and light-activated heterotrophic growth. Our findings suggest that the SQDG-defect allows construction of the photosynthetic machinery at an

  11. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

    OpenAIRE

    Yehezkel, Tuval Ben; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2015-01-01

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and a...

  12. Epitope DNA vaccines against tuberculosis: spacers and ubiquitin modulates cellular immune responses elicited by epitope DNA vaccine

    Institute of Scientific and Technical Information of China (English)

    Wang QM; Sun SH; Hu ZL; Zhou FJ; Yin M; Xiao CJ; Zhang JC

    2005-01-01

    Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed epitope DNA vaccines (p3-M-38) encoding cytotoxic T lymphocyte (CTL) epitopes of MPT64 and 38 kDa proteins of Mycobacterium tuberculosis. In order to observe the influence of spacer sequence (Ala-Ala-Tyr) or ubiquitin (UbGR) on the efficacy of the two CTL epitopes, we also constructed DNA vaccines, p3-M-S(spacer)-38, p3-Ub (UbGR)-M-S-38 and p3-Ub-M-38. The immune responses elicited by the four DNA vaccines were tested in C57BL/6 (H-2b) mice. The cytotoxicity of T cells was detected by LDH-release method and by enzyme-linked immunospot assay for epitope-specific cells secreting interferon-gamma. The results showed that DNA immunization with p3-M-38 vaccine could induce epitope-specific CD8+ CTL response and that the spacer sequence (AAY) only enhanced M epitope presentation. The protein-targeting sequence (UbGR) enhanced the immunogenicity of the two epitopes. The finding that defined spacer sequences at C-terminus and protein-targeting degradation modulated the immune response of epitope string DNA vaccines will be of importance for the further development of multi-epitope DNA vaccines against tuberculosis.

  13. Dynamic modeling of cellular response to DNA damage based on p53 stress response networks

    Institute of Scientific and Technical Information of China (English)

    Jinpeng Qi; Yongsheng Ding; Shihuang Shao

    2009-01-01

    Under acute perturbations from the outside, cells can trigger self-defensive mechanisms to fight against genome stress. To investigate the cellular response to continuous ion radiation (IR), a dynamic model for p53 stress response networks at the cellular level is proposed. The model can successfully be used to simulate the dynamic processes of double-strand breaks (DSBs) generation and their repair, switch-like ataxia telangiectasia mutated (ATM) activation, oscillations occurring in the p53-MDM2 feedback loop, as well as toxins elimination triggered by p53 stress response networks. Especially, the model can predict the plausible outcomes of cellular response under different IR dose regimes.

  14. Synergistic and additive effects of cimetidine and levamisole on cellular immune responses to hepatitis B virus DNA vaccine in mice.

    Science.gov (United States)

    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. PMID:23298196

  15. Cell-free assay measuring repair DNA synthesis in human fibroblasts

    International Nuclear Information System (INIS)

    Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

  16. 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

    Science.gov (United States)

    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

  17. Three Novel cis-Acting Elements Required for Efficient Plus-Strand DNA Synthesis of the Hepatitis B Virus Genome

    OpenAIRE

    Lee, Jehan; Shin, Myeong-Kyun; Lee, Hye-Jin; Yoon, Gyesoon; Ryu, Wang-Shick

    2004-01-01

    Synthesis of the relaxed-circular (RC) DNA genomes of hepadnaviruses by reverse transcriptase involves two template switches during plus-strand DNA synthesis. These template switches require repeat sequences (so-called donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. To determine cis-acting elements apart from the donor and acceptor sites that are required for plus-strand RC DNA synthesis by hepatitis B virus (HBV), a series of mutants bearing a s...

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Saleem Bani Hani

    2014-05-01

    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.

  1. Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant

    Institute of Scientific and Technical Information of China (English)

    陈建忠; 朱海红; 刘克洲; 陈智

    2004-01-01

    Objective:To investigate the effect of interleukin-18 (IL-18) on immune response induced by plasmid encoding hepatitis B virus middle protein antigen and to explore new strategies for prophylactic and therapeutic HBV DNA vaccines.Methods:BALB/c mice were immunized with pCMV-M alone or co-immunized with pcDNA3-18 and pCMV-M and then their sera were collected for analysing anti-HBsAg antibody by ELISA;splenocytes were isolated for detecting specific CTL response and cytokine assay in vitro.Results:The anti-HBs antibody level of mice co-immunized with pcDNA3-18 and pCMV-M was slightly higher than that of mice immunized with pCMV-M alone,but there was not significantly different (P>0.05).Compared with mice injected with pCMV-M, the specific CTL cytotoxity activity of mice immunized with pcDNA3-18 and pCMV-M was significantly enhanced (P0.05).Conclusion:The plasmid encoding IL-18 together with HBV M gene DNA vaccines may enhance specific TH1 cells and CTL cellular immune response induced in mice, so that IL-18 is a promising immune adjuvant.

  2. Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant

    Institute of Scientific and Technical Information of China (English)

    陈建忠; 朱海红; 刘克洲; 陈智

    2004-01-01

    Objective: To investigate the effect of interleukin-18 (IL-18) on immune response induced by plasmid encoding hepatitis B virus middle protein antigen and to explore new strategies for prophylactic and therapeutic HBV DNA vaccines. Methods: BALB/c mice were immunized with pCMV-M alone or co-immunized with pcDNA3-18 and pCMV-M and then their sera were collected for analysing anti-HBsAg antibody by ELISA; splenocytes were isolated for detecting specific CTL response and cytokine assay in vitro. Results: The anti-HBs antibody level of mice co-immunized with pcDNA3-18 and pCMV-M was slightly higher than that of mice immunized with pCMV-M alone, but there was not significantly different (P>0.05). Compared with mice injected with pCMV-M, the specific CTL cytotoxity activity of mice immunized with pcDNA3-18 and pCMV-M was significantly enhanced (P0.05). Conclusion: The plasmid encoding IL-18 together with HBV M gene DNA vaccines may enhance specific TH1 cells and CTL cellular immune response induced in mice, so that IL-18 is a promising immune adjuvant.

  3. DNA repair pathways in radiation induced cellular damage: a molecular approach

    NARCIS (Netherlands)

    L.R. van Veelen (Lieneke)

    2005-01-01

    markdownabstract__Abstract__ DNA damage, especially double-strand breaks, can be induced by endogenous or exogenous darnaging agents, such as ionizing radiation. Repair of DNA damage is very important in maintaining genomic stability. Incorrect repair may lead to chromosomal aberrations, translocat

  4. Cellular target of UVB-induced DNA damage resulting in local suppression of contact hypersensitivity

    NARCIS (Netherlands)

    Vink, A.A.; Shreedhar, V.; Roza, L.; Krutmann, J.; Kripke, M.L.

    1998-01-01

    Experimental data are reviewed that lend support to the hypothesis that formation of DNA damage is the initiation event of local suppression of contact hypersensitivity (CHS) after exposure to ultraviolet (UV) radiaton and that the antigen-presenting cell (APC) is an important traget for this DNA da

  5. Methods to assess the nucleocytoplasmic shuttling of the HPV E1 helicase and its effects on cellular proliferation and induction of a DNA damage response.

    Science.gov (United States)

    Lehoux, Michaël; Fradet-Turcotte, Amélie; Archambault, Jacques

    2015-01-01

    Replication of the human papillomavirus (HPV) double-stranded DNA genome in the nucleus of infected cells relies on the viral proteins E1 and E2 in conjunction with the host DNA replication machinery. This process is tightly linked to the replication of cellular DNA, in part through the cyclin-dependent phosphorylation of E1, which inhibits its export out of the nucleus to promote its accumulation in this compartment during S-phase. It has been recently shown that accumulation of E1 in the nucleus, while a prerequisite for viral DNA replication, leads to the inhibition of cellular proliferation and the activation of a DNA damage response (DDR). Here we describe methods to monitor the subcellular localization of E1 and to assess the deleterious effects of its nuclear accumulation on cellular proliferation, cell cycle progression and the induction of a DDR, using a combination of colony formation assays, immunofluorescence microcopy, and flow cytometry approaches. PMID:25348298

  6. Correlation between the levels of N6-(Δ2-isopentenyl)-adenosine and synthesis of DNA in germinating rice seeds

    International Nuclear Information System (INIS)

    Levels of N6(Δ2-isopentenyl) adenosine as determined by radioimmunoassay increased up to 18 hr, then decreased till 24 hr and again increased up to 48 hr, during the germination of rice seeds. The synthesis of isopentenyl-adenosine as followed by the incorporation of 14C-adenine and precipitation with the antibodies for the hormone showed no significant change up to 24 hr. This may imply that the hormone was generated from the stored precursors. Synthesis of DNA proceeded in cycles with the peaks of incorporation of 3H-thymidine at 18 and 36 hr suggesting a correlation between the levels of cytokinin and the synthesis of DNA up to 24 hr. Cordycopin inhibited the synthesis of isopentenyladenosine and DNA and the inhibition of the synthesis of DNA was reversed by the presence of the hormone. This indicated the involvement of cytokinin in the synthesis of DNA. (author)

  7. Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

    International Nuclear Information System (INIS)

    Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated [3H] uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity. (Author)

  8. DNA Synthesis, Assembly and Applications in Synthetic Biology

    OpenAIRE

    Ma, Siying; Tang, Nicholas; Tian, Jingdong

    2012-01-01

    The past couple of years saw exciting new developments in microchip-based gene synthesis technologies. Such technologies hold the potential for significantly increasing the throughput and decreasing the cost of gene synthesis. Together with more efficient enzymatic error correction and genome assembly methods, these new technologies are pushing the field of synthetic biology to a higher level.

  9. The effect of nitroimidazole and nitroxyl radiosensitizers on the post-irradiation synthesis of DNA

    International Nuclear Information System (INIS)

    The modification of DNA damage by three radiosensitizing drugs, present during γ-irradiation of hypoxic Chinese hamster cells, was investigated. Both 2-methyl-5-nitroimidazole-1-ethanol (metronidazole) and 1-(2-nitro-1-imidazole)-3-methoxy-2-propanol (Ro-07-0582) were found to cause large increases in the yield of DNA single-strand breaks (SSB); triacetoneamine-N-oxyl (TAN) was found to have only a small effect on SSB production. The three drugs tested did not inhibit the rejoining of SSB. A pulse label and chase procedure was used to examine post-irradiation DNA synthesis. TAN present during irradiation under hypoxia was found to cause interruptions in subsequent DNA synthesis. Metronidazole and Ro-07-0582 had no effect on post-irradiation DNA synthesis. In addition, the effects of pre- and post-irradiation exposure to TAN were investigated, since these treatments have shown increased cell-killing in survival studies. TAN pre- and post-treatments were found to have no significant effect on subsequent DNA synthesis. (author)

  10. Cockayne's syndrome: correlation of clinical features with cellular sensitivity of RNA synthesis to UV irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Lehmann, A.R.; Thompson, A.F.; Harcourt, S.A. (Medical Research Council, Brighton (United Kingdom). Cell Mutation Unit); Stefanini, Miria (Consiglio Nazionale delle Ricerche, Pavia (Italy). Ist. di Genetica Biochimica ed Evoluzionistica); Norris, P.G. (Addenbrooke' s Hospital, Cambridge (United Kingdom))

    1993-08-01

    Cockayne's syndrome (CS) is a rare autosomal recessive disorder with dwarfism, mental retardation, and otherwise clinically heterogeneous features. In cultured CS fibroblasts, the failure of RNA synthesis to recover to normal rates after UV-C irradiation provides a useful and relatively simple diagnostic test. We have measured post-UV-C RNA synthesis in 52 patients for whom a clinical diagnosis of CS was considered a possibility. Twenty-nine patients showed the defect characteristic of CS cells, and 23 had a normal response. We have attempted to correlate the cellular diagnosis with the different clinical features of the disorder. Clinical details of the patients were obtained from referring clinicians in the form of a questionnaire. Our results show that, apart from the cardinal features of dwarfism and mental retardation, sun sensitivity correlated best with a positive cellular diagnosis. Pigmentary retinopathy, gait defects, and dental caries were also good positive indicators, although several patients with a positive cellular diagnosis did not have these features. (Author).

  11. Flexible double-headed cytosine-linked 2'-deoxycytidine nucleotides. Synthesis, polymerase incorporation to DNA and interaction with DNA methyltransferases.

    Science.gov (United States)

    Kielkowski, Pavel; Cahová, Hana; Pohl, Radek; Hocek, Michal

    2016-03-15

    New types of double-headed 2'-deoxycytidine 5'-O-triphosphates (dC(XC)TPs) bearing another cytosine or 5-fluorocytosine linked through a flexible propargyl, homopropargyl or pent-1-ynyl linker to position 5 were prepared by the aqueous Sonogashira cross-coupling reactions of 5-iodo-dCTP with the corresponding (fluoro)cytosine-alkynes. The modified dC(XC)TPs were good substrates for DNA polymerases and were used for enzymatic synthesis of cytosine-functionalized DNA by primer extension or PCR. The cytosine- or fluorocytosine-linked DNA probes did not significantly inhibit DNA methyltransferases and did not cross-link to these proteins. PMID:26899597

  12. Regulation of chloroplast number and DNA synthesis in higher plants. Final report, August 1995--August 1996

    Energy Technology Data Exchange (ETDEWEB)

    Mullet, J.E.

    1997-06-17

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focused on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The research focused on the isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

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

    Science.gov (United States)

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

    2011-12-27

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

  14. Ribonucleotide reductase activity is coupled to DNA synthesis via proliferating cell nuclear antigen.

    Science.gov (United States)

    Salguero, Israel; Guarino, Estrella; Shepherd, Marianne E A; Deegan, Tom D; Havens, Courtney G; MacNeill, Stuart A; Walter, Johannes C; Kearsey, Stephen E

    2012-04-24

    Synthesis of deoxynucleoside triphosphates (dNTPs) is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimizing the mutation rate [3-7], and this is achieved by tight regulation of RNR [2, 8, 9]. In fission yeast, RNR is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow upregulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4(Cdt2) ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels, which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 level fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor proliferating cell nuclear antigen (PCNA), complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and RNR regulation. PMID:22464192

  15. Exposure to environmental polycyclic aromatic hydrocarbons: Influences on cellular susceptibility to DNA damage (sampling Kosice and Sofia)

    International Nuclear Information System (INIS)

    The aim of this study was to investigate a possible influence of occupational exposure to carcinogenic environmental polycyclic aromatic hydrocarbons (c-PAHs) on cellular susceptibility to the induction of the DNA damage. Monitoring was performed and blood samples were collected from two groups of male subjects: occupationally exposed and matched controls. The group exposed to c-PAHs (average age of 35.1 years) consisted of 52 policemen from Kosice and 26 policemen and 25 bus drivers (51 altogether) from Sofia. The control group (average age of 36.4 years) consisted of 54 unexposed subjects from Kosice and 24 from Sofia. In the investigated groups 52.5% of exposed subjects and 45.3% of control were current smokers. A challenging dose of X-rays (3 Gy) and an alkaline version of the single cell gel electrophoresis (SCGE) assay, known as Comet assay, were used to evaluate levels of induced DNA damage and repair kinetics in isolated human blood lymphocytes. DNA damage detected in lymphocytes prior to or after irradiation did not differ significantly between exposed and unexposed subjects. A significant decrease in repair efficiency due to exposure to PAHs was observed in the exposed individuals from Kosice and Sofia, when analysed separately or together. A negative influence of tobacco smoking on the efficiency of DNA repair was observed. Statistically significant differences were found between subgroups stratified according to education level in Sofia: the half times for DNA repair declined with the increasing level of education. These results confirm that environmental exposure to c-PAHs can alter the ability of blood lymphocytes to repair DNA damage and, as a result could potentially lead to effects that are hazardous to human health

  16. Typical xeroderma pigmentosum complementation group A fibroblasts have detectable ultraviolet light-induced unscheduled DNA synthesis

    International Nuclear Information System (INIS)

    Ultraviolet-induced nuclear uptake of tritiated thymidine [3H]dThd demonstrable by autoradiography in non-synthesis phases of the cell cycle is known as unscheduled DNA synthesis and reflects repair replication of ultraviolet-damaged DNA. We have reported that the rate of any such unscheduled DNA synthesis in typical group A xeroderma pigmentosum fibroblasts, if present, is less than 2% of the normal rate. We have now performed experiments to determine whether these fibroblasts have any unscheduled DNA synthesis. Fibroblast coverslip cultures of four xeroderma pigmentosum group A strains were prepared. Irradiated (254 nm ultraviolet light) and unirradiated cultures from each strain were incubated with [3H]dThd at 37degC, and autoradiograms were prepared using NTB-3 emulsion. A nuclear grain count was made of 100 consecutive nuclei of non-S-phase irradiated and unirradiated cells. A slide background grain count was simultaneously made from an acellular area adjacent to each cell analyzed. When a strain's irradiated and unirradiated autoradiograms having similar slide background grain count averages were compared, the nuclear grain count average of the irradiated cells was always higher than that of the unirradiated cells. This ultraviolet-induced increase in the mean nuclear grain count ranged from 0.4 to 1.3% of that given by normal non-xeroderma pigmentosum fibroblasts and was not reduced by 10-2M hydroxyurea. Planimetric studies showed that the ultraviolet-induced increase in nuclear grain count is not due to an increased nuclear area in irradiated cells. We conclude that these typical group A xeroderma pigmentosum strains perform very low, but detectable, ultraviolet-induced unscheduled DNA synthesis which probably reflects repair replication. We cannot, however, determine if there are significantly different rates of ultraviolet-induced unscheduled DNA synthesis among these ultraviolet strains

  17. A cell-free system for DNA repair synthesis using purified enzymes from the Novikoff hepatoma

    International Nuclear Information System (INIS)

    Novikoff DNA polymerase-β and Novikoff DNase V have been used in a cell-free DNA excision repair system for UV-irradiated substrates to determine their DNA repair capabilities. The repair system was shown to depend upon UV-irradiated DNA, incision by phage T4 UV-endonuclease, excision by DNase V and synthesis by DNA polymerase-β; ligation was not included. Highly purified calf thymus DNA was UV-irradiated at 500-750 J/m2 and incised by T4 UV-endonuclease. The repair system was used to follow the purification of DNase V and DNA polymerase-β. For increased specificity, the parameters of UV-irradiation, incision, excision and synthesis were confirmed on highly supercoiled, covalently closed, phage PM2 DNA. Optimal DNA and Mg2+ concentrations were determined for the repair assay, which was shown to be linear with respect to time. Excision of the 3'-apyrimidinic site and the 5'-pyrimidine dimer by bidirectional DNase V, presumed to occur from the above experiments, was studied more thoroughly using lightly UV-irradiated [3H]poly(dT)poly (dA), labeled in both the base and the sugar, and incised with T4 UV-endonuclease

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

    Science.gov (United States)

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

    2016-09-01

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

  19. Effect of α-Particle and X-Ray Irradiation on DNA Synthesis in Tissue Cultures

    International Nuclear Information System (INIS)

    The effect of both a-particle and X-ray irradiation on the rate of DNA synthesis in mouse fibroblast and HeLa cells in tissue culture is described. Tritiated thymidine micro autoradiography was used to indicate the rate of synthesis in the single layer cultures used. The results of the experiments show that: (1) The fraction of cells in a culture synthesizing DNA is unaffected by α-particles and X-rays in the doses used in the experiment. (2) The effect of either type of radiation is to reduce the rate of synthesis of DNA of the irradiated cells in synthesis. (3) The effect of a given dose of either type of radiation is to reduce the rate of synthesis of all the cells to a constant fraction of what it was in the unirradiated cells. (4) The rate of DNA synthesis is reduced to 37% (1/e) by a dose of ca. 25 α/μ2 or an X-ray dose of 14000 rad for mouse fibroblast cultures. In Hela cell cultures a dose of ca. 90000 rad is needed to reduce the rate of DNA synthesis to 37% of the initial value. (5) The reduction in synthesis occurs not more than a half hour after irradiation and may be an immediate effect. From (4) above the target shape can be roughly calculated and if it is assumed to be cylindrical it appears to have dimensions ca. 16 Å in one direction and 16 000 Å in the other, i. e. a long thin thread with a MW of ca. 5 * 107 in the case of the mouse fibroblast experiments. In the case of the Hela cell experiments the target volume gives a MW of ca. 107. The results are consistent with the view that the target may possibly be the DNA template (or maybe DNAP because of the high MW in one case). If the effects described reflect damage to the DNA (or DNAP) template during the exponential phase of synthesis then observations (1), (2) and (3) above follow as obvious correlatives. (author)

  20. Synthesis and characterization of a lamellar hydroxyapatite/DNA nanohybrid

    International Nuclear Information System (INIS)

    Research highlights: → A lamellar hydroxyapatite (HAp)/DNA nanohybrid was prepared as a novel gene delivering vector. → Gel electrophoresis analysis confirmed that the lamellar HAp could protect DNA from degradation of DNase I. → The protected DNA in the HAp/DNA nanohybrid could be recovered readily under acid conditions. - Abstract: Two-dimensional layered materials exhibit desired functionalities when being used as gene delivery materials. In this study, a novel gene delivering vector, lamellar hydroxyapatite (HAp)/DNA nanohybrid was prepared. The structure of HAp/DNA nanohybrid was investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Fourier transform infrared (FT-IR) spectroscopy analysis revealed that ion-exchange occurred during the process. Gel electrophoresis analysis confirmed that the lamellar HAp could protect DNA from degradation of DNase I and the protected DNA could be recovered readily under acid conditions. Furthermore, the integrity of released DNA was confirmed by UV-vis spectra.

  1. Damage to cellular and isolated DNA induced by a metabolite of aspirin

    Energy Technology Data Exchange (ETDEWEB)

    Oikawa, Shinji [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan)], E-mail: s-oikawa@doc.medic.mie-u.ac.jp; Kobayashi, Hatasu; Tada-Oikawa, Saeko [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); JSPS Research Fellow (Japan); Isono, Yoshiaki [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); Kawanishi, Shosuke [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie 513-8670 (Japan)

    2009-02-10

    Aspirin has been proposed as a possible chemopreventive agent. On the other hand, a recent cohort study showed that aspirin may increase the risk for pancreatic cancer. To clarify whether aspirin is potentially carcinogenic, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is correlated with the incidence of cancer, in cultured cells treated with 2,3-dihydroxybenzoic acid (2,3-DHBA), a metabolite of aspirin. 2,3-DHBA induced 8-oxodG formation in the PANC-1 human pancreatic cancer cell line. 2,3-DHBA-induced DNA single-strand breaks were also revealed by comet assay using PANC-1 cells. Flow cytometric analyses showed that 2,3-DHBA increased the levels of intracellular reactive oxygen species (ROS) in PANC-1 cells. The 8-oxodG formation and ROS generation were also observed in the HL-60 leukemia cell line, but not in the hydrogen peroxide (H{sub 2}O{sub 2})-resistant clone HP100 cells, suggesting the involvement of H{sub 2}O{sub 2}. In addition, an hprt mutation assay supported the mutagenicity of 2,3-DHBA. We investigated the mechanism underlying the 2,3-DHBA-induced DNA damage using {sup 32}P-labeled DNA fragments of human tumor suppressor genes. 2,3-DHBA induced DNA damage in the presence of Cu(II) and NADH. DNA damage induced by 2,3-DHBA was enhanced by the addition of histone peptide-6 [AKRHRK]. Interestingly, 2,3-DHBA and histone peptide-6 caused base damage in the 5'-ACG-3' and 5'-CCG-3' sequences, hotspots of the p53 gene. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Typical hydroxyl radical scavengers did not inhibit the DNA damage. These results suggest that ROS derived from the reaction of H{sub 2}O{sub 2} with Cu(I) participate in the DNA damage. In conclusion, 2,3-DHBA induces oxidative DNA damage and mutations, which may result in carcinogenesis.

  2. Recovery from DNA synthesis in V 79 chinese hamster cells irradiated with UV light

    International Nuclear Information System (INIS)

    Mammalian cells recover from DNA synthesis inhibition by UV light before most of the pyrimidine dimers have been removed from the genome. Most of the rodent cells show a deficient dimer excision repair compared with normal human fibroblasts. Despite this fact they recover efficiently from DNA synthesis inhibition after UV. In Chinese hamster V 79 cells was found that this recovery takes place in the absence of a significant excision repair, and it seems to be directly coupled to a recovery in the rate of movement of the replication fork. 120 refs, 31 figs. (author)

  3. Assessment of potential damage to DNA in urine of coke oven workers: an assay of unscheduled DNA synthesis.

    OpenAIRE

    Roos, F.; Renier, A.; Ettlinger, J; Iwatsubo, Y; Letourneux, M; Haguenoer, J M; Jaurand, M C; Pairon, J C

    1997-01-01

    OBJECTIVES: A study was conducted in coke oven workers to evaluate the biological consequences of the exposure of these workers, particularly production of potential genotoxic factors. METHODS: 60 coke oven workers and 40 controls were recruited in the same iron and steel works. Exposure to polycyclic aromatic hydrocarbons (PAHs) was assessed by job and measurement of 1-hydroxypyrene (1OHP) in urine samples. An unscheduled DNA synthesis assay was performed on rat pleural mesothelial cells use...

  4. Stimulation of DNA synthesis in cultured rat alveolar type II cells

    Energy Technology Data Exchange (ETDEWEB)

    Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

    1985-01-01

    Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated /sup 3/H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of /sup 3/H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture.

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

    Science.gov (United States)

    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

  6. Capture of a third Mg²⁺ is essential for catalyzing DNA synthesis.

    Science.gov (United States)

    Gao, Yang; Yang, Wei

    2016-06-10

    It is generally assumed that an enzyme-substrate (ES) complex contains all components necessary for catalysis and that conversion to products occurs by rearrangement of atoms, protons, and electrons. However, we find that DNA synthesis does not occur in a fully assembled DNA polymerase-DNA-deoxynucleoside triphosphate complex with two canonical metal ions bound. Using time-resolved x-ray crystallography, we show that the phosphoryltransfer reaction takes place only after the ES complex captures a third divalent cation that is not coordinated by the enzyme. Binding of the third cation is incompatible with the basal ES complex and requires thermal activation of the ES for entry. It is likely that the third cation provides the ultimate boost over the energy barrier to catalysis of DNA synthesis.

  7. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

    Science.gov (United States)

    Ben Yehezkel, Tuval; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-02-29

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

  8. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

    Science.gov (United States)

    Yehezkel, Tuval Ben; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-01-01

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

  9. Quantitative Transcript Analysis in Plants: Improved First-strand cDNA Synthesis

    Institute of Scientific and Technical Information of China (English)

    Nai-Zhong XIAO; Lei BA; Preben Bach HOLM; Xing-Zhi WANG; Steve BOWRA

    2005-01-01

    The quantity and quality of first-strand cDNA directly influence the accuracy of transcriptional analysis and quantification. Using a plant-derived α-tubulin as a model system, the effect of oligo sequence and DTT on the quality and quantity of first-strand cDNA synthesis was assessed via a combination of semi-quantitative PCR and real-time PCR. The results indicated that anchored oligo dT significantly improved the quantity and quality of α-tubulin cDNA compared to the conventional oligo dT. Similarly, omitting DTT from the first-strand cDNA synthesis also enhanced the levels of transcript. This is the first time that a comparative analysis has been undertaken for a plant system and it shows conclusively that small changes to current protocols can have very significant impact on transcript analysis.

  10. Bacterial Intoxication Evokes Cellular Senescence with Persistent DNA Damage and Cytokine Signaling

    DEFF Research Database (Denmark)

    Blazkova, Hana; Krejcikova, Katerina; Moudry, Pavel;

    2009-01-01

    Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram-negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type-dependent cell-cycle arrest or apoptosis; however the cell fate responses to suc...... of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.......Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram-negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type-dependent cell-cycle arrest or apoptosis; however the cell fate responses...... features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may...

  11. Translesion synthesis by yeast DNA polymerase ζ from templates containing lesions of ultraviolet radiation and acetylaminofluorene

    OpenAIRE

    Guo, Dongyu; Wu, Xiaohua; Deepak K Rajpal; Taylor, John-Stephen; Wang, Zhigang

    2001-01-01

    In the yeast Saccharomyces cerevisiae, DNA polymerase ζ (Polζ) is required in a major lesion bypass pathway. To help understand the role of Polζ in lesion bypass, we have performed in vitro biochemical analyses of this polymerase in response to several DNA lesions. Purified yeast Polζ performed limited translesion synthesis opposite a template TT (6-4) photoproduct, incorporating A or T with similar efficiencies (and less frequently G) opposite the 3′ T, and pr...

  12. Differential regulation of the cellular response to DNA double-strand breaks in G1

    DEFF Research Database (Denmark)

    Barlow, Jacqueline H; Lisby, Michael; Rothstein, Rodney

    2008-01-01

    Double-strand breaks (DSBs) are potentially lethal DNA lesions that can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that DSBs induced by ionizing radiation (IR) are efficiently processed for HR and bound by Rfa1 during G1, while endonuclease...

  13. Chromosome number distribution and cellular DNA content in colorectal adenomas from polyposis and nonpolyposis patients

    DEFF Research Database (Denmark)

    Petersen, S E; Madsen, A L; Bak, Martin

    1991-01-01

    Ploidy analyses of colorectal adenomas were performed by combined flow cytometric DNA analysis of unfixed isolated nuclei and direct chromosome preparation after Colcemid incubation for 9-20 hours. Ten of 18 adenomas from nonpolyposis patients and 4 of 13 adenomas from patients with familial aden...

  14. Harmonising measurements of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA and urine

    DEFF Research Database (Denmark)

    Møller, Peter; Cooke, Marcus S; Collins, Andrew;

    2012-01-01

    protocols. Recent attention on optimal conditions for the comet assay may lead to better understanding of the most critical steps in procedure, which generate variation in DNA damage levels between laboratories. Measurements of urinary excretion of oxidatively generated 8-oxo-7,8-dihydro-2'-deoxyguanosine...

  15. [Analysis of effectiveness of cDNA synthesis, induced using complementary primers and primers containing a noncomplementary base matrix].

    Science.gov (United States)

    D'iachenko, L B; Chenchik, A A; Khaspekov, G L; Tatarenko, A O; Bibilashvili, R Sh

    1994-01-01

    We have studied the efficiency of DNA synthesis catalyzed by M-MLV reverse transcriptase or Thermus aquaticus DNA polymerase for primers (4-17 nucleotides long) either completely matched or possessing a single mismatched base pair at all possible positions in the primer. It has been shown that DNA synthesis efficiency depends not only on the position of mismatched base pair but on the length and primary structure of the primer. The enzyme, template, and primer concentrations determine the relative level of mismatched DNA synthesis.

  16. Preparation of fluorescent DNA probe by solid-phase organic synthesis

    Directory of Open Access Journals (Sweden)

    2009-08-01

    Full Text Available Fluorescent DNA probe based on fluorescence resonance energy transfer (FRET was prepared by solid-phase organic synthesis when CdTe quantum dots (QDs were as energy donors and Au nanoparticles (AuNPs were as energy accepters. The poly(divinylbenzene core/poly(4-vinylpyridine shell microspheres, as solid-phase carriers, were prepared by seeds distillation-precipitation polymerization with 2,2′-azobisisobutyronitrile (AIBN as initiator in neat acetonitrile. The CdTe QDs and AuNPs were self-assembled on the surface of core/shell microspheres, and then the linkage of CdTe QDs with oligonucleotides (CdTe-DNA and AuNPs with complementary single-stranded DNA (Au-DNA was on the solid-phase carriers instead of in aqueous solution. The hybridization of complementary double stranded DNA (dsDNA bonded to the QDs and AuNPs (CdTe-dsDNA-Au determined the FRET distance of CdTe QDs and AuNPs. Compared with the fluorescence of CdTe-DNA, the fluorescence of CdTe-dsDNA-Au conjugates (DNA probes decreased extremely, which indicated that the FRET occurred between CdTe QDs and AuNPs. The probe system would have a certain degree recovery of fluorescence when the complementary single stranded DNA was introduced into this system, which showed that the distance between CdTe QDs and AuNPs was increased.

  17. Role of amidation in bile acid effect on DNA synthesis by regenerating mouse liver.

    Science.gov (United States)

    Barbero, E R; Herrera, M C; Monte, M J; Serrano, M A; Marin, J J

    1995-06-01

    Effect of bile acids on DNA synthesis by the regenerating liver was investigated in mice in vivo after partial hepatectomy (PH). Radioactivity incorporation into DNA after [14C]thymidine intraperitoneal administration peaked at 48 h after PH. At this time a significant taurocholate-induced dose-dependent reduction in DNA synthesis without changes in total liver radioactivity content was found (half-maximal effect at approximately 0.1 mumol/g body wt). Effect of taurocholate (0.5 mumol/g body wt) was mimicked by chocolate, ursodeoxycholate, deoxycholate, dehydrocholate, tauroursodeoxycholate, taurochenodeoxycholate, and taurodeoxycholate. In contrast, chenodeoxycholate, glycocholate, glycochenodeoxycholate, glycoursodeoxycholate, glycodeoxycholate, 5 beta-cholestane, bromosulfophthalein, and free taurine lacked this effect. No relationship between hydrophobic-hydrophilic balance and inhibitory effect was observed. Analysis by high-performance liquid chromatography indicated that inhibition of thymidine incorporation into DNA was not accompanied by an accumulation of phosphorylated DNA precursors in the liver but rather by a parallel increase in nucleotide catabolism. Bile acid-induced modifications in DNA synthesis were observed in vivo even in the absence of changes in toxicity tests, which suggests that the inhibitory effect shared by most unconjugated and tauroconjugated bile acids but not by glycoconjugated bile acids should be accounted for by mechanisms other than nonselective liver cell injury. PMID:7611405

  18. Synthesis, DNA interaction and antimicrobial activities of three rimantadine analogues

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bing-Mi; Zhang, Jun [Department of Pharmacy, Liaoning University, Shenyang 110036 (China); Wang, Xin, E-mail: wangxinlnu@163.com [Department of Pharmacy, Liaoning University, Shenyang 110036 (China); Zhang, Li-Ping; Liu, Yang [Department of Pharmacy, Liaoning University, Shenyang 110036 (China); Niu, Hua-Ying [Jinan Dachpharm Development Co., Ltd., Jinan 250100 (China); Liu, Bin, E-mail: liubinzehao@163.com [Department of Pharmacy, Liaoning University, Shenyang 110036 (China)

    2015-03-15

    The interactions of three rimantadine analogues (RAs) with calf thymus deoxyribonucleic acid (ct-DNA) in buffer solution (pH 7.4) were investigated using berberine (BR) as a probe by various methods. Fluorescence studies revealed that the RAs interacted with DNA in vitro and the quenchings were all static. Furthermore, the binding modes of these compounds to DNA were disclosed as groove binding supported by absorption spectroscopy, viscosity measurement and denatured DNA experiment. The antimicrobial activities of the RAs were also evaluated in Staphylococcus aureus and Escherichia coli, and they all exhibited good bacteriostasic effects. The results might provide an important reference for investigation of the molecular mechanism associated with the DNA binding of the RAs. - Highlights: • Three rimantadine analogues were synthesized. • The RAs effectively quenched the intrinsic fluorescence of DNA via a static combination. • These analogues can bind to DNA via groove binding mode. • The antimicrobial activities of three analogues were also evaluated by the disk diffusion method.

  19. Novel synthesis of O 6-alkylguanine containing oligodeoxyribonucleotides as substrates for the human DNA repair protein, O 6-methylguanine DNA methyltransferase (MGMT)

    OpenAIRE

    Shibata, Takayuki; Glynn, Nicola; McMurry, T. Brian H.; McElhinney, R. Stanley; Margison, Geoffrey P.; David M. Williams

    2006-01-01

    The human DNA repair protein O 6-methylguanine DNA methyltransferase (MGMT) dealkylates mutagenic O 6-alkylguanine lesions within DNA in an irreversible reaction which results in inactivation of the protein. MGMT also provides resistance of tumours to alkylating agents used in cancer chemotherapy and its inactivation is therefore of particular clinical importance. We describe a post-DNA synthesis strategy which exploits the novel, modified base 2-amino-6-methylsulfonylpurine and allows access...

  20. Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair.

    Science.gov (United States)

    Han, Chunhua; Wani, Gulzar; Zhao, Ran; Qian, Jiang; Sharma, Nidhi; He, Jinshan; Zhu, Qianzheng; Wang, Qi-En; Wani, Altaf A

    2015-01-01

    Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3' side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4(Cdt2). Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4(Cdt2) for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER. PMID:25483071

  1. Deoxyribonucleotide synthesis and the emergence of DNA in molecular evolution

    Science.gov (United States)

    Follmann, Hartmut

    1982-02-01

    DNA replication requires monomeric deoxyribonucleotides, which cannot be regarded as primary products of organic syntheses on a primitive earth. However, the present biosynthetic pathway — reductive elimination of the 2'-OH group from ribonucleotides, catalyzed by ribonucleotide reductases and thioredoxins — suggests an early, polyphyletic combination of protein-nucleotide interactions and metal catalysis. That key process had to precede the upcome of RNA-DNA dualism on the way from RNA-protein protocells to true organisms.

  2. DNA demethylation upregulated Nrf2 expression in Alzheimer's disease cellular model

    Directory of Open Access Journals (Sweden)

    Huimin eCao

    2016-01-01

    Full Text Available 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 mRNA and protein levels via down-regulating 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(PH:quinone oxidoreductas (NQO1. Our study showed that DNA demethylation promoted the Nrf2 cell signaling pathway, which may enhance the antioxidant system against AD development.

  3. Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Binladen, Jonas; Miller, Webb;

    2007-01-01

    Although ancient DNA (aDNA) miscoding lesions have been studied since the earliest days of the field, their nature remains a source of debate. A variety of conflicting hypotheses exist about which miscoding lesions constitute true aDNA damage as opposed to PCR polymerase amplification error...... strand of origin of observed damage events. With the advent of emulsion-based clonal amplification (emPCR) and the sequencing-by-synthesis technology this has changed. In this paper we demonstrate how data produced on the Roche GS20 genome sequencer can determine miscoding lesion strands of origin...

  4. Dissociation between insulin secretion and DNA synthesis in cultured pancreatic islets

    DEFF Research Database (Denmark)

    Nielsen, Jens Høiriis

    1985-01-01

    Glucose has been suggested to be the most important stimulus for beta cell replication in vivo and in vitro. In order to study the relationship between insulin secretion and DNA synthesis, newborn rat islets were cultured in the presence of different concentrations of glucose, theophylline and 3-...

  5. Labelling of Cells Engaged in DNA Synthesis: Autoradiography and BrdU Staining

    DEFF Research Database (Denmark)

    Madsen, Peder Søndergaard

    2010-01-01

    The cell cycle is divided in four phases: G1 phase, S phase (DNA-synthesis), G2 phase (together termed interphase) and M phase (mitosis). Cells that have ceased proliferation enter a state of quiescence called G0. M phase is itself composed of two tightly coupled processes: mitosis, in which...

  6. Human CD4+ T cells require exogenous cystine for glutathione and DNA synthesis

    DEFF Research Database (Denmark)

    Levring, Trine B; Kongsbak-Wismann, Martin; Rode, Anna Kathrine Obelitz;

    2015-01-01

    aim of this study was to elucidate why activated human T cells require exogenous Cys2 in order to proliferate. We activated purified naïve human CD4+ T cells and found that glutathione (GSH) levels and DNA synthesis were dependent on Cys2 and increased in parallel with increasing concentrations of Cys...

  7. Inhibitory effect of syphilitic rabbit serum on DNA synthesis in rabbit cells in vitro.

    OpenAIRE

    Wong, G H; Steiner, B; Strugnell, R; Faine, S.; Graves, S.

    1984-01-01

    A previously described toxic factor associated with Treponema pallidum (Nichols) and found in extracts of syphilitic rabbit testes has now also been detected in syphilitic rabbit serum. The toxic factor, which inhibits DNA synthesis in baby rabbit genital organ (BRGO) cells in vitro, is present in rabbit serum up to 30 days after infection with T pallidum.

  8. Unscheduled DNA synthesis in xeroderma pigmentosum cells after microinjection of yeast photoreactivating enzyme.

    NARCIS (Netherlands)

    J.C.M. Zwetsloot; J.H.J. Hoeijmakers (Jan); W. Vermeulen (Wim); A.P.M. Eker (André); D. Bootsma (Dirk)

    1986-01-01

    textabstractPhotoreactivating enzyme (PRE) from yeast causes a light-dependent reduction of UV-induced unscheduled DNA synthesis (UDS) when injected into the cytoplasm of repair-proficieint human fibroblasts (Zwetsloot et al., 1985). This result indicates that the exogenous PRE monomerizers UV-induc

  9. Ruthenium(II) arene complexes with oligocationic triarylphosphine ligands: synthesis, DNA interactions and in vitro properties

    NARCIS (Netherlands)

    Snelders, D.J.M.; Casini, A.; Edafe, F.; van Koten, G.; Klein Gebbink, R.J.M.; Dyson, P.J.

    2011-01-01

    The synthesis, DNA binding properties and cytotoxicity of a series of Ru(II)-arene complexes containing oligocationic ammonium-functionalized triarylphosphines, of the type Ru(p-cymene)Cl2(L) (L ¼ oligocationic phosphine), are reported. The complexes are highly charged (the overall charge states bei

  10. Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

    International Nuclear Information System (INIS)

    Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells

  11. Free electron laser irradiation at 200 microns affects DNA synthesis in living cells

    International Nuclear Information System (INIS)

    We describe the effect of a 200-microns wavelength free electron laser beam on the ability of asynchronized and synchronized mammalian tissue culture cells to incorporate tritiated thymidine. Compared to controls (unexposed cells), a significant proportion of exposed cells exhibited a reduction in isotope incorporation. The results suggest that this wavelength may affect DNA synthesis

  12. Design, synthesis, and characterization of nucleosomes containing site-specific DNA damage.

    Science.gov (United States)

    Taylor, John-Stephen

    2015-12-01

    How DNA damaged is formed, recognized, and repaired in chromatin is an area of intense study. To better understand the structure activity relationships of damaged chromatin, mono and dinucleosomes containing site-specific damage have been prepared and studied. This review will focus on the design, synthesis, and characterization of model systems of damaged chromatin for structural, physical, and enzymatic studies.

  13. DNA Polymerases Drive DNA Sequencing-by-Synthesis Technologies: Both Past and Present

    Directory of Open Access Journals (Sweden)

    Cheng-Yao eChen

    2014-06-01

    Full Text Available Next-generation sequencing (NGS technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. E. coli DNA polymerase I proteolytic (Klenow fragment was originally utilized in Sanger's dideoxy chain terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ⱷ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ⱷ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.

  14. In vivo effects of T-2 mycotoxin on synthesis of proteins and DNA in rat tissues

    International Nuclear Information System (INIS)

    Rats were given an ip injection of T-2 mycotoxin (T-2), the T-2 metabolite, T-2 tetraol (tetraol), or cycloheximide. Serum, liver, heart, kidney, spleen, muscle, and intestine were collected at 3, 6, and 9 hr postinjection after a 2-hr pulse at each time with [14C]leucine and [3H]thymidine. Protein and DNA synthesis levels in rats were determined by dual-label counting of the acid-precipitable fraction of tissue homogenates. Rats given a lethal dose of T-2, tetraol, or cycloheximide died between 14 and 20 hr. Maximum inhibition of protein synthesis at the earliest time period was observed in additional rats given the same lethal dose of the three treatments and continued for the duration of the study (9 hr). With sublethal doses of T-2 or tetraol, the same early decrease in protein synthesis was observed but, in most of the tissues, recovery was seen with time. In the T-2-treated rats. DNA synthesis in the six tissues studied was also suppressed, although to a lesser degree. With sublethal doses, complete recovery of DNA synthesis took place in four of the six tissues by 9 hr after toxin exposure. The appearance of newly translated serum proteins did not occur in the animals treated with T-2 mycotoxin or cycloheximide, as evidenced by total and PCA-soluble serum levels of labeled leucine. An increase in tissue-pool levels of free leucine and thymidine in response to T-2 mycotoxin was also noted. T-2 mycotoxin, its metabolite, T-2 tetraol, and cycloheximide cause a rapid inhibition of protein and DNA synthesis in all tissue types studied. These results are compared with the responses seen in in vitro studies

  15. Synthesis of heterocycles: Indolo (2,1-a) isoquinolines, renewables, and aptamer ligands for cellular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Beasley, Jonathan [Ames Laboratory (AMES), Ames, IA (United States)

    2013-01-01

    In this thesis, we explore both total syntheses and methodologies of several aromatic heterocyclic molecules. Extensions of the Kraus indole synthesis toward 2-substituted and 2,3-disubstituted indoles, as well as biologically attractive indolo[2,1-a]isoquinolines are described. Recent renewable efforts directed to commodity maleic acid and the first reported furan-based ionic liquids are described. Our total synthesis of mRNA aptamer ligand PDC-Gly, and its dye coupled forms, plus aminoglycoside dye coupled ligands used in molecular imaging, are described.

  16. Synthesis and NMR of {sup 15}N-labeled DNA fragments

    Energy Technology Data Exchange (ETDEWEB)

    Jones, R.A. [Rutgers, The State Univ. of New Jersey, Piscataway, NJ (United States)

    1994-12-01

    DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

  17. Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA.

    Science.gov (United States)

    Bullard, Whitney; Cliffe, Laura; Wang, Pengcheng; Wang, Yinsheng; Sabatini, Robert

    2015-12-01

    Telomeric DNA of trypanosomatids possesses a modified thymine base, called base J, that is synthesized in a two-step process; the base is hydroxylated by a thymidine hydroxylase forming hydroxymethyluracil (hmU) and a glucose moiety is then attached by the J-associated glucosyltransferase (JGT). To examine the importance of JGT in modifiying specific thymine in DNA, we used a Leishmania episome system to demonstrate that the telomeric repeat (GGGTTA) stimulates J synthesis in vivo while mutant telomeric sequences (GGGTTT, GGGATT, and GGGAAA) do not. Utilizing an in vitro GT assay we find that JGT can glycosylate hmU within any sequence with no significant change in Km or kcat, even mutant telomeric sequences that are unable to be J-modified in vivo. The data suggests that JGT possesses no DNA sequence specificity in vitro, lending support to the hypothesis that the specificity of base J synthesis is not at the level of the JGT reaction.

  18. Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA.

    Science.gov (United States)

    Bullard, Whitney; Cliffe, Laura; Wang, Pengcheng; Wang, Yinsheng; Sabatini, Robert

    2015-12-01

    Telomeric DNA of trypanosomatids possesses a modified thymine base, called base J, that is synthesized in a two-step process; the base is hydroxylated by a thymidine hydroxylase forming hydroxymethyluracil (hmU) and a glucose moiety is then attached by the J-associated glucosyltransferase (JGT). To examine the importance of JGT in modifiying specific thymine in DNA, we used a Leishmania episome system to demonstrate that the telomeric repeat (GGGTTA) stimulates J synthesis in vivo while mutant telomeric sequences (GGGTTT, GGGATT, and GGGAAA) do not. Utilizing an in vitro GT assay we find that JGT can glycosylate hmU within any sequence with no significant change in Km or kcat, even mutant telomeric sequences that are unable to be J-modified in vivo. The data suggests that JGT possesses no DNA sequence specificity in vitro, lending support to the hypothesis that the specificity of base J synthesis is not at the level of the JGT reaction. PMID:26815240

  19. DNA damage and autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States); Panayiotidis, Mihalis I. [School of Community Health Sciences, University of Nevada, Reno, NV 89557 (United States); Franco, Rodrigo, E-mail: rfrancocruz2@unl.edu [Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 (United States)

    2011-06-03

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  20. DNA damage and cellular death in oral mucosa cells of children who have undergone panoramic dental radiography

    Energy Technology Data Exchange (ETDEWEB)

    Angelieri, Fernanda; Oliveira, Gabriela R. de [Sao Paulo Metodista University (UMESP), Department of Orthodontics, Sao Bernardo do Campo, Sao Paulo (Brazil); Sannomiya, Eduardo K. [Sao Paulo Metodista University (UMESP), Department of Dento-Maxillofacial Radiology, Sao Bernardo do Campo, Sao Paulo (Brazil); Ribeiro, Daniel A. [Federal University of Sao Paulo (UNIFESP), Department of Health Sciences, Santos, Sao Paulo (Brazil); Universidade Federal de Sao Paulo (UNIFESP), Departamento de Ciencias da Saude, Santos, Sao Paulo (Brazil)

    2007-06-15

    Despite wide use as a diagnostic tool in medical and dental practice, radiography can induce cytotoxic effects and genetic damage. To evaluate DNA damage (micronucleus) and cellular death (pyknosis, karyolysis and karyorrhexis) in exfoliated buccal mucosa cells taken from healthy children following exposure to radiation during dental radiography. A total of 17 children who had undergone panoramic dental radiography were included. We found no statistically significant differences (P > 0.05) between micronucleated oral mucosa cells in children before and after exposure to radiation. On the other hand, radiation did cause other nuclear alterations closely related to cytotoxicity including karyorrhexis, pyknosis and karyolysis. Taken together, these results indicate that panoramic dental radiography might not induce chromosomal damage, but may be cytotoxic. Overall, the results reinforce the importance of evaluating the health side effects of radiography and contribute to the micronucleus database, which will improve our understanding and practice of this methodology in children. (orig.)

  1. Design and Synthesis of Triangulated DNA Origami Trusses.

    Science.gov (United States)

    Matthies, Michael; Agarwal, Nayan P; Schmidt, Thorsten L

    2016-03-01

    DNA nanotechnology offers unique control over matter on the nanoscale. Here, we extend the DNA origami method to cover a range of wireframe truss structures composed of equilateral triangles, which use less material per volume than standard multiple-helix bundles. From a flat truss design, we folded tetrahedral, octahedral, or irregular dodecahedral trusses by exchanging few connector strands. Other than standard origami designs, the trusses can be folded in low-salt buffers that make them compatible with cell culture buffers. The structures also have defined cavities that may in the future be used to precisely position functional elements such as metallic nanoparticles or enzymes. Our graph routing program and a simple design pipeline will enable other laboratories to make use of this valuable and potent new construction principle for DNA-based nanoengineering.

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

    Science.gov (United States)

    Wallace, Nicholas A; Galloway, Denise A

    2014-06-01

    In general, the interplay among viruses and DNA damage repair (DDR) pathways can be divided based on whether the interaction promotes or inhibits the viral lifecycle. The propagation of human papillomaviruses is both promoted and inhibited by DDR proteins. As a result, HPV proteins both activate repair pathways, such as the ATM and ATR pathways, and inhibit other pathways, most notably the p53 signaling pathway. Indeed, the role of HPV proteins, with regard to the DDR pathways, can be divided into two broad categories. The first set of viral proteins, HPV E1 and E2 activate a DNA damage response and recruit repair proteins to viral replication centers, where these proteins are likely usurped to replicate the viral genome. Because the activation of the DDR response typically elicits a cell cycle arrest that would impeded the viral lifecycle, the second set of HPV proteins, HPV E6 and E7, prevents the DDR response from pausing cell cycle progression or inducing apoptosis. This review provides a detailed account of the interactions among HPV proteins and DDR proteins that facilitate HPV propagation. PMID:24412279

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

    Science.gov (United States)

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

    2016-04-01

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

  4. Cellular immune responses of BALB/c mice induced by intramuscular injection of PRRSV-ORF5 DNA vaccine with different doses

    Institute of Scientific and Technical Information of China (English)

    CHENG Anchun; WANG Mingshu; CHEN Xiwen; XINI Nigen; DOU Wenbo; LI Xuemei; LIU Wumei; WANG Gang; ZHANG Pingying

    2007-01-01

    BALB/c mice were immunized with 50 μg,100 μg,200 μg of pcDNA-PRRSV-ORF5 DNA vaccine respectively by intramuscular injection,with PBS and pcDNA3.1(+)as controls.Fluorescence activated cell Sorter (FACS)was used to detect the number of CD4+ and CD8+T-lymphocytes.T-lymphocyte proliferation test was used to detect proliferation of the T-lymphocyte cells in peripheral blood lymphocytes of mice vaccinated with pcDNA-PRRSV-ORF5 DNA vaccine.The results showed that the difference in ConA response to T-lymphocytes in blood was highly significant between all experimental groups and the control group(P<0.01).The number of CD4+T-lymphocytes in experimental groups was significantly higher than that of the control group 7d after vaccination.The number of CD8+ T-lymphocytes in the experimental groups was higher than that of the control group 28 d after vaccination.Mice immunized with a higher dose(200 μg)of DNA vaccine demonstrated higher cellular immune response than those immunized with a lower dose(100 μg,50 μg)of DNA vaccine.The results demonstrated that pcDNA-PRRSV-ORF5 DNA vaccine could induce a good cellular immune response which may be dose-dependent.

  5. Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

    Directory of Open Access Journals (Sweden)

    Agbavwe Christy

    2011-12-01

    Full Text Available Abstract Background Light-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate. Results Stepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast. Conclusions Maskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies.

  6. Radioautographic DNA synthesis study on mice Mus musculus gingival epithelium

    International Nuclear Information System (INIS)

    The DNA-synthetizing cells frequency in the gingival epithelium basal layer of the first lower molar region in young and adult mice were studied. The 3H-thymidine and radioautography were used. The labeled cells frequency was determined by calculating their proportions. The data were statiscally analysed. (M.A.C.)

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

    Science.gov (United States)

    Griffin, Brian; Kilfoil, Maria L.

    2013-03-01

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

  8. Acute dosing and p53-deficiency promote cellular sensitivity to DNA methylating agents.

    Science.gov (United States)

    Chapman, Katherine E; Doak, Shareen H; Jenkins, Gareth J S

    2015-04-01

    Risk assessment of human exposure to chemicals is crucial for understanding whether such agents can cause cancer. The current emphasis on avoidance of animal testing has placed greater importance on in vitro tests for the identification of genotoxicants. Selection of an appropriate in vitro dosing regime is imperative in determining the genotoxic effects of test chemicals. Here, the issue of dosing approaches was addressed by comparing acute and chronic dosing, uniquely using low-dose experiments. Acute 24 h exposures were compared with equivalent dosing every 24 h over 5-day, fractionated treatment periods. The in vitro micronucleus assay was used to measure clastogenicity induced by methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU) in human lymphoblastoid cell line, TK6. Quantitative real-time (qRT) PCR was used to measure mRNA level induction of DNA repair enzymes. Lowest observed genotoxic effect levels (LOGELs) for MMS were obtained at 0.7 µg/ml for the acute study and 1.0 µg/ml for the chronic study. For acute MNU dosing, a LOGEL was observed at 0.46 µg/ml, yet genotoxicity was completely removed following the chronic study. Interestingly, acute MNU dosing demonstrated a statistically significant decrease at 0.009 µg/ml. Levels of selected DNA repair enzymes did not change significantly following doses tested. However, p53 deficiency (using the TK6-isogenic cell line, NH32) increased sensitivity to MMS during chronic dosing, causing this LOGEL to equate to the acute treatment LOGEL. In the context of the present data for 2 alkylating agents, chronic dosing could be a valuable in vitro supplement to acute dosing and could contribute to reduction of unnecessary in vivo follow-up tests. PMID:25595616

  9. Phenolic compounds isolated from Pilea microphylla prevent radiation-induced cellular DNA damage

    Directory of Open Access Journals (Sweden)

    Punit Bansal

    2011-12-01

    Full Text Available Six phenolic compounds namely, quercetin-3-O-rutinoside (1, 3-O-caffeoylquinic acid (2, luteolin-7-O-glucoside (3, apigenin-7-O-rutinoside (4, apigenin-7-O-β-d-glucopyranoside (5 and quercetin (6 were isolated from the whole plant of Pilea microphylla using conventional open-silica gel column chromatography and preparative HPLC. Further, these compounds were characterized by 1D, 2D NMR techniques and high-resolution LC–MS. Compounds 1–3 and 6 exhibited significant antioxidant potential in scavenging free radicals such as DPPH, ABTS and SOD with IC50 of 3.3–20.4 μmol/L. The same compounds also prevented lipid peroxidation with IC50 of 10.4–32.2 μmol/L. The compounds also significantly prevented the Fenton reagent-induced calf thymus DNA damage. Pre-treatment with compounds 1–3 and 6 in V79 cells attenuated radiation-induced formation of reactive oxygen species, lipid peroxidation, cytotoxicity and DNA damage, correlating the antioxidant activity of polyphenols with their radioprotective effects. Compounds 1, 3 and 6 significantly inhibited lipid peroxidation, presumably due to 3′,4′-catechol ortho-dihydroxy moiety in the B-ring, which has a strong affinity for phospholipid membranes. Oxidation of flavonoids, with catechol structure on B-ring, yields a fairly stable ortho-semiquinone radical by facilitating electron delocalization, which is involved in antioxidant mechanism. Hence, the flavonoid structure, number and location of hydroxyl groups together determine the antioxidant and radioprotection mechanism.

  10. Synthesis of DNA Oligodeoxynucleotides Containing Site-Specific 1,3-Butadiene- Deoxyadenosine Lesions

    Science.gov (United States)

    Wickramaratne, Susith; Seiler, Christopher L.

    2016-01-01

    Post-oligomerization synthesis is a useful technique for preparing site-specifically modified DNA oligomers. This approach involves site-specific incorporation of inherently reactive halogenated nucleobases into DNA strands using standard solid phase synthesis, followed by post-oligomerization nucleophilic aromatic substitution (SNAr) reactions with carcinogen-derived synthons. In these reactions, the inherent reactivities of DNA and carcinogen-derived species are reversed: the modified DNA nucleobase acts as an electrophile, while the carcinogen-derived species acts as a nucleophile. In the present protocol, we describe the use of the post-oligomerization approach to prepare DNA strands containing site- and stereospecific N6-adenine and N1, N6-adenine adducts induced by epoxide metabolites of the known human and animal carcinogen, 1,3-butadiene (BD). The resulting oligomers containing site specific, structurally defined DNA adducts can be used in structural and biological studies to reveal the roles of specific BD adducts in carcinogenesis and mutagenesis. PMID:26344227

  11. THE HUMORAL AND CELLULAR IMMUNE RESPONSES INDUCED BY HPV18L1-E6/E7 DNA VACCINES IN MICE

    Institute of Scientific and Technical Information of China (English)

    Yang Jin; Li Xu; Li Ang; Wang Yili; Si Lüsheng

    2006-01-01

    Objective To construct eukaryotic expression vector of HPV18 L1- E6, E7 chimeric gene and examine the humoral and cellular immune responses induced by this DNA vaccines in mice. Methods The C-terminal of major capsid protein L1 gene and mutant zinc finger domains of early E6/7 oncogenes in HPV18 were integrated and inserted into eukaryotic expression vector pVAX1 to generate vaccines pVAX1-L1E6Mxx, E7Mxx. CHO cells were transiently transfected with the individual construct. Target protein expressions in the lysate of the transfected cells were measured by ELISA and immunocytochemistry. After BALB/c mice were vaccinated with various recombinant plasmids(pVAX1-L1-E6M3 or pVAX1-L1-E7M3) and immunie adjuvants (pLXHDmB7-2 or LTB) through different administration routes (intramuscular or intranasal) , the great cellular immune responses were produced as revealed by delayed-type hypersensitivity (DTH) and lymphocyte proliferation, and the expression of IL-4 and IFN- γ cells in CD4+ and CD8+subpopulations. Results The highly efficient expression of pVAX1-L1E6Mxx, E7Mxx vector in host eukaryotic cells were demonstrated both by ELISA and immunocytochemistry. The level of specific serum IgG against HPV in experiment groups mice was much higher than that of control group, and intranuscular immunization group had the highest antibody level. Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8+ IFN-γ + cells number, but CD4+ IL4+ cell number was higher in intranasal immunization groups. The immunization groups using pLXHDmB7-2 as adjuvant were superior to other groups in immunoresponse. Conclusion These DNA vaccines produce remarkable cellular and humoral immuneresponses in the mouse and may provide as prophylatic and therapeutic candidates for HPV induced cancer treatment.

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

    Directory of Open Access Journals (Sweden)

    Alessia Lamolinara

    2015-01-01

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    1987-01-01

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

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

    International Nuclear Information System (INIS)

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

  15. Epigallocatechin gallate inhibits HBV DNA synthesis in a viral replication - inducible cell line

    Institute of Scientific and Technical Information of China (English)

    Wei He; Li-Xia Li; Qing-Jiao Liao; Chun-Lan Liu; Xu-Lin Chen

    2011-01-01

    AIM: To analyze the antiviral mechanism of Epigallocatechin gallate (EGCG) against hepatitis B virus (HBV) replication. METHODS: In this research, the HBV-replicating cell line HepG2.117 was used to investigate the antiviral mechanism of EGCG. Cytotoxicity of EGCG was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hepatitis B virus e antigen (HBeAg) and hepatitis B virus surface antigen (HBsAg) in the supernatant were detected by enzyme-linked immunosorbent assay. Precore mRNA and pregenomic RNA (pgRNA) levels were determined by semi-quantitative reverse transcription polymerase chain reaction (PCR) assay. The effect of EGCG on HBV core promoter activity was measured by dual luciferase reporter assay. HBV covalently closed circular DNA and replicative intermediates of DNA were quantified by real-time PCR assay. RESULTS: When HepG2.117 cells were grown in the presence of EGCG, the expression of HBeAg was suppressed, however, the expression of HBsAg was not affected. HBV precore mRNA level was also downregulated by EGCG, while the transcription of precore mRNA was not impaired. The synthesis of both HBV covalently closed circular DNA and replicative intermediates of DNA were reduced by EGCG treatment to a similar extent, however, HBV pgRNA transcripted from chromosome-integrated HBV genome was not affected by EGCG treatment, indicating that EGCG targets only replicative intermediates of DNA synthesis. CONCLUSION: In HepG2.117 cells, EGCG inhibits HBV replication by impairing HBV replicative intermediates of DNA synthesis and such inhibition results in reduced production of HBV covalently closed circular DNA.

  16. The effect of caffeine and adenine on radiation induced suppression of DNA synthesis, and cell survival

    International Nuclear Information System (INIS)

    Exposure of cultured mammalian cells to ionizing radiation or UV light results in a transient decrease in the rate of DNA synthesis. This depression in synthetic rate may be attenuated or deferred via a post-irradiation treatment with caffeine or adenine. It has been suggested that this attenuation may increase the fixation of damage and, therefore, increase radiation sensitivity. However, it has been previously reported that, for V79 cells treated with caffeine or adenine, no correlation exists between the extent of depression and cell survival. The present investigation expands upon these findings by examining the effect of caffeine or adenine post-irradiation treatment on two cell lines with normal UV sensitivity, mouse 3T3 and CHO AA8 cells, and one UV sensitive cell line, CHO UV5 cells. Both caffeine and adenine have been found to reduce, or delay, the suppression in DNA synthesis in all three cell lines. Surprisingly, caffeine appeared to induced even the UV5 cells to recover DNA synthetic ability. The amount of reduction in suppression of DNA synthesis, however, varies between the different cell lines and no consistent relationship with cell survival has emerged

  17. Synthesis of Cross-Linked DNA Containing Oxidized Abasic Site Analogues

    Science.gov (United States)

    2015-01-01

    DNA interstrand cross-links are an important family of DNA damage that block replication and transcription. Recently, it was discovered that oxidized abasic sites react with the opposing strand of DNA to produce interstrand cross-links. Some of the cross-links between 2′-deoxyadenosine and the oxidized abasic sites, 5′-(2-phosphoryl-1,4-dioxobutane) (DOB) and the C4-hydroxylated abasic site (C4-AP), are formed reversibly. Chemical instability hinders biochemical, structural, and physicochemical characterization of these cross-linked duplexes. To overcome these limitations, we developed methods for preparing stabilized analogues of DOB and C4-AP cross-links via solid-phase oligonucleotide synthesis. Oligonucleotides of any sequence are attainable by synthesizing phosphoramidites in which the hydroxyl groups of the cross-linked product were orthogonally protected using photochemically labile and hydrazine labile groups. Selective unmasking of a single hydroxyl group precedes solid-phase synthesis of one arm of the cross-linked DNA. The method is compatible with commercially available phosphoramidites and other oligonucleotide synthesis reagents. Cross-linked duplexes containing as many as 54 nt were synthesized on solid-phase supports. Subsequent enzyme ligation of one cross-link product provided a 60 bp duplex, which is suitable for nucleotide excision repair studies. PMID:24949656

  18. A new paradigm of DNA synthesis: three-metal-ion catalysis.

    Science.gov (United States)

    Yang, Wei; Weng, Peter J; Gao, Yang

    2016-01-01

    Enzyme catalysis has been studied for over a century. How it actually occurs has not been visualized until recently. By combining in crystallo reaction and X-ray diffraction analysis of reaction intermediates, we have obtained unprecedented atomic details of the DNA synthesis process. Contrary to the established theory that enzyme-substrate complexes and transition states have identical atomic composition and catalysis occurs by the two-metal-ion mechanism, we have discovered that an additional divalent cation has to be captured en route to product formation. Unlike the canonical two metal ions, which are coordinated by DNA polymerases, this third metal ion is free of enzyme coordination. Its location between the α- and β-phosphates of dNTP suggests that the third metal ion may drive the phosphoryltransfer from the leaving group opposite to the 3'-OH nucleophile. Experimental data indicate that binding of the third metal ion may be the rate-limiting step in DNA synthesis and the free energy associated with the metal-ion binding can overcome the activation barrier to the DNA synthesis reaction. PMID:27602203

  19. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    Science.gov (United States)

    Gardner, Shea N.; Mariella, Jr., Raymond P.; Christian, Allen T.; Young, Jennifer A.; Clague, David S.

    2011-01-18

    A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.

  20. Synthesis, Characterization, and DNA Binding Studies of Nanoplumbagin

    Directory of Open Access Journals (Sweden)

    Sheik Dawood Shahida Parveen

    2014-01-01

    Full Text Available The traditional anticancer medicine plumbagin (PLN was prepared as nanostructured material (nanoplumbagin, NPn1 from its commercial counterparts, simultaneously coencapsulating with cetyltrimethylammonium bromide or cyclodextrin as stabilizers using ultrasonication technique. Surface morphology of NPn analysed from atomic force microscopy (AFM indicates that NPn has tunable size between 75 nm and 100 nm with narrow particle size distribution. Its binding efficiency with herring sperm DNA was studied using spectral and electrochemical techniques and its efficiency was found to be more compared to the commercial microcrystalline plumbagin (PLN. DNA cleavage was also studied by gel electrophoresis. The observed results indicate that NPn1 has better solubility in aqueous medium and hence showed better bioavailability compared to its commercial counterparts.

  1. Synthesis, DNA binding and topoisomerase inhibition of mononaphthalimide homospermidine derivatives

    Institute of Scientific and Technical Information of China (English)

    Zhi Yong Tian; Hong Xia Ma; Song Qiang Xie; Xue Wang; Jin Zhao; Chao Jie Wang; Wen Yuan Gao

    2008-01-01

    Two novel mononaphthalimide homospermidine derivatives (2a, 2b) with three or four methylene unit as linkages weresynthesized and evaluated for cytotoxicity against human leukemia K562, murine melanoma B 16 and Chinese hamster ovary CHOcell lines. The presence of homospermidine motif could greatly elevate the potency of 1,8-naphthalimide. Conjugate 2b with longerspacer exhibited higher in vitro cytotoxicity than 2a. The DNA binding experiments indicated that conjugates 2b could bind toherring sperm DNA. The topoisomerase Ⅱ poison trials revealed that 2b could inhibit the activity of top. Ⅱ.2008 Chao Jie Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  2. Synthesis and biological activity of benzamide DNA minor groove binders.

    Science.gov (United States)

    Khan, Gul Shahzada; Pilkington, Lisa I; Barker, David

    2016-02-01

    A range of di- and triaryl benzamides were synthesised to investigate the effect of the presence and nature of a polar sidechain, bonding and substitution patterns and functionalisation of benzylic substituents. These compounds were tested for their antiproliferative activity as well as their DNA binding activity. The most active compounds in all assays were unsymmetrical triaryl benzamides with a bulky or alkylating benzylic substituent and a polar amino sidechain.

  3. Simple Laboratory methods to measure cell proliferation using DNA synthesis property

    Directory of Open Access Journals (Sweden)

    Madhavan H N

    2007-01-01

    Full Text Available This is a mini-review on the techniques to measure proliferation of cells by estimation of DNA synthesis. This is not an exhaustive review of literature, but a bird’s eye view of a few selected articles which may provide the technical details to the readers.The nucleus of a cell occupies about 10-30% of the cells space, depends on the type of genetic material (DNA -DeoxyriboNucleic Acid. DNA is a long, double-stranded, helical molecule which carries the genetic information. Duplication of the DNA takes place by the phenomena of replication. One copy of double-stranded DNA molecule forms two double-stranded DNA molecules. DNA replication is the fundamental process used in all living organisms as it is the basis for biological inheritance. This process is known also as Mitosis in somatic cells. In Mitosis, the duplication process results in two genetically identical "daughter" cells from a single "parent" cell. The resulting double-stranded DNA molecules are identical; proof reading and error-checking mechanisms exist to ensure near perfect pair. Mitosis is divided into six phases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis.

  4. Retroviral DNA integration: viral and cellular determinants of target-site selection.

    Directory of Open Access Journals (Sweden)

    Mary K Lewinski

    2006-06-01

    Full Text Available Retroviruses differ in their preferences for sites for viral DNA integration in the chromosomes of infected cells. Human immunodeficiency virus (HIV integrates preferentially within active transcription units, whereas murine leukemia virus (MLV integrates preferentially near transcription start sites and CpG islands. We investigated the viral determinants of integration-site selection using HIV chimeras with MLV genes substituted for their HIV counterparts. We found that transferring the MLV integrase (IN coding region into HIV (to make HIVmIN caused the hybrid to integrate with a specificity close to that of MLV. Addition of MLV gag (to make HIVmGagmIN further increased the similarity of target-site selection to that of MLV. A chimeric virus with MLV Gag only (HIVmGag displayed targeting preferences different from that of both HIV and MLV, further implicating Gag proteins in targeting as well as IN. We also report a genome-wide analysis indicating that MLV, but not HIV, favors integration near DNase I-hypersensitive sites (i.e., +/- 1 kb, and that HIVmIN and HIVmGagmIN also favored integration near these features. These findings reveal that IN is the principal viral determinant of integration specificity; they also reveal a new role for Gag-derived proteins, and strengthen models for integration targeting based on tethering of viral IN proteins to host proteins.

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

    Science.gov (United States)

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

    2016-09-01

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

  6. Synthesis, photochemistry, DNA cleavage/binding and cytotoxic properties of fluorescent quinoxaline and quinoline hydroperoxides.

    Science.gov (United States)

    Chowdhury, Nilanjana; Gangopadhyay, Moumita; Karthik, S; Pradeep Singh, N D; Baidya, Mithu; Ghosh, S K

    2014-01-01

    Novel fluorescent quinoxaline and quinoline hydroperoxides were shown to perform dual role as both fluorophores for cell imaging and photoinduced DNA cleaving agents. Photophysical studies of newly synthesized quinoxaline and quinoline hydroperoxides showed that they all exhibited moderate to good fluorescence. Photolysis of quinoxaline and quinoline hydroperoxides in acetonitrile using UV light above 350nm resulted in the formation of corresponding ester compounds via γ-hydrogen abstraction by excited carbonyl chromophore. Single strand DNA cleavage was achieved on irradiation of newly synthesized hydroperoxides by UV light (⩾350nm). Both hydroxyl radicals and singlet oxygen were identified as reactive oxygen species (ROS) responsible for the DNA cleavage. Further, we showed quinoline hydroperoxide binds to ct-DNA via intercalative mode. In vitro biological studies revealed that quinoline hydroperoxide has good biocompatibility, cellular uptake property and cell imaging ability. Finally, we showed that quinoline hydroperoxide can permeate into cells efficiently and may cause cytotoxicity upon irradiation by UV light.

  7. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailing description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  8. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  9. Studies on a Novel Minor-groove Targeting Artificial Nuclease: Synthesis and DNA Binding Behavior

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nucleases play an important role in molecular biology, for example, in DNA sequencing. Synthetic polyamide conjugates can be considered as a novel tool for the selective inhibition of gene expressions and also as potential drugs in anticancer or antiviral chemotherapy. In this article, the synthesis of a novel minor-groove targeting artificial nuclease, an oligopyrrol-containing compound, has been reported. It was found that this novel compound can bind DNA in AT-rich minor groove with high affinity and site specificity. DNA binding behavior was determined by using UV-Vis and CD. It is indicated that compound 6 can enhance the Tm of DNA from 80. 4 C to 84. 4 ℃ and that it possesses a high binding constant value(Kb = 3.05×104 L/mol).

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

    Science.gov (United States)

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

    2014-06-01

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

  11. Synthesis, photochemical properties and DNA binding studies of dna cleaving agents based on chiral dipyridine dihydrodioxins salts

    Science.gov (United States)

    Shamaev, Alexei

    activated by UV-light. The mechanism of o-quinone release and intramolecular ET was studied in detail by methods of Ultrafast Transient Absortion Spectroscopy and supported by high-level quantum mechanical calculations. The binding properties of chiral intercalators based on PDHD to various DNA oligonucleotides were studied by various methods and DNA cleavage properties indicating strong binding and cleaving ability of the synthesized PDHDs. Also, a new method for synthesis of cyclohexa[e]pyrenes which possibly capable of intramolecular ET and electron transfer-oxidative stress (ET-OS) DNA cleavage was developed and partially accomplished.

  12. Synthesis of a duplex oligonucleotide containing a nitrogen mustard interstrand DNA-DNA cross-link.

    Science.gov (United States)

    Ojwang, J O; Grueneberg, D A; Loechler, E L

    1989-12-01

    Many cancer chemotherapeutic agents react with DNA and give adducts that block DNA replication, which is thought to result in cytotoxicity, especially in rapidly proliferating cells such as cancer cells. One class of these agents is bifunctionally reactive (e.g., the nitrogen mustards) and forms DNA-DNA cross-links. It is unknown whether inter- or intrastrand cross-links are more effective at blocking DNA replication. To evaluate this, a DNA shuttle vector is being constructed with an interstrand cross-link at a unique site. In the first step of this project, a duplex oligonucleotide containing an interstrand cross-link is isolated by denaturing polyacrylamide gel electrophoresis from the reaction of nitrogen mustard with two partially complementary oligodeoxynucleotides. The purified oligonucleotide product is characterized and shown to be cross-linked in a 5'-GAC-3' 3'-CTG-5' sequence by a nitrogen mustard moiety that is bound at the N(7)-position of the guanines in the opposing strands; the glycosylic bonds of these guanine adducts are stabilized in their corresponding imidazole ring-opened form. Nitrogen mustard is shown to react with a variety of oligonucleotides and, based upon these results, its preferred targets for interstrand cross-linking are 5'-GXC-3' sequences, where X can be any of the four deoxyribonucleotide bases. PMID:2819709

  13. The assembly and properties of protobiological structures - The beginnings of cellular peptide synthesis

    Science.gov (United States)

    Fox, S. W.; Nakashima, T.

    1980-01-01

    New data indicate that lysine-rich proteinoids have the ability to catalyze the synthesis of peptide bonds from a variety of amino acids and ATP. This capacity is evident in aqueous solution, in suspension of phase-separated complexes of lysine-rich proteinoid with acidic proteinoids, and in suspension of phase-separated particles composed of lysine-rich proteinoids with polynucleotides. Since the proteinoid complexes can contain other catalytic activities, including ability to catalyze internucleotide bond formation, it is inferred that the first protocells on earth already had a number of biological types of activity.

  14. Influence of nitroimidazole derivatives and irradiation on the DNA synthesis of L5178Y cells and human lymphocytes

    International Nuclear Information System (INIS)

    The radiosensitizing effect of 4-nitroimidazoles a further substance similar to the known 2-nitroderivative misonidazole was involved in the testing. The investigation with four different nitroimidazole derivatives carried out under the same conditions permit a good comparison of their effectiveness for the selected criteria. The influence of these substances on the DNA synthesis before and after irradiation was examined in vitro in L5178Y cells and human lymphocytes. Regarding their inhibitory effect on DNA synthesis, all substances in a radiotherapeutically relevant concentration are without effect for well oxygenated cells. This also applies to hypoxic cultures, except the 2-nitroimidazole. The radiation-induced inhibition of the DNA synthesis is considerably intensified by all derivatives only under hypoxia. The radiation-induced DNA synthesis in lymphocytes is not influenced. (author)

  15. Human cellular protein patterns and their link to genome DNA mapping and sequencing data: towards an integrated approach to the study of gene expression

    DEFF Research Database (Denmark)

    Celis, J E; Rasmussen, H H; Leffers, H;

    1993-01-01

    Analysis of cellular protein patterns by computer-aided two-dimensional gel electrophoresis together with recent advances in protein sequence analysis and expression systems have made possible the establishment of comprehensive two-dimensional gel protein databases that may link protein and DNA m...

  16. Protein synthesis directly from PCR: progress and applications of cell-free protein synthesis with linear DNA.

    Science.gov (United States)

    Schinn, Song-Min; Broadbent, Andrew; Bradley, William T; Bundy, Bradley C

    2016-06-25

    A rapid, versatile method of protein expression and screening can greatly facilitate the future development of therapeutic biologics, proteomic drug targets and biocatalysts. An attractive candidate is cell-free protein synthesis (CFPS), a cell-lysate-based in vitro expression system, which can utilize linear DNA as expression templates, bypassing time-consuming cloning steps of plasmid-based methods. Traditionally, such linear DNA expression templates (LET) have been vulnerable to degradation by nucleases present in the cell lysate, leading to lower yields. This challenge has been significantly addressed in the recent past, propelling LET-based CFPS as a useful tool for studying, screening and engineering proteins in a high-throughput manner. Currently, LET-based CFPS has promise in fields such as functional proteomics, protein microarrays, and the optimization of complex biological systems. PMID:27085957

  17. Regulatory nexus of synthesis and degradation deciphers cellular Nrf2 expression levels.

    Science.gov (United States)

    Suzuki, Takafumi; Shibata, Tatsuhiro; Takaya, Kai; Shiraishi, Kouya; Kohno, Takashi; Kunitoh, Hideo; Tsuta, Koji; Furuta, Koh; Goto, Koichi; Hosoda, Fumie; Sakamoto, Hiromi; Motohashi, Hozumi; Yamamoto, Masayuki

    2013-06-01

    Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine. PMID:23572560

  18. Chemoenzymatic Synthesis of Cellular Adhesion Tripeptide RGD Precursor in Organic Media

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Chemoenzymatic synthesis of tripeptide Bz-RGD-(OEt)2 was conducted in this study. First, the free dipeptide Gly-Asp was synthesized via a novel chemical method, wherein only L-aspartic acid was used and was followed by the esterification of Gly-Asp. The formation of the linkage between the third amino acid Bz-Arg-OEt and Gly-Asp-(OEt)2 was completed by using the enzymatic method in organic media. The effects of several factors such as pH, the water content, triethylamine(TEA), the molar ratio of the substrates, and the reaction time on the yield of Bz-RGD-(OEt)2 were examined. It was obtained that the optimum conditions for Bz-RGD-(OEt)2 synthesis in an ethanol/Tris-HCl buffer system(volume ratio 93:7) were as follows; pH=8.0; temperature, 30 ℃; reaction time, 7 h. The tripeptide yield was 75.2%.

  19. The Foundry: the DNA synthesis and construction Foundry at Imperial College.

    Science.gov (United States)

    Chambers, Stephen; Kitney, Richard; Freemont, Paul

    2016-06-15

    The establishment of a DNA synthesis and construction foundry at Imperial College in London heralds a new chapter in the development of synthetic biology to meet new global challenges. The Foundry employs the latest technology to make the process of engineering biology easier, faster and scalable. The integration of advanced software, automation and analytics allows the rapid design, build and testing of engineered organisms. PMID:27284027

  20. The Foundry: the DNA synthesis and construction Foundry at Imperial College

    Science.gov (United States)

    Chambers, Stephen; Kitney, Richard; Freemont, Paul

    2016-01-01

    The establishment of a DNA synthesis and construction foundry at Imperial College in London heralds a new chapter in the development of synthetic biology to meet new global challenges. The Foundry employs the latest technology to make the process of engineering biology easier, faster and scalable. The integration of advanced software, automation and analytics allows the rapid design, build and testing of engineered organisms. PMID:27284027

  1. Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs: Synthesis and DNA Binding.

    Directory of Open Access Journals (Sweden)

    Yuliya Kirillova

    Full Text Available New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA.

  2. Divalent cation ionophores stimulate resorption and inhibit DNA synthesis in cultured fetal rat bone

    Energy Technology Data Exchange (ETDEWEB)

    Lorenzo, J.A.; Raisz, L.G.

    1981-06-01

    Two divalent cation ionophores, A23187 and Ionomycin, which are selective for calcium, stimulated the resorption of fetal rat long bones in organ culture at 0.1 to 1 micromolar but not at higher concentrations. Both agents inhibited DNA synthesis at concentrations that stimulated resorption. These results might explain the differences in ionophore effects on bone previously reported, and they imply that cell replication is not required for osteoclast formation in fetal rat long bone cultures.

  3. Detection, characterization and measure of a new radiation-induced damage in isolated and cellular DNA; Detection, caracterisation et mesure d'un nouveau dommage radio-induit de l'ADN isole et cellulaire

    Energy Technology Data Exchange (ETDEWEB)

    Regulus, P

    2006-10-15

    Deoxyribonucleic acid (DNA) contains the genetic information and chemical injury to this macromolecule may have severe biological consequences. We report here the detection of 4 new radiation-induced DNA lesions by using a high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) approach. For that purpose, the characteristic fragmentation of most 2'-deoxy-ribo nucleosides, the loss of 116 Da corresponding to the loss of the 2-deoxyribose moiety, was used in the so-called neutral loss mode of the HPLC-MS/MS. One of the newly detected lesions, named dCyd341 because it is a 2'-deoxycytidine modification exhibiting a molecular weight of 341 Da, was also detected in cellular DNA. Characterization of this modified nucleoside was performed using NMR and exact mass determination of the product obtained by chemical synthesis. A mechanism of formation was then proposed, in which the first event is the H-abstraction at the C4 position of a 2-deoxyribose moiety. Then, the sugar modification produced exhibits a reactive aldehyde that, through reaction with a vicinal cytosine base, gives rise to dCyd341. dCyd341 could be considered as a complex damage since its formation involves a DNA strand break and a cross-link between a damaged sugar residue and a vicinal cytosine base located most probably on the complementary DNA strand. In addition to its characterization, preliminary biological studies revealed that cells are able to remove the lesion from DNA. Repair studies have revealed the ability of cells to excise the lesion. Identification of the repair systems involved could represent an interesting challenge. (author)

  4. DNA damage-processing in E. coli: on-going protein synthesis is required for fixation of UV-induced lethality and mutation.

    Science.gov (United States)

    Burger, Amanda; Raymer, Jenny; Bockrath, R

    2002-10-01

    UV irradiation of E. coli produces photoproducts in the DNA genome. In consequence, some bacteria lose viability (colony-forming ability) or remain viable as mutant cells. However, the end-points of viability inactivation (lethality) or mutation are determined by cellular processes that act on the UV-damaged DNA. We have investigated the in vivo time course for processes that deal with cyclobutane pyrimidine dimers (CPD) which can be specifically removed by photoreactivation (PR). At different times during post-UV incubation, samples were challenged with PR and assayed for viability or mutation. We used excision-defective E. coli B/r cells and worked under yellow light to avoid background PR. During post-UV incubation (0-100min) in fully supplemented defined medium, inactivation and mutation were initially significantly reversed by PR but the extent of this reversal decreased during continued incubation defining "fixation" of lethality or mutation, respectively. In contrast, if protein synthesis was restricted during the post-UV incubation, no fixation developed. When chloramphenicol was added to inhibit protein synthesis after 30min of supplemented post-UV incubation, at a time sufficient for expression of UV-induced protein(s), fixation of lethality or mutation was still annulled (no change in the effectiveness of PR developed). Lethality fixation did progress when protein synthesis was restricted and the cells were incubated in the presence of puromycin or were either clpP or clpX defective. We discuss these and related results to suggest (1) on-going protein synthesis is required in the fixation process for lethality and mutation to sustain an effective level of a hypothetical protein sensitive to ClpXP proteolysis and (2) this protein plays a critical role in the process leading to exchange between Pol III activity and alternative polymerase activities required as each cell deals with damage in template DNA.

  5. Mechanism of ultraviolet-induced mutagenesis: extent and fidelity of in vitro DNA synthesis on irradiated templates

    International Nuclear Information System (INIS)

    The effect of uv irradiation on the extent and fidelity of DNA synthesis in vitro was studied by using homopolymers and primed single-stranded phi X174 phage DNA as substrates. Unfractionated and fractionated cell-free extracts from Escherichia coli pol+ and polA1 mutants as well as purified DNA polymerase I were used as sources of enzymatic activity. (DNA polymerases, as used here, refer to deoxynucleosidetriphosphate : DNA deoxynucleotidyltransferase, EC 2.7.7.7.) The extent of inhibition of DNA synthesis on uv-irradiated phi X174 DNA suggested that pyrimidine dimers act as an absolute block for chain elongation by DNA polymerases I and III. Experiments with an irradiated poly(dC) template failed to detect incorporation of noncomplementary bases due to pyrimidine dimers. A large increase in the turnover of nucleoside triphosphates to free monophosphates during synthesis by DNA polymerase I on irradiated phi X174 DNA has been observed. We propose that this nucleotide turnover is due to idling by DNA polymerase (i.e., incorporation and subsequent excision of nucleotides opposite uv photolesions, by the 3' to 5' ''proofreading'' exonuclease) thus preventing replication past pyrimidine dimers and the potentially mutagenic event that should result

  6. Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis

    International Nuclear Information System (INIS)

    Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability. (authors)

  7. Deoxyadenosine family: improved synthesis, DNA damage and repair, analogs as drugs.

    Science.gov (United States)

    Biswas, Himadri; Kar, Indrani; Chattopadhyaya, Rajagopal

    2013-08-01

    Improved synthesis of 2'-deoxyadenosine using Escherichia coli overexpressing some enzymes and gram-scale chemical synthesis of 2'-deoxynucleoside 5'-triphosphates reported recently are described in this review. Other topics include DNA damage induced by chromium(VI), Fenton chemistry, photoinduction with lumazine, or by ultrasound in neutral solution; 8,5'-cyclo-2'-deoxyadenosine isomers as potential biomarkers; and a recapitulation of purine 5',8-cyclonucleoside studies. The mutagenicities of some products generated by oxidizing 2'-deoxyadenosine 5'-triphosphate, nucleotide pool sanitization, and translesion synthesis are also reviewed. Characterizing cross-linking between nucleosides in opposite strands of DNA and endonuclease V-mediated deoxyinosine excision repair are discussed. The use of purine nucleoside analogs in the treatment of rarer chronic lymphoid leukemias is reviewed. Some analogs at the C8 position induced delayed polymerization arrest during HIV-1 reverse transcription. The susceptibility of clinically metronidazole-resistant Trichomonas vaginalis to two analogs, toyocamycin and 2-fluoro-2'-deoxyadenosine, were tested in vitro. GS-9148, a dAMP analog, was translocated to the priming site in a complex with reverse transcriptase and double-stranded DNA to gain insight into the mechanism of reverse transcriptase inhibition. PMID:25436589

  8. Profiling of Epstein-Barr virus latent RNA expression in clinical specimens by gene-specific multiprimed cDNA synthesis and PCR.

    Science.gov (United States)

    Stevens, Servi J C; Brink, Antoinette A T P; Middeldorp, Jaap M

    2005-01-01

    We describe a two-step RT-PCR method for simultaneous detection of EBNA-1 (QK and Y3K splice variants), EBNA-2, LMP-1, LMP-2a and -2b, ZEBRA, and BARTs RNA encoded by Epstein-Barr virus. As a control for RNA integrity, the low-copy-number transcript derived from U1A snRNP, a cellular housekeeping gene, is coamplified. Copy DNA (cDNA) for these nine targets is simultaneously synthesized in a gene-specific, multiprimed cDNA reaction, which strongly reduces the amount of required clinical specimen and allows more sensitive detection than random hexamer or oligo-dT priming. For amplification, cDNA synthesis is followed by nine separate PCRs for the mentioned targets. Primers were designed either as intron-flanking, to avoid background DNA amplification, or in different exons, allowing identification of differentially spliced RNA molecules. To increase specificity, PCR products are detected by autoradiography after hybridization with radiolabeled internal oligonucleotide probes. The method described is highly suitable for profiling EBV latent RNA expression in tissue biopsies, cultured or isolated cells, and unfractionated whole blood and for definition of EBV latency type I, II, or III gene expression in these samples.

  9. DNA repair after ultraviolet irradiation of ICR 2A frog cells: pyrimidine dimers are long acting blocks to nascent DNA synthesis

    International Nuclear Information System (INIS)

    The ability of ICR 2A frog cells to repair DNA damage induced by ultraviolet irradiation was examined. These cells are capable of photoreactivation but are nearly totally deficient in excision repair. They have the ability to convert the small molecular weight DNA made after irradiation into large molecules but do not show an enhancement in this process when the UV dose is delivered in two separate exposures separated by a 3- or 24-h incubation. Total DNA synthesis is depressed and low molecular weight DNA continues to be synthesized during pulse-labeling as long as 48 h after irradiation. The effects of pyrimidine dimer removal through exposure of UV irradiated cells to photoreactivating light indicate that dimers act as the critical lesions blocking DNA synthesis

  10. DNA synthesis and cell division in the adult primate brain

    International Nuclear Information System (INIS)

    It is generally accepted that the adult human brain is incapable of producing new neuron. Even cursory examination of neurologic, neuropathologic, or neurobiological textbooks published during the past 50 years will testify that this belief is deeply entrenched. In his classification of cell populations on the basis of their proliferative behavior, Leblond regarded neurons of the central nervous system as belonging to a category of static, nonrenewing epithelial tissue incapable of expanding or replenishing itself. This belief, however needs to re reexamined for two major reasons: First, as reviewed below, a number of reports have provided evidence of neurogenesis in adult brain of several vertebrate species. Second, the capacity for neurogenesis in the adult primate central nervous system has never been examined by modern methods. In this article the author described recent results from an extensive autoradiographic analysis performed on twelve rhesus monkeys injected with the specific DNA precursor [3H] thymidine at ages ranging from 6 postnatal months to 17 years

  11. Synthesis and cellular cytotoxicities of new -substituted indole-3-carbaldehyde and their indolylchalcones

    Indian Academy of Sciences (India)

    Magdy A H Zahran; Atef M Ibrahim

    2009-07-01

    A simple and efficient method for -alkylation of indole-3-carbaldehyde derivatives using a mixture of different bases in DMF under conventional and microwave irradiation conditions to afford -substituted indole-3-carbaldehyde derivatives 3a-o is reported. These derivatives which undergo Claisen-Schmidt condensation with 1-biphenyl-4-yl-ethanone yielded the corresponding indolylchalcone derivatives 5a-h. A comparative study showed that the microwave irradiation condition afforded excellent yield and shorten reaction time of all the synthesized indole derivatives which possess promising antitumor activity as well as interchelation bioactivity of indolylchalcones 5a-h with DNA.

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

    Science.gov (United States)

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

    2016-01-01

    Cell-free DNA (cfDNA) has significant potential in the diagnosis and monitoring of clinical conditions. However, accurately and easily distinguishing the relative proportion of DNA molecules in a mixture derived from two different sources (i.e., donor and recipient tissues after transplantation) is challenging. In human cellular transplantation, there is currently no useable method to detect in vivo engraftment, and blood-based non-invasive tests for allograft rejection in solid organ transplantation are either non-specific or absent. Elevated levels of donor cfDNA have been shown to correlate with solid organ rejection, but complex methodology limits implementation of this promising biomarker. We describe a cost-effective method to quantify donor cfDNA in recipient plasma using a panel of high-frequency single nucleotide polymorphisms, next-generation (semiconductor) sequencing, and a novel mixture model algorithm. In vitro, our method accurately and rapidly determined donor:recipient DNA admixture. For in vivo testing, donor cfDNA was serially quantified in an infant with a urea cycle disorder after receiving six daily infusions of donor liver cells. Donor cfDNA isolated from 1 to 2 ml of recipient plasma was detected as late as 24 weeks after infusion suggesting engraftment. The percentage of circulating donor cfDNA was also assessed in pediatric and adult heart transplant recipients undergoing routine endomyocardial biopsy with levels observed to be stable over time and generally measuring rejection. Unlike existing non-invasive methods used to define the proportion of donor cfDNA in solid organ transplant patients, our assay does not require sex mismatch, donor genotyping, or whole-genome sequencing and potentially has broad application to detect cellular engraftment or allograft injury after transplantation.

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

    Science.gov (United States)

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

    2016-06-01

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

  14. Positive impact of sucrose supplementation during slow freezing of cat ovarian tissues on cellular viability, follicle morphology, and DNA integrity.

    Science.gov (United States)

    Tanpradit, Nae; Comizzoli, Pierre; Srisuwatanasagul, Sayamon; Chatdarong, Kaywalee

    2015-06-01

    The objectives of the study were to (1) examine and optimize the impact of sucrose during slow freezing and (2) compare the results of two freezing methods (slow freezing and vitrification) on cellular viability (germinal and stromal cells), follicle morphology, DNA integrity, and gap junction protein expression (connexin 43 [Cx 43]). Different sucrose supplementations (0, 0.1, and 0.3 M) in standard freezing medium were compared before and after slow freezing. Ovarian tissue slow frozen using 0.1- (4.0 ± 0.4) or 0.3-M sucrose (3.9 ± 0.5) yielded better follicular viability (number of positive follicles per 0.0625 mm(2)) than the group without sucrose (1.9 ± 0.2; P sucrose-treated groups (0.1 M, 47.4% and 0.3 M, 43.5%) than the group without sucrose (0 M, 33.8%; P sucrose groups (0.1 M, 1.2% and 0.3 M, 1.9%) than the group without sucrose (7.7%; P sucrose concentrations. In terms of the freezing methods used, vitrified ovarian tissues had fewer viable follicles (3.2 ± 0.6) than the slow-freezing method (4.6 ± 0.6; P sucrose supplementation and slow-freezing method on the follicular viability, follicular histologic appearances of follicles, and apoptosis of the follicles and stromal cells in cat ovarian tissues.

  15. Defect in UV-induced unscheduled DNA synthesis in cultured epidermal keratinocytes from xeroderma pigmentosum

    International Nuclear Information System (INIS)

    DNA repair synthesis in 8 explant-outgrowth cultures of epidermal cells isolated from variant and complementation groups A and E of xeroderma pigmentosum (XP) was examined by measuring unscheduled DNA synthesis (UDS) on autoradiographs. The extents of UDS in XP epidermal cells were compared with those in normal epidermal cells obtained from 26 subjects. In both normal and XP epidermal cells, UDS was induced dose-dependently by radiation at doses of 5-20 J/m2. XP epidermal cells showed various extents of defect in DNA repair depending on the type of XP. In XP-A, the extent of UDS in epidermal cells was very low, being seen in only 3-10% of the normal epidermal cells. But epidermal cells isolated from XP-E and XP-variants exhibited relatively high levels of residual DNA repair; i.e., 69-84% of the control in XP-E and 67-85% in XP-variant. The extents of UDS in XP epidermal cells were almost the same as those in fibroblastic cells isolated from the same specimens. (Auth.)

  16. A microfluidic DNA computing processor for gene expression analysis and gene drug synthesis.

    Science.gov (United States)

    Zhang, Yu; Yu, Hao; Qin, Jianhua; Lin, Bingcheng

    2009-11-06

    Boolean logic performs a logical operation on one or more logic input and produces a single logic output. Here, we describe a microfluidic DNA computing processor performing Boolean logic operations for gene expression analysis and gene drug synthesis. Multiple cancer-related genes were used as input molecules. Their expression levels were identified by interacting with the computing related DNA strands, which were designed according to the sequences of cancer-related genes and the suicide gene. When all the expressions of the cancer-related genes fit in with the diagnostic criteria, positive diagnosis would be confirmed and then a complete suicide gene (gene drug) could be synthesized as an output molecule. Microfluidic chip was employed as an effective platform to realize the computing process by integrating multistep biochemical reactions involving hybridization, displacement, denaturalization, and ligation. By combining the specific design of the computing related molecules and the integrated functions of the microfluidics, the microfluidic DNA computing processor is able to analyze the multiple gene expressions simultaneously and realize the corresponding gene drug synthesis with simplicity and fast speed, which demonstrates the potential of this platform for DNA computing in biomedical applications.

  17. Induction of DNA repair synthesis in human monocytes/B-lymphocytes compared with T-lymphocytes after exposure to N-acetoxy-N-acetylaminofluorene and dimethylsulfate in vitro

    DEFF Research Database (Denmark)

    Knudsen, Lisbeth E.; Ryder, L P; Wassermann, K

    1992-01-01

    We have explored the induction of DNA repair synthesis in monocyte/B- and T-lymphocyte enriched cell fractions from 12 different human mononuclear blood cell populations. Unscheduled DNA synthesis was measured in monocyte/B- and T-cells after exposure to the DNA-damaging agents dimethylsulfate (D...

  18. Inhibitor of DNA synthesis is present in normal chicken serum

    International Nuclear Information System (INIS)

    The authors have found that heat-inactivated serum (570C for 1 hour) from normal chickens reduces the proliferation of mitogen-stimulated chicken and murine splenocytes as well as some transformed mammalian lymphoblastoid cell lines. Greater than a 50% reduction in 3H-thymidine incorporation was observed when concanavalin A (Con A)-activated chicken splenocytes that were cultured in the presence of 10% autologous or heterologous serum were compared to mitogen-stimulated cells cultured in the absence of serum. Normal chicken serum (10%) also caused greater than 95% suppression of 3H-thymidine incorporation by bovine (EBL-1 and BL-3) and gibbon ape (MLA 144) transformed lymphoblastoid cell lines. The only cell line tested that was not inhibited by chicken serum was an IL-2-dependent, murine cell line. Chicken serum also inhibited both 3H-thymidine incorporation and IL-2 synthesis by Con A-activated murine splenocytes. Suppression was caused by actions other than cytotoxicity because viability of chicken splenocytes was unaffected by increasing levels of chicken serum. Furthermore, dialyzed serum retained its activity, which suggested that thymidine in the serum was not inhibiting uptake of radiolabeled thymidine. Suppressive activity was not due to adrenal glucocorticoids circulating in plasma because neither physiologic nor pharmacologic doses of corticosterone had inhibitory effects on mitogen-stimulated chicken splenocytes. These data demonstrate that an endogenous factor that is found in normal chicken serum inhibits proliferation of T-cells from chickens and mice as well as some transformed mammalian lymphoblastoid cell lines

  19. Synthesis and Crystal Structure of 2’-Se-modified guanosine Containing DNA

    Energy Technology Data Exchange (ETDEWEB)

    Salon, J.; Sheng, J; Gan, J; Huang, Z

    2010-01-01

    Selenium modification of nucleic acids is of great importance in X-ray crystal structure determination and functional study of nucleic acids. Herein, we describe a convenient synthesis of a new building block, the 2{prime}-SeMe-modified guanosine (G{sub Se}) phosphoramidite, and report the first incorporation of the 2{prime}-Se-G moiety into DNA. The X-ray crystal structure of the 2{prime}-Se-modified octamer DNA (5{prime}-GTG{sub Se}TACAC-3{prime}) was determined at a resolution of 1.20 {angstrom}. We also found that the 2{prime}-Se modification points to the minor groove and that the modified and native structures are virtually identical. Furthermore, we observed that the 2{prime}-Se-G modification can significantly facilitate the crystal growth with respect to the corresponding native DNA.

  20. Oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by dietary polyphenols: implications for a cancer preventive mechanism.

    Science.gov (United States)

    Khan, Husain Y; Zubair, Haseeb; Ullah, Mohd F; Ahmad, Aamir; Hadi, Sheikh M

    2011-12-01

    To account for the observed anticancer properties of plant polyphenols, we have earlier proposed a mechanism which involves the mobilization of endogenous copper ions by polyphenols leading to the generation of reactive oxygen species (ROS) that serve as proximal DNA cleaving agents and lead to cell death. Over the last decade we have proceeded to validate our hypothesis with considerable success. As a further confirmation of our hypothesis, in this paper we first show that oral administration of copper to rats leads to elevated copper levels in lymphocytes. When such lymphocytes with a copper overload were isolated and treated with polyphenols EGCG, genistein and resveratrol, an increased level of DNA breakage was observed. Further, preincubation of lymphocytes having elevated copper levels with the membrane permeable copper chelator neocuproine, resulted in inhibition of polyphenol induced DNA degradation. However, membrane impermeable chelator of copper bathocuproine, as well as iron and zinc chelators were ineffective in causing such inhibition in DNA breakage, confirming the involvement of endogenous copper in polyphenol induced cellular DNA degradation. It is well established that serum and tissue concentrations of copper are greatly increased in various malignancies. In view of this fact, the present results further confirm our earlier findings and strengthen our hypothesis that an important anticancer mechanism of plant polyphenols could be the mobilization of intracellular copper leading to ROS-mediated cellular DNA breakage. In this context, it may be noted that cancer cells are under considerable oxidative stress and increasing such stress to cytotoxic levels could be a successful anticancer approach.

  1. The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

    Institute of Scientific and Technical Information of China (English)

    Scott D McCulloch; Thomas A Kunkel

    2008-01-01

    In their seminal publication describing the structure of the DNA double helix [1], Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.

  2. Synthesis of Carbohydrate Capped Silicon Nanoparticles and their Reduced Cytotoxicity, In Vivo Toxicity, and Cellular Uptake.

    Science.gov (United States)

    Ahire, Jayshree H; Behray, Mehrnaz; Webster, Carl A; Wang, Qi; Sherwood, Victoria; Saengkrit, Nattika; Ruktanonchai, Uracha; Woramongkolchai, Noppawan; Chao, Yimin

    2015-08-26

    The development of smart targeted nanoparticles (NPs) that can identify and deliver drugs at a sustained rate directly to cancer cells may provide better efficacy and lower toxicity for treating primary and advanced metastatic tumors. Obtaining knowledge of the diseases at the molecular level can facilitate the identification of biological targets. In particular, carbohydrate-mediated molecular recognitions using nano-vehicles are likely to increasingly affect cancer treatment methods, opening a new area in biomedical applications. Here, silicon NPs (SiNPs) capped with carbohydrates including galactose, glucose, mannose, and lactose are successfully synthesized from amine terminated SiNPs. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] analysis shows an extensive reduction in toxicity of SiNPs by functionalizing with carbohydrate moiety both in vitro and in vivo. Cellular uptake is investigated with flow cytometry and confocal fluorescence microscope. The results show the carbohydrate capped SiNPs can be internalized in the cells within 24 h of incubation, and can be taken up more readily by cancer cells than noncancerous cells. Moreover, these results reinforce the use of carbohydrates for the internalization of a variety of similar compounds into cancer cells. PMID:26121084

  3. Synthesis of Carbohydrate Capped Silicon Nanoparticles and their Reduced Cytotoxicity, In Vivo Toxicity, and Cellular Uptake.

    Science.gov (United States)

    Ahire, Jayshree H; Behray, Mehrnaz; Webster, Carl A; Wang, Qi; Sherwood, Victoria; Saengkrit, Nattika; Ruktanonchai, Uracha; Woramongkolchai, Noppawan; Chao, Yimin

    2015-08-26

    The development of smart targeted nanoparticles (NPs) that can identify and deliver drugs at a sustained rate directly to cancer cells may provide better efficacy and lower toxicity for treating primary and advanced metastatic tumors. Obtaining knowledge of the diseases at the molecular level can facilitate the identification of biological targets. In particular, carbohydrate-mediated molecular recognitions using nano-vehicles are likely to increasingly affect cancer treatment methods, opening a new area in biomedical applications. Here, silicon NPs (SiNPs) capped with carbohydrates including galactose, glucose, mannose, and lactose are successfully synthesized from amine terminated SiNPs. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] analysis shows an extensive reduction in toxicity of SiNPs by functionalizing with carbohydrate moiety both in vitro and in vivo. Cellular uptake is investigated with flow cytometry and confocal fluorescence microscope. The results show the carbohydrate capped SiNPs can be internalized in the cells within 24 h of incubation, and can be taken up more readily by cancer cells than noncancerous cells. Moreover, these results reinforce the use of carbohydrates for the internalization of a variety of similar compounds into cancer cells.

  4. Biomimetic synthesis of cellular SiC based ceramics from plant precursor

    Indian Academy of Sciences (India)

    O P Chakrabarti; H S Maiti; R Majumdar

    2004-10-01

    A novel biomimetic approach in designing and fabricating engineering ceramic materials has gained much interest in recent times. Following this approach, synthesis has been made of dense Si–SiC duplex ceramic composites and highly porous SiC ceramics in the image of the morphological features inherent in the caudex stem of a local monocotyledonous plant. The process route involves making of a carbonaceous biopreform and its subsequent reaction with an infiltrating silicon melt to yield the biomorphic Si–SiC ceramic composites with flexural strength and Young’s modulus of 264 MPa and 247 Gpa, respectively and loss in weight of only ∼ 9% during oxidative heating up to 1200°C in flowing air. The Si–SiC composites were transformed into porous (49 vol.%) SiC ceramics with complete preservation of microcellular anatomy of the parent plant, by depleting residual silicon phase in channel pores through reaction with carbon. SiC based materials so derived can be used in structural applications and in designing high temperature filters and catalyst supports.

  5. Iron may induce both DNA synthesis and repair in rat hepatocytes stimulated by EGF/pyruvate

    Energy Technology Data Exchange (ETDEWEB)

    Chenoufi, N.; Loreal, O.; Cariou, S.; Hubert, N.; Lescoat, G. [Univ. Hospital Pontchaillou, Unite de Recherches Hepatologiques, INSERM U 49, Rennes (France); Drenou, B. [Univ. Hospital Pontchaillou, Lab. d`Hematologie et d`Immunologie, Rennes (France); Leroyer, P.; Brissot, P. [Univ. Hospital Pontchaillou, Clinique des Maladies du Foie, Rennes (France)

    1997-03-01

    Background/Aims: Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested. Methods: The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex. Results: In EGF/pyruvate-stimulated cultures, the level of [{sup 3}H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [{sup 3}H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However,the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [{sup 3}H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures. Conclusions: Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma. (au) 61 refs.

  6. Synthesis and in vitro cellular interactions of superparamagnetic iron nanoparticles with a crystalline gold shell

    Energy Technology Data Exchange (ETDEWEB)

    Bandyopadhyay, Sulalit, E-mail: sulalit.bandyopadhyay@ntnu.no [Ugelstad Laboratory, Department of Chemical Engineering (Norway); Singh, Gurvinder [Ugelstad Laboratory, Department of Chemical Engineering (Norway); Sandvig, Ioanna [MI Lab and Department of Circulation and Medical Imaging, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim (Norway); Sandvig, Axel [MI Lab and Department of Circulation and Medical Imaging, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim (Norway); Department of Neurosurgery, Umeå University Hospital, Umeå (Sweden); Mathieu, Roland; Anil Kumar, P. [Department of Engineering Sciences, Uppsala University, Box 534, SE-75121 Uppsala (Sweden); Glomm, Wilhelm Robert [Ugelstad Laboratory, Department of Chemical Engineering (Norway); Sector for Biotechnology and Nanomedicine, SINTEF Materials and Chemistry, N-7465 Trondheim (Norway)

    2014-10-15

    Graphical abstract: - Highlights: • A novel synthetic protocol for Fe@Au nanoparticles (NPs) has been optimized. • Surface functionalization and characterization of Fe@Au NPs. • NPs retain superparamagnetic properties after Au coating. • No toxic effects on two different cell types. • NPs suitable for theranostic applications. - Abstract: Fe@Au core–shell nanoparticles (NPs) exhibit multiple functionalities enabling their effective use in applications such as medical imaging and drug delivery. In this work, a novel synthetic method was developed and optimized for the synthesis of highly stable, monodisperse Fe@Au NPs of average diameter ∼24 nm exhibiting magneto-plasmonic characteristics. Fe@Au NPs were characterized by a wide range of experimental techniques, including scanning (transmission) electron microscopy (S(T)EM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS) and UV–vis spectroscopy. The formed particles comprise an amorphous iron core with a crystalline Au shell of tunable thickness, and retain the superparamagnetic properties at room temperature after formation of a crystalline Au shell. After surface modification, PEGylated Fe@Au NPs were used for in vitro studies on olfactory ensheathing cells (OECs) and human neural stem cells (hNSCs). No adverse effects of the Fe@Au particles were observed post-labeling, both cell types retaining normal morphology, viability, proliferation, and motility. It can be concluded that no appreciable toxic effects on both cell types, coupled with multifunctionality and chemical stability make them ideal candidates for therapeutic as well as diagnostic applications.

  7. Effects of gastrin, epidermal growth factor, and somatostatin on DNA synthesis in a small intestinal crypt cell line (IEC-6)

    International Nuclear Information System (INIS)

    Exposure of IEC-6 cells for 24 hr to either gastrin (50-500 ng/ml) or EGF (100-500 ng/ml) significantly (100-165%) the rate of [3H]thymidine incorporation into DNA (referred to as DNA synthesis) when compared with the corresponding basal levels. Somatostatin (10-500 ng/ml) produced no apparent change in DNA synthesis in IEC cells. On the other hand, somatostatin completely inhibited the EGF-induced rise in DNA synthesis. The gastrin-mediated stimulation in DNA synthesis was not affected by somatostatin. The rate of DNA synthesis in IEC cells in the presence of both gastrin and EGF was found to be greater (additive) than that caused by either of the peptides alone. A similar but less dramatic change in the actual number of cells (assessment of cell replication) was observed when the IEC cells were exposed for 24 hr to gastrin, EGF, and somatostatin, either alone or in combination. Whereas gastrin (250 ng/ml) and EGF (250 ng/ml) by themselves increased the number of cells significantly by 29 and 37%, respectively, together they caused a 72% stimulation, when compared with the basal levels. Somatostatin by itself caused no apparent change in IEC cell population, but it significantly inhibited the EGF- but not the gastrin-induced stimulation in IEC cell replication. It is concluded that both gastrin and EGF exert a direct proliferative effect on IEC cells, and the EGF action is regulated by somatostatin

  8. Glycosaminoglycan-functionalized poly-lactide-co-glycolide nanoparticles: synthesis, characterization, cytocompatibility, and cellular uptake

    Directory of Open Access Journals (Sweden)

    Lamichhane SP

    2015-01-01

    Full Text Available Surya P Lamichhane,1 Neha Arya,1,2 Nirdesh Ojha,3 Esther Kohler,1 V Prasad Shastri1,2,41Institute for Macromolecular Chemistry, University of Freiburg, Freiburg, 2Helmholtz Virtual Institute on “Multifunctional Biomaterials for Medicine”, 3Laboratory for Process Technology, Department of Microsystems Engineering, University of Freiburg, Freiburg, 4Centre for Biological Signaling Studies (BIOSS, University of Freiburg, Freiburg, GermanyAbstract: The efficient delivery of chemotherapeutics to the tumor via nanoparticle (NP-based delivery systems remains a significant challenge. This is compounded by the fact that the tumor is highly dynamic and complex environment composed of a plurality of cell types and extracellular matrix. Since glycosaminoglycan (GAG production is altered in many diseases (or pathologies, NPs bearing GAG moieties on the surface may confer some unique advantages in interrogating the tumor microenvironment. In order to explore this premise, in the study reported here poly-lactide-co-glycolide (PLGA NPs in the range of 100–150 nm bearing various proteoglycans were synthesized by a single-step nanoprecipitation and characterized. The surface functionalization of the NPs with GAG moieties was verified using zeta potential measurements and X-ray photoelectron spectroscopy. To establish these GAG-bearing NPs as carriers of therapeutics, cellular toxicity assays were undertaken in lung epithelial adenocarcinoma (A549 cells, human pulmonary microvascular endothelial cells (HPMEC, and renal proximal tubular epithelial cells. In general NPs were well tolerated over a wide concentration range (100–600 µg/mL by all cell types and were taken up to appreciable extents without any adverse cell response in A549 cells and HPMEC. Further, GAG-functionalized PLGA NPs were taken up to different extents in A459 cells and HPMEC. In both cell systems, the uptake of heparin-modified NPs was diminished by 50%–65% in comparison to that of

  9. Synthesis, characterization; DNA binding and antitumor activity of ruthenium(II) polypyridyl complexes.

    Science.gov (United States)

    Srishailam, A; Gabra, Nazar Mohammed; Kumar, Yata Praveen; Reddy, Kotha Laxma; Devi, C Shobha; Anil Kumar, D; Singh, Surya S; Satyanarayana, S

    2014-12-01

    Three new ruthenium(II) polypyridyl complexes [Ru(phen)2BrIPC](2+) (1), [Ru(bpy)2 BrIPC](2+) (2) and [Ru(dmb)2BrIPC](2+) (3) where, BrIPC = (6-bromo-3-(1H-imidazo[4,5-f] [1,10]-phenanthroline, phen = 1,10-phenanthroline, bpy = 2,2' bipyridine, dmb = 4,4'-dimethyl 2,2' bipyridine, were synthesised and characterised. DNA-binding nature was investigated by spectroscopic titrations and mode of binding was assessed by viscosity measurements. The DNA-binding constants Kb of complexes 1, 2 and 3 were determined to be in the order of 10(5). Experimental results showed that these complexes interact with CT-DNA by intercalative mode. Photocleavage and antimicrobial activities were complex concentration dependent, at high concentration, high activity and vice versa. MTT assay was performed on HeLa cell lines, IC50 values of complexes in the order of 3 > 2 > 1 > cisplatin. From comet assay, cellular uptake studies, we observed that complexes could enter into the cell membrane and accumulate inside the nucleus. Molecular docking studies support the DNA binding affinity with hydrogen bonding and van der Waals attractions between base pairs and phosphate backbone of DNA with metal complexes. PMID:25318017

  10. Studies on the Synthesis, Characterization, DNA Binding, Cytotoxicity and Antioxidant activity of 2-methyl-4-nitrophenylferrocene

    International Nuclear Information System (INIS)

    We report herein the synthesis, structural characterization, DNA binding, BamH1 digestion, cytotoxicity and antioxidant activity of 2-methyl-4-nitrophenylferrocene. Structural characterization is based on multinuclear (1H and 13C) NMR, FT-IR spectroscopy and elemental analysis. Interaction of 2-methyl-4-nitrophenylferrocene with pBR322 plasmid DNA shows noncovalent interactions however these noncovalent interactions reveal the prevention of BamH1 restriction site (g/ggtcc). In the voltammogram, a negative shift in peak potential has been observed on addition of increasing concentration of CT-DNA, which shows electrostatic interaction for 2-methyl-4-nitrophenylferro with negatively charged phosphate of DNA backbone. The binding ratio, binding constant, binding free energy and diffusion coefficient of free and bound drug were calculated to understand the mechanism. The high negative value of -delta G signifies the spontaneity and high conformational stability of 2-methyl-4-nitrophenylferro with CT-DNA. The compound has the ability to scavenge free radicals as have been revealed by DPPH findings. (author)

  11. Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities

    OpenAIRE

    Brunet, Erika; Corgnali, Maddalena; Perrouault, Loïc; Roig, Victoria; Asseline, Ulysse; Sørensen, Mads D.; Babu, B. Ravindra; Wengel, Jesper; Giovannangeli, Carine

    2005-01-01

    Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities; to this end, ...

  12. Inhibition by 2-deoxy-D-ribose of DNA synthesis and growth in Raji cells

    International Nuclear Information System (INIS)

    When Raji cells were cultured for 3 days in serum-free medium, addition of 2-deoxy-D-ribose at the start of culture inhibited incorporation of [3H]thymidine and cell division. At deoxyribose concentrations between 1 and 5 mM, viability was 80% or greater after 3 days of culture even though 5 mM deoxyribose inhibited thymidine incorporation 95-99%. Inhibition by deoxyribose could be completely reversed if the culture medium was replaced with fresh medium up to 8 hr after the start of culture. The inhibition was specific for deoxyribose since other monosaccharides had no effect. Inhibition of DNA synthesis did not appear to be due to depletion of essential nutrients in the medium since the percentage inhibition of thymidine incorporation by cells cultured either in suboptimal serum-free media or in media supplemented with 0.025-5% human AB serum was similar. When DNA repair synthesis was measured as hydroxyurea-resistant thymidine incorporation, addition of deoxyribose to Raji cultures caused increased thymidine incorporation. These results, together with data from others,suggest that deoxyribose damages DNA

  13. Arsenic Trioxide Modulates DNA Synthesis and Apoptosis in Lung Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Kenneth Ndebele

    2010-04-01

    Full Text Available Arsenic trioxide, the trade name Trisenox, is a drug used to treat acute promyleocytic leukemia (APL. Studies have demonstrated that arsenic trioxide slows cancer cells growth. Although arsenic influences numerous signal-transduction pathways, cell-cycle progression, and/or apoptosis, its apoptotic mechanisms are complex and not entirely delineated. The primary objective of this research was to evaluate the effects of arsenic trioxide on DNA synthesis and to determine whether arsenic-induced apoptosis is mediated via caspase activation, p38 mitogen–activated protein kinase (MAPK, and cell cycle arrest. To achieve this goal, lung cancer cells (A549 were exposed to various concentrations (0, 2, 4, 6, 8, and 10 µg/mL of arsenic trioxide for 48 h. The effect of arsenic trioxide on DNA synthesis was determined by the [3H]thymidine incorporation assay. Apoptosis was determined by the caspase-3 fluorescein isothiocyanate (FITC assay, p38 MAP kinase activity was determined by an immunoblot assay, and cell-cycle analysis was evaluated by the propidium iodide assay. The [3H]thymidine-incorporation assay revealed a dose-related cytotoxic response at high levels of exposure. Furthermore, arsenic trioxide modulated caspase 3 activity and induced p38 MAP kinase activation in A549 cells. However, cell-cycle studies showed no statistically significant differences in DNA content at subG1 check point between control and arsenic trioxide treated cells.

  14. Assembly fabrication of linkers on glass surface and their effect on DNA synthesis and hybridization

    Institute of Scientific and Technical Information of China (English)

    ShenJiayao; XiaoPengfeng; HouPeng; JiMeiju; SunXiao; HeNongyue

    2003-01-01

    Linkers were assembled on a glass surface based on the hydrolysis and condensation of 3-glycidoxy-propyltrimethoxysilane (GPS). After the assembly of GPS, four approaches were tried to open the ending epoxide group of GPS or to further elongate the linkers. The effect of these approaches on DNA in situ synthesis and hybridization was investigated. For the spacing of the synthesis initiation sites, the wettability of the support and the length of the linking group that attaches the initiation site to the surface have direct influences on the yield of coupling reactions and the subsequent hybridization events. X-ray photoelectron spectroscopy (XPS) and mean contact angles of deionized water of the above slides were measured to assess the linker's characteristics in each procedure. It was proved that the glass slides were successfully modified and became excellent supports for the oligonucleotides synthesis. In addition, it proved best for the in situ oligonueleotides synthesis that a glass slide was in turn treated with ethylenediamine, glutaradehyde, ethanolamine and sodium borohydride solution at ambient temperature after silanized with GPS.

  15. DNA Binding and Photocleavage Properties, Cellular Uptake and Localization, and in-Vitro Cytotoxicity of Dinuclear Ruthenium(II) Complexes with Varying Lengths in Bridging Alkyl Linkers.

    Science.gov (United States)

    Liu, Ping; Wu, Bao-Yan; Liu, Jin; Dai, Yong-Cheng; Wang, You-Jun; Wang, Ke-Zhi

    2016-02-15

    Two new dinuclear Ru(II) polypyridyl complexes containing three and ten methylene chains in their bridging linkers are synthesized and characterized. Their calf thymus DNA-binding and plasmid DNA photocleavage behaviors are comparatively studied with a previously reported, six-methylene-containing analog by absorption and luminescence spectroscopy, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, DNA viscosity measurements, DNA thermal denaturation, and agarose gel electrophoresis analyses. Theoretical calculations applying the density functional theory (DFT) method for the three complexes are also performed to understand experimentally observed DNA binding properties. The results show that the two complexes partially intercalate between the base pairs of DNA. Cellular uptake and colocalization studies have demonstrated that the complexes could enter HeLa cells efficiently and localize within lysosomes. The in-vitro antitumor activity against HeLa and MCF-7 tumor cells of the complexes are studied by MTT cytotoxic analysis. A new method, high-content analysis (HCA), is also used to assess cytotoxicity, apoptosis and cell cycle arrest of the three complexes. The results show that the lengths of the alkyl linkers could effectively tune their biological properties and that HCA is suitable for rapidly identifying cytotoxicity and can be substituted for MTT assays to evaluate the cell cytotoxicity of chemotherapeutic agents.

  16. Assessment of DNA synthesis in Islet-1+ cells in the adult murine heart

    International Nuclear Information System (INIS)

    Highlights: • Islet-1 was expressed in the adult heart. • Islet-1-positive cells did not proliferate in the adult heart. • Sinoatrial node cells did not proliferate in the adult heart. - Abstract: Rationale: Islet-1 positive (Islet-1+) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1+ cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart. Methods and results: DNA synthesis was analyzed by the incorporation of tritiated thymidine (3H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of 3H-thymidine for 5 days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1+ cells. Whereas Islet− non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. Conclusions: Islet-1+ cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes

  17. Assessment of DNA synthesis in Islet-1{sup +} cells in the adult murine heart

    Energy Technology Data Exchange (ETDEWEB)

    Weinberger, Florian, E-mail: f.weinberger@uke.de; Mehrkens, Dennis, E-mail: dennis.mehrkens@uk-koeln.de; Starbatty, Jutta, E-mail: starbatty@uke.uni-hamburg.de; Nicol, Philipp, E-mail: Philipp.Nicol@gmx.de; Eschenhagen, Thomas, E-mail: t.eschenhagen@uke.de

    2015-01-02

    Highlights: • Islet-1 was expressed in the adult heart. • Islet-1-positive cells did not proliferate in the adult heart. • Sinoatrial node cells did not proliferate in the adult heart. - Abstract: Rationale: Islet-1 positive (Islet-1{sup +}) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1{sup +} cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart. Methods and results: DNA synthesis was analyzed by the incorporation of tritiated thymidine ({sup 3}H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of {sup 3}H-thymidine for 5 days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1{sup +} cells. Whereas Islet{sup −} non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. Conclusions: Islet-1{sup +} cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes.

  18. Organometallic B12-DNA conjugate

    DEFF Research Database (Denmark)

    Hunger, Miriam; Mutti, Elena; Rieder, Alexander;

    2014-01-01

    Design, synthesis, and structural characterization of a B12-octadecanucleotide are presented herein, a new organometallic B12-DNA conjugate. In such covalent conjugates, the natural B12 moiety may be a versatile vector for controlled in vivo delivery of oligonucleotides to cellular targets in hum...

  19. Determination of radioinduced delay in DNA synthesis in two-garlic-clones cells (Allium Sativum L.)

    International Nuclear Information System (INIS)

    To contribute to tech improvement of the use of ionizing radiations as an auxiliary tool in the fitoimprovement, dose-effect curves for the 'Martinez' and 'Sancti Spiritus-3' clones were stablished by using as effect the delay induced by radiations in DNA synthesis determined by the 'Martinez' clone which induces a delay of 50% in reference to the control is approximately 11 Gy, while the dose value for the 'Sancti Spiritus-3' clone is 18 Gy, thus the 'Martinez' clones has a higher sensitivity to radiations than the other clone, therefore it coincides with what we found for these clones other indexes are used as radiosensitivity criteria

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

    Institute of Scientific and Technical Information of China (English)

    Saleem Bani Hani; Mekki Bayachou

    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.

  1. Fabrication of polyurethane molecular stamps for the synthesis of DNA microarray

    Science.gov (United States)

    Liu, Zhengchun; He, Quanguo; Xiao, Pengfeng; He, Nongyao; Lu, Zuhong; Bo, Liang

    2001-10-01

    Polyurethane based on polypropylene glycol (PPG) and Toluene diisocyanate (TDI) using 3,3'-dichloride-4,4'- methylenedianiline (MOCA) as the crosslinker is presented for the first time to fabricate molecular stamps (PU stamps) for the synthesis of DNA microarray with contact procedure. The predictability of the process is achieved by utilizing commercially available starting materials. SEM analysis of the morphology of PU stamps and master showed that PU elastometer could replicate subtly the motherboard's patterns with high fidelity. It was proved from the contact angle measurement that PU stamps surface has good affinity with acetonitrile, which guarantee the well-distribution of DNA monomers on patterned stamps. Laser confocal fluorescence microscopy images of oligonucleotide arrays confirmed polyurethane is an excellent material for molecular stamps.

  2. DNA microarray synthesis by using PDMS molecular stamps (Ⅲ)-- Optimization for the reaction conditions

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Optimization for the technological processes of fabricating oligonucleotide microarray by the molecular stamping method is studied in this note. Three factors that affect the pressing coupling reactions of the nucleosides are focused on: the stability of the chemical activities of the reaction solutions, the contamination of the remain of the reactive nucleotides among the different spots on the chip, and the influence of the capping reaction on the hybridization result. The experiments show that the acetonitrile solution of tetrazole and nucleoside monomer could maintain sufficient reactive activity for more than 10 h. An effective method has been used and proved to eliminate the residual reactive nucleosides on chip with small molecules containing hydroxyl group. Finally, the capping step-- a regular step in the conventional DNA chemical synthesis can be neglected in our on-chip DNA synthetic process, which would not affect its hybridization results.

  3. Estimations of the DNA Synthesis Rate of Bone Marrow Cells after Administration of Labelled Thymidine In Vitro

    International Nuclear Information System (INIS)

    Bone marrow cells are incubated with labelled thymidine under varying in vitro conditions. The incorporation rate of labelled thymidine into DNA is influenced by the condition and duration of. the in vitro incubation. Similar influences operate on the pool size of labelled thymidine phosphates. Up to concentrations of 10-6 M thymidine in the incubation medium there is a linear relation of thymidine concentration and thymidine incorporation into DNA. Concentrations of thymidine exceeding 10-6 M lead to increasing inhibition of the thymidine kinase. The endogenous formation of thymidylate cannot be inhibited entirely by exogenous thymidine supply. Consequently, determinations of the DNA synthesis rate from the incorporated amount of labelled thymidine have to be corrected for the respective endogenous thymidylate contribution. A better procedure is to block the formation of endogenous thymidylate by means of amethopterin. Standard conditions are described, under which an undisturbed synthesis of DNA thymine from exogenous thymidine only takes place. Determinations can be performed by means of autoradiographic or biochemical techniques. By application of the semi-automatic grain counting technique, after sufficient autoradiographic standardization, evaluations of DNA synthesis rates and DNA synthesis times of different cell types in the bone marrow become practicable. (author)

  4. Xeroderma pigmentosum variants have a slow recovery of DNA synthesis after irradiation with ultraviolet light

    International Nuclear Information System (INIS)

    Human cells (normal and xeroderma pigmentosum variant) irradiated with ultraviolet light and pulse-labelled with [3H]thymidine underwent transient decline and recovery of molecular weights of newly synthesized DNA and rates of [3H]thymidine incorporation. The ability of synthesize normal-sized DNA recovered more rapidly in both cell types than thymidine incorporation. During recovery cells steadily increased in their ability to replicate normalsized DNA on damaged templates. The molecular weight versus time curves fitted exponential functions with similar rate constants in normal and heterozygous xeroderma pigmentosum cells, but with a slower rate in two xeroderma pigmentosum variant cell lines. Caffeine added during the post-irradiation period eliminated the recovery of molecular weights in xeroderma pigmentsoum variant but not in normal cells. The recovery of the ability to synthesize normal-sized DNA represents a combination of a number of cellular regulatory processes, some of which are constitutive, and one of which is altered in the xeroderma pigmentosum variant such that recovery becomes slow and caffeine sensitive. (Auth.)

  5. RNA polymerase motors on DNA track: effects of traffic congestion on RNA synthesis

    CERN Document Server

    Tripathi, Tripti

    2007-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by a ssDNA. In some circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track. We refer to such collective movements of the RNAPs as RNAP traffic because of the similarities between the collective dynamics of the RNAPs on ssDNA track and that of vehicles in highway traffic. In this paper we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechano-chemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the ssDNA track. We also suggest novel experiments for testing o...

  6. Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis.

    Science.gov (United States)

    Hedglin, Mark; Pandey, Binod; Benkovic, Stephen J

    2016-03-29

    In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicating the lagging strand template and anchors to the proliferating cell nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The stability of this complex is integral to every aspect of lagging strand replication. Most of our understanding comes from Saccharomyces cerevisae where the extreme stability of the pol δ holoenzyme ensures that every nucleobase within an Okazaki fragment is faithfully duplicated before dissociation but also necessitates an active displacement mechanism for polymerase recycling and exchange. However, the stability of the human pol δ holoenzyme is unknown. We designed unique kinetic assays to analyze the processivity and stability of the pol δ holoenzyme. Surprisingly, the results indicate that human pol δ maintains a loose association with PCNA while replicating DNA. Such behavior has profound implications on Okazaki fragment synthesis in humans as it limits the processivity of pol δ on undamaged DNA and promotes the rapid dissociation of pol δ from PCNA on stalling at a DNA lesion.

  7. Structural, molecular and cellular functions of MSH2 and MSH6 during DNA mismatch repair, damage signaling and other noncanonical activities

    International Nuclear Information System (INIS)

    The field of DNA mismatch repair (MMR) has rapidly expanded after the discovery of the MutHLS repair system in bacteria. By the mid 1990s yeast and human homologues to bacterial MutL and MutS had been identified and their contribution to hereditary non-polyposis colorectal cancer (HNPCC; Lynch syndrome) was under intense investigation. The human MutS homologue 6 protein (hMSH6), was first reported in 1995 as a G:T binding partner (GTBP) of hMSH2, forming the hMutSα mismatch-binding complex. Signal transduction from each DNA-bound hMutSα complex is accomplished by the hMutLα heterodimer (hMLH1 and hPMS2). Molecular mechanisms and cellular regulation of individual MMR proteins are now areas of intensive research. This review will focus on molecular mechanisms associated with mismatch binding, as well as emerging evidence that MutSα, and in particular, MSH6, is a key protein in MMR-dependent DNA damage response and communication with other DNA repair pathways within the cell. MSH6 is unstable in the absence of MSH2, however it is the DNA lesion-binding partner of this heterodimer. MSH6, but not MSH2, has a conserved Phe-X-Glu motif that recognizes and binds several different DNA structural distortions, initiating different cellular responses. hMSH6 also contains the nuclear localization sequences required to shuttle hMutSα into the nucleus. For example, upon binding to O6meG:T, MSH6 triggers a DNA damage response that involves altered phosphorylation within the N-terminal disordered domain of this unique protein. While many investigations have focused on MMR as a post-replication DNA repair mechanism, MMR proteins are expressed and active in all phases of the cell cycle. There is much more to be discovered about regulatory cellular roles that require the presence of MutSα and, in particular, MSH6

  8. EFFECT OF ASCORBIC ACID ON DNA SYNTHESIS, INTRACELLULAR ACCUMULATION OF ADM AND ADM RESISTANCE OF TUMOR CELL LINES

    Institute of Scientific and Technical Information of China (English)

    Xie Zuofu; Lin Xiandong; Zhou Dongmei; Lin Sheng

    1998-01-01

    Objective: To determine the effect of ascorbic acid (AA) on DNA synthesis, intracellular accumulation of ADM and ADM resistance of tumor cell lines.Methods: K562, K562/ADM and KB cell lines were used to study the effect of ascorbic acid on DNA synthesis,intracellular accumulation of ADM and ADM resistance by fluid scintillometry, MTT method, spectrofluorophotometry and immunocytochemistry. Results: Results showed that AA was capable of inhibiting DNA synthesis of K562 and K562/ADM in a dose-dependence fashion,but not KB cell line, and significantly reducing ADM sensitivity in K562 and KB cell lines, as well as potentiating obviously ADM resistance in K562/ADM cell line. Conclusion: These effects of AA may be closely correlated with significant elevation of intracellular accumulation of ADM in KB cell line, and significant reduction of that in K562 and K562/ADM cell lines but possibly not correlated with the expression of Pglycoprotein.

  9. Cellular and molecular mechanistic insight into the DNA-damaging potential of few-layer graphene in human primary endothelial cells.

    Science.gov (United States)

    Sasidharan, Abhilash; Swaroop, Siddharth; Chandran, Parwathy; Nair, Shantikumar; Koyakutty, Manzoor

    2016-07-01

    Despite graphene being proposed for a multitude of biomedical applications, there is a dearth in the fundamental cellular and molecular level understanding of how few-layer graphene (FLG) interacts with human primary cells. Herein, using human primary umbilical vein endothelial cells as model of vascular transport, we investigated the basic mechanism underlying the biological behavior of graphene. Mechanistic toxicity studies using a battery of cell based assays revealed an organized oxidative stress paradigm involving cytosolic reactive oxygen stress, mitochondrial superoxide generation, lipid peroxidation, glutathione oxidation, mitochondrial membrane depolarization, enhanced calcium efflux, all leading to cell death by apoptosis/necrosis. We further investigated the effect of graphene interactions using cDNA microarray analysis and identified potential adverse effects by down regulating key genes involved in DNA damage response and repair mechanisms. Single cell gel electrophoresis assay/Comet assay confirmed the DNA damaging potential of graphene towards human primary cells. PMID:26970024

  10. Dancing on damaged chromatin. Functions of ATM and the RAD50/MRE11/NBS1 complex in cellular responses to DNA damage

    International Nuclear Information System (INIS)

    In order to preserve and protect genetic information, eukaryotic cells have developed a signaling or communications network to help the cell respond to DNA damage, and ATM and NBS1 are key players in this network. ATM is a protein kinase which is activated immediately after a DNA double strand break (DSB) is formed, and the resulting signal cascade generated in response to cellular DSBs is regulated by post-translational protein modifications such as phosphorylation and acetylation. In addition, to ensure the efficient functioning of DNA repair and cell cycle checkpoints, the highly ordered structure of eukaryotic chromatin must be appropriately altered to permit access of repair-related factors to DNA. These alterations are termed chromatin remodeling, and are executed by a specific remodeling complex in conjunction with histone modifications. Current advances in the molecular analysis of DNA damage responses have shown that the auto-phosphorylation of ATM and the interaction between ATM and NBS1 are key steps for ATM activation, and that the association of ATM and NBS1 is involved in chromatin remodeling. Identification of novel factors which function in ubiquitination (RNF8, Ubc13, Rap80, etc.) has also enabled us to understand more details of the early stages in DNA repair pathways which respond to DSBs. In this review, the focus is on the role of ATM and the RAD50/MRE11/NBS1 complex in DSB response pathways, and their role in DSB repair and in the regulation of chromatin remodeling. (author)

  11. Error-prone translesion synthesis past DNA-peptide cross-links conjugated to the major groove of DNA via C5 of thymidine.

    Science.gov (United States)

    Wickramaratne, Susith; Boldry, Emily J; Buehler, Charles; Wang, Yen-Chih; Distefano, Mark D; Tretyakova, Natalia Y

    2015-01-01

    DNA-protein cross-links (DPCs) are exceptionally bulky, structurally diverse DNA adducts formed in cells upon exposure to endogenous and exogenous bis-electrophiles, reactive oxygen species, and ionizing radiation. If not repaired, DPCs can induce toxicity and mutations. It has been proposed that the protein component of a DPC is proteolytically degraded, giving rise to smaller DNA-peptide conjugates, which can be subject to nucleotide excision repair and replication bypass. In this study, polymerase bypass of model DNA-peptide conjugates structurally analogous to the lesions induced by reactive oxygen species and DNA methyltransferase inhibitors was examined. DNA oligomers containing site-specific DNA-peptide conjugates were generated by copper-catalyzed [3 + 2] Huisgen cyclo-addition between an alkyne-functionalized C5-thymidine in DNA and an azide-containing 10-mer peptide. The resulting DNA-peptide conjugates were subjected to steady-state kinetic experiments in the presence of recombinant human lesion bypass polymerases κ and η, followed by PAGE-based assays to determine the catalytic efficiency and the misinsertion frequency opposite the lesion. We found that human polymerase κ and η can incorporate A, G, C, or T opposite the C5-dT-conjugated DNA-peptide conjugates, whereas human polymerase η preferentially inserts G opposite the lesion. Furthermore, HPLC-ESI(-)-MS/MS sequencing of the extension products has revealed that post-lesion synthesis was highly error-prone, resulting in mutations opposite the adducted site or at the +1 position from the adduct and multiple deletions. Collectively, our results indicate that replication bypass of peptides conjugated to the C5 position of thymine by human translesion synthesis polymerases leads to large numbers of base substitution and frameshift mutations.

  12. Effect of different BNCT protocols on DNA synthesis in precancerous and normal tissues in an experimental model of oral cancer

    International Nuclear Information System (INIS)

    We previously reported the therapeutic success of different BNCT protocols in the treatment of oral cancer, employing the hamster cheek pouch model. The aim of the present study was to evaluate the effect of these BNCT protocols on DNA synthesis in precancerous and normal tissue in this model and assess the potential lag in the development of second primary tumors in precancerous tissue. The data are relevant to potential control of field cancerized tissue and tolerance of normal tissue. We evaluated DNA synthesis in precancerous and normal pouch tissue 1-30 days post-BNCT mediated by BPA, GB-10 or BPA + GB-10 employing incorporation of bromo-deoxyuridine as an end-point. The BNCT-induced potential lag in the development of second primary tumors in precancerous tissue was monitored. A drastic, statistically significant reduction in DNA synthesis occurred in pacancerous tissue as early as 1 day post-BNCT and was sustained at virtually all time points until 30 days post-BNCT for all protocols. The histological categories evaluated individually within precancerous tissue (dysplasia, hyperplasia and NUMF [no unusual microscopic features]) responded similarly. DNA synthesis in normal tissue treated with BNCT oscillated around the very low pre-treatment values. A BNCT-induced lag in the development of second primary tumors was observed. BNCT induced a drastic fall in DNA synthesis in precancerous tissue that would be associated to the observed lag in the development of second primary tumors. The minimum variations in DNA synthesis in BNCT-treated normal tissue would correlate with the absence of normal tissue radiotoxicity. The present data would contribute to optimize therapeutic efficacy in the treatment of field-cancerized areas. (author)

  13. The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes

    International Nuclear Information System (INIS)

    The in vitro unscheduled DNA synthesis (UDS) assay was evaluated for inclusion in a battery of assays used at The Upjohn Company for evaluation of lead compounds in the development of new and existing drug entities. This evaluation process uncompassed aspects of the isolation of hepatocytes and tests of reference mutagens and genotoxins. The flow rate of perfusion solutions and their temperatures were critical in the isolation of high viability hepatocytes in good yield. The attachment of freshly isolated hepatocytes to coverslips was greatly enhanced by coating the coverslips with type III colagen. Results of testing 12 known genotoxic agents (UV light, cyclophosphamide, 7,12-dimethylbenzanthracene, dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, benzo[a]pyrene, methyl methanesulfonate, ethyl methanesulfonate, N-propyl-N'-nitro-N-nitrosoguanidine, benzidine and 4-aminobiphenyl) were in agreement with the literature. The use of X-ray did not induce unscheduled DNA synthesis in hepatocytes. This latter finding draws attention to the inability of this assay to detect agents which result in 'short-patch' repair of damage. (author). 35 refs.; 8 tabs

  14. Amino acids attached to 2'-amino-LNA: Synthesis of DNA mixmer oligonucleotides with increased duplex stability

    DEFF Research Database (Denmark)

    Johannsen, Marie Willaing; Wengel, Jesper; Wamberg, Michael Chr.;

    2010-01-01

    The synthesis of 2'-amino-LNA (locked nucleic acid) opens up exciting possibilities for modification of nucleic acids by conjugation to the 2'-nitrogen. Incorporation of unmodified and N-functionalized 2'-amino-LNA nucleotides improve duplex stability compared to unmodified DNA. 2'-Amino......-LNA nucleosides derivatized with amino acids have been synthesized and incorporated into DNA oligonucleotides. Following oligonucleotide synthesis, peptides have been added using solid phase peptide coupling chem. Modification of oligonucleotides with pos. charged residues greatly improves thermal stability....

  15. Cell type-dependent induction of DNA damage by 1800 MHz radiofrequency electromagnetic fields does not result in significant cellular dysfunctions.

    Directory of Open Access Journals (Sweden)

    Shanshan Xu

    Full Text Available BACKGROUND: Although IARC clarifies radiofrequency electromagnetic fields (RF-EMF as possible human carcinogen, the debate on its health impact continues due to the inconsistent results. Genotoxic effect has been considered as a golden standard to determine if an environmental factor is a carcinogen, but the currently available data for RF-EMF remain controversial. As an environmental stimulus, the effect of RF-EMF on cellular DNA may be subtle. Therefore, more sensitive method and systematic research strategy are warranted to evaluate its genotoxicity. OBJECTIVES: To determine whether RF-EMF does induce DNA damage and if the effect is cell-type dependent by adopting a more sensitive method γH2AX foci formation; and to investigate the biological consequences if RF-EMF does increase γH2AX foci formation. METHODS: Six different types of cells were intermittently exposed to GSM 1800 MHz RF-EMF at a specific absorption rate of 3.0 W/kg for 1 h or 24 h, then subjected to immunostaining with anti-γH2AX antibody. The biological consequences in γH2AX-elevated cell type were further explored with comet and TUNEL assays, flow cytometry, and cell growth assay. RESULTS: Exposure to RF-EMF for 24 h significantly induced γH2AX foci formation in Chinese hamster lung cells and Human skin fibroblasts (HSFs, but not the other cells. However, RF-EMF-elevated γH2AX foci formation in HSF cells did not result in detectable DNA fragmentation, sustainable cell cycle arrest, cell proliferation or viability change. RF-EMF exposure slightly but not significantly increased the cellular ROS level. CONCLUSIONS: RF-EMF induces DNA damage in a cell type-dependent manner, but the elevated γH2AX foci formation in HSF cells does not result in significant cellular dysfunctions.

  16. Synchronization of DNA array replication kinetics

    Science.gov (United States)

    Manturov, Alexey O.; Grigoryev, Anton V.

    2016-04-01

    In the present work we discuss the features of the DNA replication kinetics at the case of multiplicity of simultaneously elongated DNA fragments. The interaction between replicated DNA fragments is carried out by free protons that appears at the every nucleotide attachment at the free end of elongated DNA fragment. So there is feedback between free protons concentration and DNA-polymerase activity that appears as elongation rate dependence. We develop the numerical model based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) for DNA elongation process with conditions pointed above and we study the possibility of the DNA polymerases movement synchronization. The results obtained numerically can be useful for DNA polymerase movement detection and visualization of the elongation process in the case of massive DNA replication, eg, under PCR condition or for DNA "sequencing by synthesis" sequencing devices evaluation.

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

    OpenAIRE

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

    2007-01-01

    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 cycl...

  18. Phospholipase C-delta1 expression is linked to proliferation, DNA synthesis, and cyclin E levels.

    Science.gov (United States)

    Stallings, Jonathan D; Zeng, Yue X; Narvaez, Francisco; Rebecchi, Mario J

    2008-05-16

    We previously reported that phospholipase C-delta1 (PLC-delta1) accumulates in the nucleus at the G1/S transition, which is largely dependent on its binding to phosphatidylinositol 4,5-bisphosphate ( Stallings, J. D., Tall, E. G., Pentyala, S., and Rebecchi, M. J. (2005) J. Biol. Chem. 280, 22060-22069 ). Here, using small interfering RNA (siRNA) that specifically targets rat PLC-delta1, we investigated whether this enzyme plays a role in cell cycle control. Inhibiting expression of PLC-delta1 significantly decreased proliferation of rat C6 glioma cells and altered S phase progression. [3H]Thymidine labeling and fluorescence-activated cell sorting analysis indicated that the rates of G1/S transition and DNA synthesis were enhanced. On the other hand, knockdown cultures released from the G1/S boundary were slower to reach full G2/M DNA content, consistent with a delay in S phase. The levels of cyclin E, a key regulator of the G1/S transition and DNA synthesis, were elevated in asynchronous cultures as well as those blocked at the G1/S boundary. Epifluorescence imaging showed that transient expression of human phospholipase C-delta1, resistant to these siRNA, suppressed expression of cyclin E at the G1/S boundary despite treatment of cultures with rat-specific siRNA. Although whole cell levels of phosphatidylinositol 4,5-bisphosphate were unchanged, suppression of PLC-delta1 led to a significant rise in the nuclear levels of this phospholipid at the G1/S boundary. These results support a role for PLC-delta1 and nuclear phospholipid metabolism in regulating cell cycle progression.

  19. Synthesis of a multibranched porphyrin-oligonucleotide scaffold for the construction of DNA-based nano-architectures.

    Science.gov (United States)

    Clavé, Guillaume; Chatelain, Grégory; Filoramo, Arianna; Gasparutto, Didier; Saint-Pierre, Christine; Le Cam, Eric; Piétrement, Olivier; Guérineau, Vincent; Campidelli, Stéphane

    2014-05-01

    The interest in the functionalization of oligonucleotides with organic molecules has grown considerably over the last decade. In this work, we report on the synthesis and characterization of porphyrin-oligonucleotide hybrids containing one to four DNA strands (P1-P4). The hybrid P4, which inserts one porphyrin and four DNA fragments, was combined with gold nanoparticles and imaged by transmission electron microscopy.

  20. 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

    Energy Technology Data Exchange (ETDEWEB)

    Kultti, Anne, E-mail: anne.kultti@uku.fi [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland); Pasonen-Seppaenen, Sanna [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland); Jauhiainen, Marjo [Department of Pharmaceutical Chemistry, University of Kuopio, FIN-70211 Kuopio (Finland); Rilla, Kirsi J.; Kaernae, Riikka; Pyoeriae, Emma; Tammi, Raija H.; Tammi, Markku I. [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland)

    2009-07-01

    Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

  1. Enzymatic synthesis of modified oligonucleotides by PEAR using Phusion and KOD DNA polymerases.

    Science.gov (United States)

    Wang, Xuxiang; Zhang, Jianye; Li, Yingjia; Chen, Gang; Wang, Xiaolong

    2015-02-01

    Antisense synthetic oligonucleotides have been developed as potential gene-targeted therapeutics. We previously reported polymerase-endonuclease amplification reaction (PEAR) for amplification of natural and 5'-O-(1-thiotriphosphate) (S)-modified oligonucleotides. Here, we extended the PEAR technique for enzymatic preparation of 2'-deoxy-2'-fluoro-(2'-F) and 2'-F/S double-modified oligonucleotides. The result showed that KOD and Phusion DNA polymerase could synthesize oligonucleotides with one or two modified nucleotides, and KOD DNA polymerase is more suitable than Phusion DNA polymerase for PEAR amplification of 2'-F and 2'-F/S double modified oligonucleotides. The composition of PEAR products were analyzed by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection and showed that the sequence of the PEAR products are maintained at an extremely high accuracy (>99.9%), and after digestion the area percent of full-length modified oligonucleotides reaches 89.24%. PEAR is suitable for synthesis of modified oligonucleotides efficiently and with high purity. PMID:25517220

  2. Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

    Institute of Scientific and Technical Information of China (English)

    Parker L Andersen; Fang Xu; Wei Xiao

    2008-01-01

    In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modi-fications of DNA. The term DNA damage tolerance (DDT) has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen (PCNA) by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubcl3-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis (TLS) with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same Ki64 residue by Mms2-Ubcl3-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic insta-bility and cancer.

  3. Mitochondrial DNA background modulates the assembly kinetics of OXPHOS complexes in a cellular model of mitochondrial disease.

    NARCIS (Netherlands)

    Pello, R.; Martin, M.A.; Carelli, V.; Nijtmans, L.G.J.; Achilli, A.; Pala, M.; Torroni, A.; Gomez-Duran, A.; Ruiz-Pesini, E.; Martinuzzi, A.; Smeitink, J.A.M.; Arenas, J.; Ugalde, C.

    2008-01-01

    Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disorder, is mostly due to three mitochondrial DNA (mtDNA) mutations in respiratory chain complex I subunit genes: 3460/ND1, 11778/ND4 and 14484/ND6. Despite considerable clinical evidences, a genetic modifying role of the m

  4. Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

    Science.gov (United States)

    Takamitsu, Emi; Otsuka, Motoaki; Haebara, Tatsuki; Yano, Manami; Matsuzaki, Kanako; Kobuchi, Hirotsugu; Moriya, Koko; Utsumi, Toshihiko

    2015-01-01

    To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources. PMID:26308446

  5. Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses.

    Directory of Open Access Journals (Sweden)

    Emi Takamitsu

    Full Text Available To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.

  6. The role of human equilibrative nucleoside transporter 1 on the cellular transport of the DNA methyltransferase inhibitors 5-azacytidine and CP-4200 in human leukemia cells.

    Science.gov (United States)

    Hummel-Eisenbeiss, Johanna; Hascher, Antje; Hals, Petter-Arnt; Sandvold, Marit Liland; Müller-Tidow, Carsten; Lyko, Frank; Rius, Maria

    2013-09-01

    The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [¹⁴C]5-azacytidine demonstrated Na⁺-independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-5'-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances. PMID:23814180

  7. A derivative of an ataxia-telangiectasia (A-T) cell line with normal radiosensitivity but A-T-like inhibition of DNA synthesis

    International Nuclear Information System (INIS)

    Ataxia-telangiectasia (A-T) cells are hypersensitive to the lethal effects of ionizing radiation and fail to inhibit DNA synthesis following radiation exposure. A cell line derived from an A-T line following DNA-mediated gene transfer has normal radiation sensitivity, but the kinetics of DNA synthesis after γ-irradiation are similar to those of A-T cells. (author)

  8. The effects of over-expressing Tip60 on cellular DNA damage repair and cell cycle progression

    International Nuclear Information System (INIS)

    To investigate the effects of Tip60 on DNA damage repair, cell cycle and the related mechanism as well, the proliferative activity, DNA double strand break (DSB) repair competency and cell cycle arrest were analyzed in stable Tip60-overexpression U2OS cells established by transfecting with exogenous Tip60 gene. It was found that the overexpression of Tip60 inhibited the proliferative activity but increased the DNA damage repair competency. The radiation-induced G2/M arrest was prolonged in Tip60 over-expressed U2OS cells, which was associated with a decreasing level of cell cycle checkpoint protein Cyclin B/CDC2 complex. (authors)

  9. Defective recovery of semi-conservative DNA synthesis in xeroderma pigmentosum cells following split-dose ultraviolet irradiation

    International Nuclear Information System (INIS)

    In normal human fibroblasts the authors observe an enhancement of the recovery of the rate of semi-conservative DNA synthesis after split-dose UV-irradation relative to a single total UV dose. The enhanced recovery is totally absent in both a xeroderma pigmentosum variant line and two xeroderma pigmentosum lines belonging to complementation groups A and C. (Auth.)

  10. Synthesis of DNA templated trifunctional electrically conducting, optical, and magnetic nanochain of Nicore-Aushell for biodevice

    Science.gov (United States)

    Mandal, Madhuri; Mandal, Kalyan

    2009-07-01

    Synthesis of trifunctional, e.g., electrically conducting, optical, and magnetic nanochains of Nicore-Aushell, has been discussed here. Properties of the materials were investigated from the view of its application in bionanodevice. Our investigation indicates that such material attached to biomolecule "DNA chain" and having three main properties in one material will have great potentiality in medical instrumentation and biocomputer device.

  11. Neuropeptide Y stimulates DNA synthesis in human vascular smooth muscle cells through neuropeptide Y Y1 receptors

    DEFF Research Database (Denmark)

    Nilsson, T; Edvinsson, L

    2000-01-01

    We investigated the mitogenic effect, measured as [3H]thymidine incorporation, of neuropeptide Y (NPY) on smooth muscle cells (SMCs) from human subcutaneous arteries (diameter: 0.4 mm). NPY stimulated DNA synthesis in a concentration-dependent manner, Emax 32 +/- 5% relative to control. The effec...

  12. ENHANCEMENT OF DNA SYNTHESIS IN CULTURED ADULT RAT HEPATOCYTES BY 5-HT THROUGH STIMULATION OF 5-HT2 RECEPTOR

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Hepatocytes were isolated from livers of adult male Sprague-Dawley rats and cultured in Williams'E Medium with [3 H] thymidine. The effect of 5-hydroxytryptamine (5-HT) was investigated through adding various concentrations (10-8~10-3 mol/L) of 5-HT to the hepatocyte cultures in the presence or absence of epidermal growth factor (EGF) and insulin. The involvement of 5-HT2 receptor was examined by adding a 5-HT2 receptor antagonist, ketanserin (10-6 mol/L), to some of the cultures containing 5-HT. The increment of DNA synthesis was measured by [3 H] thymidine incorporation. The results showed that 5-HT2 (≥10-6 mol/L) significantly (P<0.05) increased the amount of DNA synthesis induced by EGF and insulin in the cultured adult rat hepaptocytes. The effect of 5-HT in enhancing DNA synthesis began to appear at a concentration between 10-7 and 10-6 mol/L and reached maximum at concentrations of ≥10-4 mol/L. The enhancement of DNA synthesis by 5-HT was significantly (P<0.05) antagonized by ketanserin, suggesting that this effect of 5-HT was mediated by 5-HT2 receptor subtype.

  13. Synthesis and spectroscopic studies of the aminoglycoside (neomycin)--perylene conjugate binding to human telomeric DNA.

    Science.gov (United States)

    Xue, Liang; Ranjan, Nihar; Arya, Dev P

    2011-04-12

    Synthesis of a novel perylene-neomycin conjugate (3) and the properties of its binding to human telomeric G-quadruplex DNA, 5'-d[AG3(T2AG3)3] (4), are reported. Various spectroscopic techniques were employed to characterize the binding of conjugate 3 to 4. A competition dialysis assay revealed that 3 preferentially binds to 4, in the presence of other nucleic acids, including DNA, RNA, DNA-RNA hybrids, and other higher-order structures (single strands, duplexes, triplexes, other G-quadruplexes, and the i-motif). UV thermal denaturation studies showed that thermal stabilization of 4 increases as a function of the increasing concentration of 3. The fluorescence intercalator displacement (FID) assay displayed a significantly tighter binding of 3 with 4 as compared to its parent constituents [220-fold stronger than neomycin (1) and 4.5-fold stronger than perylene diamine (2), respectively]. The binding of 3 with 4 resulted in pronounced changes in the molar ellipticity of the DNA absorption region as confirmed by circular dichroism. The UV-vis absorption studies of the binding of 3 to 4 resulted in a red shift in the spectrum of 3 as well as a marked hypochromic change in the perylene absorption region, suggesting that the ligand-quadruplex interaction involves stacking of the perylene moiety. Docking studies suggest that the perylene moiety serves as a bridge that end stacks on 4, making contacts with two thymine bases in the loop, while the two neomycin moieties branch into the grooves of 4.

  14. Rational design, synthesis, and DNA binding properties of novel sequence-selective peptidyl congeners of ametantrone.

    Science.gov (United States)

    Gianoncelli, Alessandra; Basili, Serena; Scalabrin, Matteo; Sosic, Alice; Moro, Stefano; Zagotto, Giuseppe; Palumbo, Manlio; Gresh, Nohad; Gatto, Barbara

    2010-07-01

    Natural and synthetic compounds characterized by an anthraquinone nucleus represent an important class of anti-neoplastic agents, the mechanism of action of which is related to intercalation into DNA. Ametantrone (AM) is a synthetic 9,10-anthracenedione bearing two (hydroxyethylamino)ethylamino residues at positions 1 and 4; along with other anthraquinones and anthracyclines, it shares a polycyclic intercalating moiety and charged side chains that stabilize DNA binding. All these drugs elicit adverse side effects, which represent a challenge for antitumor chemotherapy. In the present work the structure of AM was augmented with appropriate groups that target well-defined base pairs in the major groove. These should endow AM with DNA sequence selectivity. We describe the rationale for the synthesis and the evaluation of activity of a new series of compounds in which the planar anthraquinone is conjugated at positions 1 and 4 through the side chains of AM or other bioisosteric linkers to appropriate dipeptides. The designed novel AM derivatives were shown to selectively stabilize two oligonucleotide duplexes that both have a palindromic GC-rich hexanucleotide core, but their stabilizing effects on a random DNA sequence was negligible. In the case of the most effective compound, the 1,4-bis-[Gly-(L-Lys)] derivative of AM, the experimental results confirm the predictions of earlier theoretical computations. In contrast, AM had equal stabilizing effects on all three sequences and showed no preferential binding. This novel peptide derivative can be classified as a strong binder regarding the sequences that it selectively targets, possibly opening the exploitation of less cytotoxic conjugates of AM to the targeted treatment of oncological and viral diseases. PMID:20458714

  15. Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases α, β, η, ι, κ, REV1 and Klenow Fragment exo-, but not by DNA polymerase ζ.

    Science.gov (United States)

    Suzuki, Masayo; Kino, Katsuhito; Kawada, Taishu; Oyoshi, Takanori; Morikawa, Masayuki; Kobayashi, Takanobu; Miyazawa, Hiroshi

    2016-03-01

    Guanine is the most easily oxidized of the four DNA bases, and contiguous guanines (GG) in a sequence are more readily oxidized than a single guanine in a sequence. Continued oxidation of GGs results in a contiguous oxidized guanine lesion. Two contiguous 2,5-diamino-4H-imidazol-4-ones, an oxidized form of guanine that hydrolyses to 2,2,4-triamino-5(2H)-oxazolone (Oz), are detected following the oxidation of GG. In this study, we analysed translesion synthesis (TLS) across two contiguous Oz molecules (OzOz) using Klenow Fragment exo(-) (KF exo(-)) and DNA polymerases (Pols) α, β, ζ, η, ι, κ and REV1. We found that KF exo(-) and Pols α, β, ι and REV1 inserted one nucleotide opposite the 3' Oz of OzOz and stalled at the subsequent extension, and that Pol κ incorporated no nucleotide. Pol η only inefficiently elongated the primer up to full-length across OzOz; the synthesis of most DNA strands stalled at the 3' or 5' Oz of OzOz. Surprisingly, however, Pol ζ efficiently extended the primer up to full-length across OzOz, unlike the other DNA polymerases, but catalysed error-prone nucleotide incorporation. We therefore believe that Pol ζ is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ζ.

  16. Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA.

    Science.gov (United States)

    Furukohri, Asako; Nishikawa, Yoshito; Akiyama, Masahiro Tatsumi; Maki, Hisaji

    2012-07-01

    DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.

  17. Phosphorylation of eIF4E Confers Resistance to Cellular Stress and DNA-Damaging Agents through an Interaction with 4E-T: A Rationale for Novel Therapeutic Approaches.

    Directory of Open Access Journals (Sweden)

    Alba Martínez

    Full Text Available Phosphorylation of the eukaryotic translation initiation factor eIF4E is associated with malignant progression and poor cancer prognosis. Accordingly, here we have analyzed the association between eIF4E phosphorylation and cellular resistance to oxidative stress, starvation, and DNA-damaging agents in vitro. Using immortalized and cancer cell lines, retroviral expression of a phosphomimetic (S209D form of eIF4E, but not phospho-dead (S209A eIF4E or GFP control, significantly increased cellular resistance to stress induced by DNA-damaging agents (cisplatin, starvation (glucose+glutamine withdrawal, and oxidative stress (arsenite. De novo accumulation of eIF4E-containing cytoplasmic bodies colocalizing with the eIF4E-binding protein 4E-T was observed after expression of phosphomimetic S209D, but not S209A or wild-type eIF4E. Increased resistance to cellular stress induced by eIF4E-S209D was lost upon knockdown of endogenous 4E-T or use of an eIF4E-W73A-S209D mutant unable to bind 4E-T. Cancer cells treated with the Mnk1/2 inhibitor CGP57380 to prevent eIF4E phosphorylation and mouse embryonic fibroblasts derived from Mnk1/2 knockout mice were also more sensitive to arsenite and cisplatin treatment. Polysome analysis revealed an 80S peak 2 hours after arsenite treatment in cells overexpressing phosphomimetic eIF4E, indicating translational stalling. Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1. We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required. Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.

  18. Synthesis of Novel Metal Ion Sensors Based on DNA-Metal Interactions

    Institute of Scientific and Technical Information of China (English)

    Akira Ono; Shiqi Cao; Humika Togashi; Yoko Miyake

    2005-01-01

    @@ 1Introduction The interactions of metal ions with nucleic acids, nucleosides, and nucleo-bases have been extensively investigated[1,2]. We have reported that thymine-thymine (T-T) and cytosine-cytosine (C- C) miss base pairs in DNA duplexes highly selectively capture HgⅡ ion and Ag Ⅰ ion, which result in formations of metal-mediated base pairs, T-HgⅡ -T and C-AgⅠ -C, in duplexes[3]. The phenomenon is expected to be useful for a variety of studies such as synthesis of nano-wires containing metal ions, developing metal-ion sensing methods, etc.Here, we report novel oligodeoxyribonucleotide (ODN)-based sensors that detect HgⅡ ions and AgⅠ ions in aqueous solutions.

  19. DNA-based vaccination induces humoral and cellular immune responses against hepatitis B virus surface antigen in mice without activation of Cmyc

    Institute of Scientific and Technical Information of China (English)

    Lian San Zhao; Shan Qin; Tao You Zhou; Hong Tang; Li Liu; Bing Jun Lei

    2000-01-01

    AIM To develop a safe and effective DNA vaccine for inducing humoral and cellular immunological responses against hepatitis B virus surface antigen (HBsAg). METHODS BALB/c mice were inoculated with NV-HB/s, a recombinant plasmid that had been inserted S gene of hepatitis B virus genome and could express HBsAg in eukaryotes. HBsAg expression was measured by ABC immunohistochemical assay, generation of anti-HBs by ELISA and cytotoxic T lymphocyte (CTL), by MTT method, existence of vaccine DNA by Southern blot hybridization and activation of oncogene C-myc by in situ hybridization.RESULTS With NV-HB/s vaccination by intramuscular injection, anti-HBs was initially positive 2 weeks after inoculation while all mice tested were HBsAg positive in the muscles. The titers and seroconversion rate of anti-HBs were steadily increasing as time went on and were dose-dependent. All the mice inoculated with 100 μg NV-HB/ s were anti-HBs positive one month after inoculation, the titer was 1:1024 or more. The humoral immune response was similar induced by either intramuscular or intradermal injection. CTL activities were much stronger (45.26%) in NV-HB/s DNA immunized mice as compared with those (only 6%) in plasmaderived HBsAg vaccine immunized mice. Two months after inoculation, all muscle samples were positive by Southern-blot hybridization for NV-HB/s DNA detection, but decreased to 25%and all were undetectable by in situ hybridization after 6 months. No oncogene Cmyc activation was found in the muscle of inoculation site. CONCLUSION NV-HB/s could generate humoral and cellular immunological responses against HBsAg that had been safely expressed in situ by NV-HB/s vaccination.

  20. Recovery of DNA synthesis after ultraviolet irradiation of xeroderma pigmentosum cells depends on excision repair and is blocked by caffeine

    International Nuclear Information System (INIS)

    Normal human and xeroderma pigmentosum (XP, excision-defective group A) cells (both SV40-transformed) pulse-labeled with [3H] thymidine at various times after irradiation with ultraviolet light showed a decline and recovery of both the molecular weights of newly synthesized DNA and the rated of synthesis per cell. At the same ultraviolet dose, both molecular weights and rates of synthesis were inhibited more in XP than in normal cells. This indicates that excision repair plays a role in minimizing the inhibition of chain growth, possibly by excision of dimers ahead of the growing point. The ability to synthesize normal-sized DNA recovered more rapidly than rates of synthesis in normal cells, but both parameters recovered in phase in XP cells. During recovery in normal cells there are therefore fewer actively replicating clusters of replicons because the single-strand breaks involved in the excision of dimers inhibit replicon initiation. XP cells have few excision repair events and therefore fewer breaks to interfere with initiation, but chain growth is blocked by unexcised dimers. In both cell types recovery of the ability to synthesize normal-sized DNA was prevented by growing cells in caffeine after irradiation, possibly because of competition between the DNA binding properties of caffeine and replication proteins. These observations imply that excision repair and semiconservative replication interact strongly in irradiated cells to produce a complex spectrum of changes in DNA replication which may be confused with parts of alternative systems such as post-replication repair. (author)

  1. Spiro[pyrrolidine-3, 3´-oxindole] as potent anti-breast cancer compounds: Their design, synthesis, biological evaluation and cellular target identification.

    Science.gov (United States)

    Hati, Santanu; Tripathy, Sayantan; Dutta, Pratip Kumar; Agarwal, Rahul; Srinivasan, Ramprasad; Singh, Ashutosh; Singh, Shailja; Sen, Subhabrata

    2016-01-01

    The spiro[pyrrolidine-3, 3´-oxindole] moiety is present as a core in number of alkaloids with substantial biological activities. Here in we report design and synthesis of a library of compounds bearing spiro[pyrrolidine-3, 3´-oxindole] motifs that demonstrated exceptional inhibitory activity against the proliferation of MCF-7 breast cancer cells. The synthesis involved a one pot Pictet Spengler-Oxidative ring contraction of tryptamine to the desired scaffolds and occurred in 1:1 THF and water with catalytic trifluoroacetic acid and stoichiometric N-bromosuccinimide as an oxidant. Phenotypic profiling indicated that these molecules induce apoptotic cell death in MCF-7 cells. Target deconvolution with most potent compound 5l from the library, using chemical proteomics indicated histone deacetylase 2 (HDAC2) and prohibitin 2 as the potential cellular binding partners. Molecular docking of 5l with HDAC2 provided insights pertinent to putative binding interactions. PMID:27573798

  2. Peptide Nucleic Acid with a Lysine Side Chain at the β-Position: Synthesis and Application for DNA Cleavage.

    Science.gov (United States)

    Sugiyama, Toru; Kuwata, Keiko; Imamura, Yasutada; Demizu, Yosuke; Kurihara, Masaaki; Takano, Masashi; Kittaka, Atsushi

    2016-01-01

    This paper reports the synthesis of new β-Lys peptide nucleic acid (PNA) monomers and their incorporation into a 10-residue PNA sequence. PNA containing β-Lys PNA units formed a stable hybrid duplex with DNA. However, incorporation of β-Lys PNA units caused destabilization of PNA-DNA duplexes to some extent. Electrostatic attractions between β-PNA and DNA could reduce this destabilization effect. Subsequently, bipyridine-conjugated β-Lys PNA was prepared and exhibited sequence selective cleavage of DNA. Based on the structures of the cleavage products and molecular modeling, we reasoned that bipyridine moiety locates within the minor groove of the PNA-DNA duplexes. The lysine side chain of β-PNA is a versatile handle for attaching various functional molecules. PMID:27373637

  3. Synthesis and crystal structure study of 2′-Se-adenosine-derivatized DNA

    Institute of Scientific and Technical Information of China (English)

    SALON; Jozef

    2010-01-01

    The selenium derivatization of nucleic acids is a novel and promising strategy for 3D structure determination of nucleic acids.Selenium can serve as an excellent anomalous scattering center to solve the phase problem,which is one of the two major bottlenecks in macromolecule X-ray crystallography.The other major bottleneck is crystallization.It has been demonstrated that the incorporated selenium functionality at the 2′-positions of the nucleosides and nucleotides is stable and does not cause significant structure perturbation.Furthermore,it was observed that the 2′-Se-derivatization could facilitate crystallization of oligonucleotides with fast crystal growth and high diffraction quality.Herein,we describe a convenient synthesis of the 2′-Se-adenosine phosphoramidite,and report the first synthesis and X-ray crystal structure determination of the DNA containing the 2′-Se-A derivatization.The 3D structure of 2′-Se-A-DNA decamer 5′-GTACGCGT(2′-Se-A)C-3′2 was determined at 1.75 ? resolution,the 2′-Se-functionality points to the minor groove,and the Se-modified and native structures are virtually identical.Moreover,we have observed that the 2′-Se-A modification can greatly facilitate the crystal growth with high diffraction quality.In conjunction with the crystallization facilitation by the 2′-Se-U and 2′-Se-T,this novel observation on the 2′-Se-A functionality suggests that the 2′-Se moiety is sole responsible for the crystallization facilitation and the identity of nucleobases does not influence the crystal growth significantly.

  4. The effect of human milk on DNA synthesis of neonatal rat hepatocytes in primary culture.

    Science.gov (United States)

    Kohno, Y; Shiraki, K; Mura, T

    1991-03-01

    We studied the effect of human milk on DNA synthesis of neonatal hepatocytes to elucidate the physiologic role of human milk in growth of the liver. Neonatal hepatocytes were isolated from 5-d-old rats and cultured in serum-free medium. Human milk stimulated DNA synthesis of these hepatocytes in a concentration-dependent manner. The stimulatory activity of 7.5% (vol/vol) human milk plus 0.1 mumol/L insulin was five times that of control and was almost the same as that of 20 micrograms/L human epidermal growth factor (hEGF) plus insulin. The effect of human milk was additive with treatment with hEGF and insulin. The milk associated with prolonged jaundice of infants was significantly more active than the milk that was not associated with jaundice, although the concentration of hEGF was not different between the two types of milk. The mitogenic activity of milk was heat-labile, inactivated by DTT and stable after treatment with trypsin. Three peaks of the activity were detected in milk by gel filtration and the fraction containing proteins of molecular weight between 36,000 and 76,000 showed the highest activity. Anti-hEGF antibody did not inhibit this activity completely. These results suggested the presence of mitogens other than hEGF or a more active form of hEGF in human milk. The milk associated with breast-milk jaundice exerts a different influence on cell growth and may affect maturation of the liver function related to bilirubin metabolism. The mitogenic activity of milk might be important for growth and development of the liver in infants.

  5. Evaluation of [methyl- 14C]4'-thio-thymidine for DNA synthesis imaging in vivo

    International Nuclear Information System (INIS)

    Objective: In order to obtain a thymidine analog that might prove simpler to use for imaging DNA synthesis and follow the same biochemistry of thymidine in vivo, we evaluated [methyl- 11C]4'-thio-thymidine ([methyl- 11C]S-dThd) by using the [14C]-labeled counterpart ([methyl- 14C]S-dThd). Methods: [methyl-14C]S-dThd was synthesized by rapid methylation of 5-trimethyl-stannyl-4' -thio-2' -deoxyuridine via a palladium mediated Stille-coupling reaction with [14C]methyl iodide. Degradation of [methyl- 14C]S-dThd when incubated in human blood was analyzed by HPLC. The in vivo potential of [methyl- 14C]S-dThd was evaluated by distribution study of EMT-6 mammary carcinoma-bearing mice. Gemcitabine, a potent inhibitor of DNA synthesis, was used to modulate cell proliferation. Tissue extraction was also performed to investigate the incorporation of [methyl-14C]S-dThd into DNA. Results: [methyl- 14C]S-dThd was obtained in 31-41% radiochemical yield (calculated from [14C]methyl iodide) at 130, 5 min reaction in N,N-dimethylforamide. After semi-preparative HPLC purification, radiochemical purity of [methyl- 14C]S-dThd was >99% and the specific activity was 2.04 GBq/mmol (according to the specific activity of [14C]methyl iodide). Incubation with human blood demonstrated rapid degradation of [2- 14C]thymidine. In contrast, [methyl- 14C]S-dThd was stable with less than 3% degradation at 60 min. In vivo distribution study showed progressive accumulation of radioactivity in proliferating tissues (spleen, thymus, duodenum and tumor). On the other hand, the washout of radioactivity by the non-proliferating tissues (lung, liver, kidney and muscle) appeared nearly exponential. The tumor uptake of [methyl- 14C]S-dThd was high (8.8%ID/g at 60 min) and selective (Tumor to blood ratio: 12.2 at 60 min). Gemicitabine pretreatment significantly reduced the tumor uptake of [methyl- 14C]S-dThd. Relative blood flow as measured by the uptake 4-[N-Methyl- 14C]iodoantipyrine was similar in the

  6. Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

    Directory of Open Access Journals (Sweden)

    Rak Janusz

    2011-08-01

    Full Text Available Abstract Background Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. Results An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb. Conclusions The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs

  7. Translesion Synthesis DNA Polymerase: A Novel DNA Polymerase%跨损伤合成的DNA聚合酶——一类新的DNA聚合酶

    Institute of Scientific and Technical Information of China (English)

    陈建明; 余应年

    2001-01-01

    although there are many repair pathways in cells, some lesions still escape repair inevitably and remain in genome. In cells, the molecular mechanism of translesion DNA synthesis has been one of the major unsolved problems in DNA repair for a long time. Recently, it was found that the members of a structurally related UmuC/DinB protein superfarnily have DNA polyrnerase function. Unlike the classical replicative DNA polymerases, these newly identified DNA polymerases can carry out translesion DNA synthesis in both error prone/mutagenic and/or error-free ways. It was also found that their functions are conserved from bacteria to human.%细胞虽然拥有多种修复途径,但有些DNA损伤仍不可避免地会逃避修复而在基因组上保留下来,细胞跨 损伤DNA合成的分子机制一直是DNA修复中主要的未解决问题之一.最近通过对一类结构相关性UmuC/DinB 蛋白质超家族成员的研究发现它们具有DNA聚合酶功能.这类新发现的DNA聚合酶不同于经典的复制性DNA 聚合酶,它们能以易误/突变(error-prone/mutagenic)或无误(error-free)方式进行跨损伤(translesion)DNA合 成,并且从细菌到人在进化上功能保守.

  8. The effect of a DNA repair gene on cellular invasiveness: XRCC3 over-expression in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Veronica L Martinez-Marignac

    Full Text Available Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3 and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion.

  9. Inhibition of semiconservative DNA synthesis in ICR 2A frog cells exposed to monochromatic uv wavelengths (252-313 nm) and photoreactivating light

    International Nuclear Information System (INIS)

    Exposure of ICR 2A frog cells to monochromatic uv wavelengths in the range 252-313 nm caused an inhibition of semiconservative DNA synthesis which was partially relieved in cells receiving a post irradiation treatment with photoreactivating light (>350 nm). Hence pyrimidine dimers acted as lesions blocking DNA synthesis in uv-irradiated cells based upon the specificity of photoreactivating enzyme for the light-dependent monomerization of dimers in DNA. Compared with the shorter wavelengths tested, however, this recovery of DNA synthesis was not as great in cells exposed to 302-nm radiation and was nearly absent in 313-nm-irradiated cells up to 12 hr after treatment. These results suggest that nondimer photoproducts also play an important role in causing DNA synthesis inhibition in cells exposed to wavelengths greater than 300 nm

  10. Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP6-Binding Proteins.

    Science.gov (United States)

    Jiao, Chensong; Summerlin, Matthew; Bruzik, Karol S; Hanakahi, Leslyn

    2015-10-20

    Inositol hexakisphosphate (IP6) is a soluble inositol polyphosphate, which is abundant in mammalian cells. Despite the participation of IP6 in critical cellular functions, few IP6-binding proteins have been characterized. We report on the synthesis, characterization, and application of biotin-labeled IP6 (IP6-biotin), which has biotin attached at position 2 of the myo-inositol ring via an aminohexyl linker. Like natural IP6, IP6-biotin stimulated DNA ligation by nonhomologous end joining (NHEJ) in vitro. The Ku protein is a required NHEJ factor that has been shown to bind IP6. We found that IP6-biotin could affinity capture Ku and other required NHEJ factors from human cell extracts, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and XLF. Direct binding studies with recombinant proteins show that Ku is the only NHEJ factor with affinity for IP6-biotin. DNA-PKcs, XLF, and the XRCC4:ligase IV complex interact with Ku in cell extracts and likely interact indirectly with IP6-biotin. IP6-biotin was used to tether streptavidin to Ku, which inhibited NHEJ in vitro. These proof-of-concept experiments suggest that molecules like IP6-biotin might be used to molecularly target biologically important proteins that bind IP6. IP6-biotin affinity capture experiments show that numerous proteins specifically bind IP6-biotin, including casein kinase 2, which is known to bind IP6, and nucleolin. Protein binding to IP6-biotin is selective, as IP3, IP4, and IP5 did not compete for binding of proteins to IP6-biotin. Our results document IP6-biotin as a useful tool for investigating the role of IP6 in biological systems. PMID:26397942

  11. Epidermal DNA synthesis in organ culture explants. A study of hairless mouse ear epidermis.

    Science.gov (United States)

    Hansteen, I L; Iversen, O H; Refsum, S B

    1979-10-01

    Explants of split mouse ear were incubated in organ culture for up to 48 h, and the cell proliferation was studied by the addition of Thymidine-methyl-3-H (3HTdR) to the medium during different time periods, mainly for the first 14 h of incubation. Cultures were started at 0900, 2130 and 2300. In all cases the labelling index remained stable for 6-8 h, and then increased. The mean grain count, however, was falling and so was the epidermal DNA-specific uptake of 3HTdR. Based on the experimental results, calculations can be made of the flux of cells through S. It is concluded that the increasing LI is not due to inherent diurnal variation in cell proliferation, and is not a sign of real growth but caused instead by a complete block of the cell exit from S, probably combined with periods of an increased entrance rate into S. Other methodological factors, however, may also contribute to the increasing LI. Hence, this system is not suited for the measurement of factors that influence epidermal DNA synthesis.

  12. Delay in maturation of the submandibular gland in Chagas disease correlates with lower DNA synthesis

    Directory of Open Access Journals (Sweden)

    José B Alves

    2008-09-01

    Full Text Available It has been demonstrated that the acute phase of Trypanosoma cruzi infection promotes several changes in the oral glands. The present study examined whether T. cruzi modulates the expression of host cell apoptotic or mitotic pathway genes. Rats were infected with T. cruzi then sacrificed after 18, 32, 64 or 97 days, after which the submandibular glands were analyzed by immunohistochemistry. Immunohistochemical analyses using an anti-bromodeoxyuridine antibody showed that, during acute T. cruzi infection, DNA synthesizing cells in rat submandibular glands were lower than in non-infected animals (p < 0.05. However, after 64 days of infection (chronic phase, the number of immunolabeled cells are similar in both groups. However, immunohistochemical analysis of Fas and Bcl-2 expression did not find any difference between infected and non-infected animals in both the acute and chronic stages. These findings suggest that the delay in ductal maturation observed at the acute phase of Chagas disease is correlated with lower expression of DNA synthesis genes, but not apoptotic genes.

  13. Characterisation of Human Keratinocytes by Measuring Cellular Repair Capacity of UVB-Induced DNA Damage and Monitoring of Cytogenetic Changes in Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Greinert, R.; Breibart, E.W.; Mitchell, D.; Smida, J.; Volkmer, B

    2000-07-01

    The molecular mechanisms for UV-induced photocarcinogenesis are far from being understood in detail, especially in the case of malignant melanoma of the skin. Nevertheless, it is known that deficiencies in cellular repair processes of UV-induced DNA damage (e.g. in the case of Xeroderma pigmentosum) represent important aetiological factors in the multistep development of skin cancer. The repair kinetics have therefore been studied of an established skin cell line (HaCaT), primary human keratinocytes, melanocytes and melanoma cell lines, using fluorescence microscopy and flow cytometry. Our data show a high degree of interindividual variability in cellular repair capacity for UV-induced DNA lesions, which might be due to individual differences in the degree of tolerable damage and/or the onsets of saturation of the enzymatic repair system. The cytogenetic analysis of melanoma cell lines, using spectral karyotyping (SKY) furthermore proves that malignant melanoma of the skin are characterised by high numbers of chromosomal aberrations. (author)

  14. RNA:protein ratio of the unicellular organism as a characteristic of phosphorous and nitrogen stoichiometry and of the cellular requirement of ribosomes for protein synthesis

    Directory of Open Access Journals (Sweden)

    Sams Carl E

    2006-09-01

    Full Text Available Abstract Background Mean phosphorous:nitrogen (P:N ratios and relationships of P:N ratios with the growth rate of organisms indicate a surprising similarity among and within microbial species, plants, and insect herbivores. To reveal the cellular mechanisms underling this similarity, the macromolecular composition of seven microorganisms and the effect of specific growth rate (SGR on RNA:protein ratio, the number of ribosomes, and peptide elongation rate (PER were analyzed under different conditions of exponential growth. Results It was found that P:N ratios calculated from RNA and protein contents in these particular organisms were in the same range as the mean ratios reported for diverse organisms and had similar positive relationships with growth rate, consistent with the growth-rate hypothesis. The efficiency of protein synthesis in microorganisms is estimated as the number of active ribosomes required for the incorporation of one amino acid into the synthesized protein. This parameter is calculated as the SGR:PER ratio. Experimental and theoretical evidence indicated that the requirement of ribosomes for protein synthesis is proportional to the RNA:protein ratio. The constant of proportionality had the same values for all organisms, and was derived mechanistically from the characteristics of the protein-synthesis machinery of the cell (the number of nucleotides per ribosome, the average masses of nucleotides and amino acids, the fraction of ribosomal RNA in the total RNA, and the fraction of active ribosomes. Impairment of the growth conditions decreased the RNA:protein ratio and increased the overall efficiency of protein synthesis in the microorganisms. Conclusion Our results suggest that the decrease in RNA:protein and estimated P:N ratios with decrease in the growth rate of the microorganism is a consequence of an increased overall efficiency of protein synthesis in the cell resulting from activation of the general stress response and

  15. Deinococcus radiodurans PprI Switches on DNA Damage Response and Cellular Survival Networks after Radiation Damage*S⃞

    OpenAIRE

    Lu, Huiming; Gao, Guanjun; Xu, Guangzhi; Fan, Lu; Yin, Longfei; Shen, Binghui; Hua, Yuejin

    2009-01-01

    Preliminary findings indicate that PprI is a regulatory protein that stimulates transcription and translation of recA and other DNA repair genes in response to DNA damage in the extremely radioresistant bacterium Deinococcus radiodurans. To define the repertoire of proteins regulated by PprI and investigate the in vivo regulatory mechanism of PprI in response to γ radiation, we performed comparative proteomics analyses on wild type (R1) and a pprI knock-out strain (YR1) under conditions of io...

  16. Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

    Directory of Open Access Journals (Sweden)

    Stougaard Magnus

    2007-11-01

    Full Text Available Abstract Background In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature. Methods Synchronized cultured cells were fixed with methanol/acetic acid to prepare chromosome spreads in teflon-coated diagnostic well-slides. Apart from the slide format and the chromosome spreading everything was done essentially according to standard protocols. Hybridization targets were detected in situ with padlock probes, which were ligated and amplified using target primed rolling circle DNA synthesis, and detected by fluorescence labeling. Results An optimized protocol for the spreading of condensed metaphase chromosomes in teflon-coated diagnostic well-slides was developed. Applying this protocol we generated specimens for target primed rolling circle DNA synthesis of padlock probes recognizing a 40 nucleotide sequence in the male specific repetitive satellite I sequence (DYZ1 on the Y-chromosome and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a gene positioned on the long arm of chromosome 6. These targets were detected with good efficiency, but the efficiency on other target sites was unsatisfactory. Conclusion Our aim was to test the applicability of the method used on mitochondrial DNA to the analysis of nuclear genomes, in particular as

  17. Protein-DNA chimeras: synthesis of two-arm chimeras and non-mechanical effects of the DNA spring

    International Nuclear Information System (INIS)

    DNA molecular springs have recently been used to control the activity of enzymes and ribozymes. In this approach, the mechanical stress exerted by the molecular spring alters the enzyme's conformation and thus the enzymatic activity. Here we describe a method alternative to our previous one to attach DNA molecular springs to proteins, where two separate DNA 'arms' are coupled to the protein and subsequently ligated. We report certain non-mechanical effects associated with the DNA spring observed in some chimeras with specific DNA sequences and the nucleotide binding enzyme guanylate kinase. If a ssDNA 'arm' is attached to the protein by one end only, we find that in some cases (depending on the DNA sequence and attachment point on the protein's surface) the unhybridized DNA arm inhibits the enzyme, while hybridization of the DNA arm leads to an apparent activation of the enzyme. One interpretation is that, in these cases, hybridization of the DNA arm removes it from the vicinity of the active site of the enzyme. We show how mechanical and non-mechanical effects of the DNA spring can be distinguished. This is important if one wants to use the protein-DNA chimeras to quantitatively study the response of the enzyme to mechanical perturbations.

  18. Remodelers organize cellular chromatin by counteracting intrinsic histone-DNA sequence preferences in a class-specific manner

    NARCIS (Netherlands)

    Y.M. Moshkin (Yuri); G.E. Chalkley (Gillian); T.W. Kan (Tsung Wai); B.A. Reddy (Ashok); Z. Özgür (Zeliha); W.F.J. van IJcken (Wilfred); D.H. Dekkers (Dick); J.A.A. Demmers (Jeroen); A.A. Travers (Andrew); C.P. Verrijzer (Peter)

    2012-01-01

    textabstractThe 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 D

  19. Measurement of DNA damage after exposure to electromagnetic radiation in the cellular phone communication frequency band (835.62 and 847.74 MHz).

    Science.gov (United States)

    Malyapa, R S; Ahern, E W; Straube, W L; Moros, E G; Pickard, W F; Roti Roti, J L

    1997-12-01

    Mouse C3H 10T1/2 fibroblasts and human glioblastoma U87MG cells were exposed to cellular phone communication frequency radiations to investigate whether such exposure produces DNA damage in in vitro cultures. Two types of frequency modulations were studied: frequency-modulated continuous-wave (FMCW), with a carrier frequency of 835.62 MHz, and code-division multiple-access (CDMA) centered on 847.74 MHz. Exponentially growing (U87MG and C3H 10T1/2 cells) and plateau-phase (C3H 10T1/2 cells) cultures were exposed to either FMCW or CDMA radiation for varying periods up to 24 h in specially designed radial transmission lines (RTLs) that provided relatively uniform exposure with a specific absorption rate (SAR) of 0.6 W/kg. Temperatures in the RTLs were monitored continuously and maintained at 37 +/- 0.3 degrees C. Sham exposure of cultures in an RTL (negative control) and 137Cs gamma-irradiated samples (positive control) were included with every experiment. The alkaline comet assay as described by Olive et al. (Exp. Cell Res. 198, 259-269, 1992) was used to measure DNA damage. No significant differences were observed between the test group exposed to FMCW or CDMA radiation and the sham-treated negative controls. Our results indicate that exposure of cultured mammalian cells to cellular phone communication frequencies under these conditions at an SAR of 0.6 W/kg does not cause DNA damage as measured by the alkaline comet assay. PMID:9399708

  20. Inhibition of semiconservative DNA synthesis in ICR 2A frog cells by pyrimidine dimers and nondimer photoproducts induced by ultraviolet radiation

    International Nuclear Information System (INIS)

    DNA synthesis was examined in ultraviolet (uv)-irradiated ICR 2A frog cells in which either pyrimidine dimers or nondimer photoproducts represented the major class of DNA lesions. In addition, cells were exposed to 60Co γ rays. The cultures were pulse-labeled and the size distribution of the DNA synthesized was estimated using both sucrose gradient sedimentation and alkaline step elution. Using either of these techniques, it was found that the presence of dimers resulted in a reduction principally in the synthesis of high molecular weight (MW) DNA. In contrast, nondimer photoproducts caused a strong inhibition in the synthesis of low MW DNA, as was also observed in γ-irradiated cells. Hence the induction of pyrimidine dimers in DNA mainly affected the elongation of replicons, whereas nondimer lesions primarily caused an inhibition of replicon initiation

  1. Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles

    Science.gov (United States)

    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

  2. Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles

    Directory of Open Access Journals (Sweden)

    Singh Jagbir

    2012-02-01

    Full Text Available Abstract 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

  3. The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2'-Deoxyuridine in Cellular DNA under Various Conditions.

    Directory of Open Access Journals (Sweden)

    Anna Ligasová

    Full Text Available We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority.

  4. The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2'-Deoxyuridine in Cellular DNA under Various Conditions

    Science.gov (United States)

    Ligasová, Anna; Liboska, Radek; Rosenberg, Ivan; Koberna, Karel

    2015-01-01

    We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority. PMID:26161977

  5. The S229L Colon Tumor-associated Variant of DNA Polymerase β Induces Cellular Transformation as a Result of Decreased Polymerization Efficiency*

    Science.gov (United States)

    Nemec, Antonia A.; Murphy, Drew L.; Donigan, Katherine A.; Sweasy, Joann B.

    2014-01-01

    DNA polymerase β (Pol β) plays a key role in base excision repair (BER) by filling in small gaps that are generated after base adducts are excised from the DNA. Pol β is mutated in a large number of colorectal tumors, and these mutations may drive carcinogenesis. In the present study, we wished to determine whether the S229L somatic Pol β variant identified in a stage 3 colorectal tumor is a driver of carcinogenesis. We show that S229L does not possess any defects in binding to either DNA or nucleotides compared with the WT enzyme, but exhibits a significant loss of polymerization efficiency, largely due to an 8-fold decrease in the polymerization rate. S229L participates in BER, but due to its lower catalytic rate, does so more slowly than WT. Expression of S229L in mammalian cells induces the accumulation of BER intermediate substrates, chromosomal aberrations, and cellular transformation. Our results are consistent with the interpretation that S229L is a driver of carcinogenesis, likely as a consequence of its slow polymerization activity during BER in vivo. PMID:24668809

  6. Synthesis, interaction with DNA, cytotoxicity, cell cycle arrest and apoptotic inducing properties of ruthenium(II) molecular "light switch" complexes.

    Science.gov (United States)

    Shobha Devi, C; Anil Kumar, D; Singh, Surya S; Gabra, Nazar; Deepika, N; Kumar, Y Praveen; Satyanarayana, S

    2013-06-01

    In an endeavor toward the development of metal-based anticancer drugs, we present here the design, synthesis and characterization of three ruthenium(II) functionalized phenanthroline complexes with extended π-conjugation. These complexes have been shown to act as promising CT-DNA intercalators as evidenced by UV-visible, luminescence, emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide and salt dependent studies. All three complexes [Ru(Hdpa)2PPIP](2+) (1), [Ru(Hdpa)2PIP](2+) (2), [Ru(Hdpa)24HEPIP](2+) (3) clearly demonstrated that they can bind to DNA through the intercalation mode. Cell viability experiments indicated that all complexes showed significant dose dependent cytotoxicity in selected cell lines. The apoptosis and cell cycle arrest were also investigated. The complexes were docked into DNA-base-pairs using the 'GOLD' (Genetic Optimization for Ligand Docking), docking program. PMID:23665797

  7. DNA mismatch repair protein MSH2 dictates cellular survival in response to low dose radiation in endometrial carcinoma cells.

    LENUS (Irish Health Repository)

    Martin, Lynn M

    2013-07-10

    DNA repair and G2-phase cell cycle checkpoint responses are involved in the manifestation of hyper-radiosensitivity (HRS). The low-dose radioresponse of MSH2 isogenic endometrial carcinoma cell lines was examined. Defects in cell cycle checkpoint activation and the DNA damage response in irradiated cells (0.2 Gy) were evaluated. HRS was expressed solely in MSH2+ cells and was associated with efficient activation of the early G2-phase cell cycle checkpoint. Maintenance of the arrest was associated with persistent MRE11, γH2AX, RAD51 foci at 2 h after irradiation. Persistent MRE11 and RAD51 foci were also evident 24 h after 0.2 Gy. MSH2 significantly enhances cell radiosensitivity to low dose IR.

  8. Pulsed Electromagnetic Field Stimulates Cellular Proliferation in Human Intervertebral Disc Cells

    OpenAIRE

    Lee, Hwan-Mo; Kwon, Un-Hye; Kim, Hyang; Kim, Ho-Joong; Kim, Boram; Park, Jin-Oh; Moon, Eun-Soo; Moon, Seong-Hwan

    2010-01-01

    Purpose The purpose of this study is to investigate the mechanism of cellular proliferation of electromagnetic field (EMF) on human intervertebral disc (IVD) cells. Materials and Methods Human IVD cells were cultured three-dimensionally in alginate beads. EMF was exposed to IVD cells with 650Ω, 1.8 millitesla magnetic flux density, 60 Hz sinusoidal wave. Cultures were divided into a control and EMF group. Cytotoxicity, DNA synthesis and proteoglycan synthesis were measured by MTT assay, [3H]-...

  9. UV-assisted photocatalytic synthesis of highly dispersed Ag nanoparticles supported on DNA decorated graphene for quantitative iodide analysis.

    Science.gov (United States)

    Kong, Fen-Ying; Li, Wei-Wei; Wang, Jing-Yi; Wang, Wei

    2015-07-15

    Herein, we report, for the first time, the synthesis of reduced graphene oxide-DNA-Ag (RGO-DNA-Ag) nanohybrids by ultraviolet (UV) irradiation of aqueous solutions of GO and Ag ions in the presence of DNA. The morphology and microstructure characterizations of the resultant nanohybrids reveal that the proposed method leads to the simultaneous reduction of GO and Ag ions together with efficient dispersion of Ag nanoparticles on the surface of RGO sheets. This simple and fast synthesis route is carried out at ambient conditions without using any additional chemical reducing agents, which has the potential to provide new avenues for the green fabrication of various RGO-based nanomaterials. Additionally, the RGO-DNA-Ag nanohybrids can be utilized as a novel sensing interfacial for direct determination of iodide by simple differential pulse voltammetry (DPV), without requiring any preceding preconcentration of the analyte. Based on the RGO-DNA-Ag nanohybrids modified electrode, a wide linear range of 1μM-1mM and a low detection limit of 0.2μM were obtained. This sensitive and direct method of analysis can be applied successfully to the determination of iodide in real samples.

  10. Rectangular Coordination Polymer Nanoplates: Large-Scale, Rapid Synthesis and Their Application as a Fluorescent Sensing Platform for DNA Detection

    OpenAIRE

    Yingwei Zhang; Yonglan Luo; Jingqi Tian; Asiri, Abdullah M.; Al-Youbi, Abdulrahman O.; Xuping Sun

    2012-01-01

    In this paper, we report on the large-scale, rapid synthesis of uniform rectangular coordination polymer nanoplates (RCPNs) assembled from Cu(II) and 4,4'-bipyridine for the first time. We further demonstrate that such RCPNs can be used as a very effective fluorescent sensing platform for multiple DNA detection with a detection limit as low as 30 pM and a high selectivity down to single-base mismatch. The DNA detection is accomplished by the following two steps: (1) RCPN binds dye-labeled sin...

  11. EFFECT OF HYPOXIA ON DNA SYNTHESIS AND C-MYC GENE EXPRESSION OF PULMONARY ARTERY SMOOTH MUSCLE CELLS

    Institute of Scientific and Technical Information of China (English)

    罗兰; 李世强; 蔡英年

    1996-01-01

    The neonate is particularly susceptible to the development of hypoxie pulmonary hypertension. The present study was undertaken to observe the effect of hypoxia on DNA synthesis and c-mye gene expressionbetween newborn calf and adult bovine PASMC in vitro DNA synthesis measured by 3H-TdR incorporation was increased after hypoxie challenge for 24h. Hypoxia enhanced the increment in 3H-TdR incorporationinduced by EGF. Northern blot analysis revealed that PASMC cultured in both normoxia and hypoxia expressed c-mye gene transcript of 2.2Kb ,but there is a higher 2.2Kb mRNA expression in hypoxie PASMC than that in normoxia. We speculate that newborn calf PASMC exhibited potential response to hypoxia than adult,which was augmented by EGF. Enhanced c-myc gene expression may lead to a great understanding of the mechanism of PASMC growth in the development of pulmonary hypertension.

  12. DNA Synthesis in the Giant Nuclei of Insects - Control Machinery and Structures Observed in the Silk-Producing Gland of Bombyx Mori

    International Nuclear Information System (INIS)

    The existence in many insect organs of giant nuclei without visible chromosomes raises the question of possible homologies between the chromatin structures of these nuclei and those of polytene nuclei or common euploid cells. Studies have been made of the nuclei in the silk-producing gland of Bombyx mori. The DNA synthesis is cyclic. During the third stage there are three successive synthesis cycles, which appear to be relatively autonomous in the individual nuclei. For more than 24 hours after moulting, however, synthesis is greatly reduced; moulting factors thus cause synchronization of all the nuclei. This leads to the conclusion that the triggering of a synthesis cycle is controlled by general factors external to the cell. At the end of larval development, DNA synthesis is suspended at the moment when large-scale secretion of silk begins. Evaluation of the pool of endogenic precursors of DNA shows that it is considerably reduced at the end of the DNA synthesis period. The hypothesis proposed is that large-scale synthesis of fibroin requires polarization of the metabolism, hence the depletion of the nucleotide pool and the end of DNA synthesis. DNA synthesis within a single nucleus is to some extent asynchronic. In particular, a well-defined, delayed-synthesis structure visible only in the female seems to be a possible homologue of a sex chromosome. Other asynchronisms are also apparent, though less clearly. Functional studies thus allow the supposition that in the giant nucleus replication units retain an individuality comparable to that of a polytene chromosome. These observations together lead to the conclusion that a nucleus in the silk-producing gland has physiological and structural characteristics similar to those of a polytene nucleus, differing from it essentially in the lesser degree of condensation of its structures. (author)

  13. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells

    OpenAIRE

    Maddocks, Oliver D. K.; Labuschagne, Christiaan F.; Adams, Peter D; Vousden, Karen H

    2016-01-01

    Summary: Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under ...

  14. Complementation of the xeroderma pigmentosum DNA repair synthesis defect with Escherichia coli UvrABC proteins in a cell-free system.

    OpenAIRE

    Hansson, J; Grossman, L; Lindahl, T; Wood, R D

    1990-01-01

    A newly developed cell-free system was used to study DNA repair synthesis carried out by extracts from human cell lines in vitro. Extracts from a normal human lymphoid cell line and from cell lines established from individuals with hereditary dysplastic nevus syndrome perform damage-dependent repair synthesis in plasmid DNA treated with cis- or trans-diamminedichloro-platinum(II) or irradiated with ultraviolet light. Cell extracts of xeroderma pigmentosum origin (complementation groups A, C, ...

  15. Rectangular coordination polymer nanoplates: large-scale, rapid synthesis and their application as a fluorescent sensing platform for DNA detection.

    Directory of Open Access Journals (Sweden)

    Yingwei Zhang

    Full Text Available In this paper, we report on the large-scale, rapid synthesis of uniform rectangular coordination polymer nanoplates (RCPNs assembled from Cu(II and 4,4'-bipyridine for the first time. We further demonstrate that such RCPNs can be used as a very effective fluorescent sensing platform for multiple DNA detection with a detection limit as low as 30 pM and a high selectivity down to single-base mismatch. The DNA detection is accomplished by the following two steps: (1 RCPN binds dye-labeled single-stranded DNA (ssDNA probe, which brings dye and RCPN into close proximity, leading to fluorescence quenching; (2 Specific hybridization of the probe with its target generates a double-stranded DNA (dsDNA which detaches from RCPN, leading to fluorescence recovery. It suggests that this sensing system can well discriminate complementary and mismatched DNA sequences. The exact mechanism of fluorescence quenching involved is elucidated experimentally and its use in a human blood serum system is also demonstrated successfully.

  16. DNA synthesis time in germinating rice and pattern of diethylsulphate induced mutations in pre-soaked seeds

    International Nuclear Information System (INIS)

    DNA synthesis pattern in germinating rice seeds, pre-soaked in water for varying periods upto 48 hr, was determined by following the pulse incorporation of 3H-thymidine into the TCA-insoluble nucleoprotein. Synthesis of DNA commenced at 24 hr, progressively increased to a first peak at about 38 hr, thereafter showed a 1/3rd drop and subsequently increased to a 2nd and still higher peak at 46 to 48 hr of pre-soaking. Treatments of diethylsulphate (dES) at a low concentration (0.2%-2hr) administered at various progressing stages of DNA synthesis resulted in decrease in seedling height and survival, and increase in mutation frequency at 45 hr. pre-soaking, maximum mutation frequencies of 20, 10 and 2% on M1 plants, M1 spikes and M2 seedling bases, respectively were observed. Higher dES concentration (0.3%-2hr) given at later periods of pre-soaking showed near lethal effects and consequently decreased mutation frequencies. Treatments of sodium fluoride given singly or in combination with dES did not show any substantially different results as compared to those of the respective controls. Mutation spectra observed after dES treatments to germinating seeds, at different pre-soaking periods, were quite dissimilar. Specific mutations of economic importance like semi-dwarf mutants were isolated from the treatment of germinating seeds pre-soaked for 37.5 hr or more when shoot apex cells were undergoing DNA synthesis. (author)

  17. Recognition and repair of chemically heterogeneous structures at DNA ends.

    Science.gov (United States)

    Andres, Sara N; Schellenberg, Matthew J; Wallace, Bret D; Tumbale, Percy; Williams, R Scott

    2015-01-01

    Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not "clean." Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini. PMID:25111769

  18. DNA repair in lymphocytes from patients with secondary leukemia as measured by strand rejoining and unscheduled DNA synthesis

    DEFF Research Database (Denmark)

    Bohr, V; Køber, L

    1985-01-01

    The ability to repair damage to DNA was compared in 2 groups of patients having undergone treatment for leukemia, one of which developed secondary leukemia (SL), and the other without signs of secondary malignancy (treated controls). Both were related to normal controls. DNA repair was assessed...

  19. Interacting RNA polymerase motors on DNA track: effects of traffic congestion and intrinsic noise on RNA synthesis

    CERN Document Server

    Tripathi, Tripti

    2007-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by a DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechano-chemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two new measures of {\\it fluctuations} in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis dep...

  20. Comparison of protein and DNA synthesis assays of guinea pig spleen lymphocytes after stimulation with influenza virus antigen and phytohemagglutinin

    International Nuclear Information System (INIS)

    Two in vitro methods for the demonstration of cell-mediated immune response are compared: Protein and DNA synthesis for detection of in vitro influenza virus antigen- and mitogen-induced lymphocyte stimulation. Guinea pig spleen lymphocytes sensitized with influenza virus antigen were tested in a microadaptation of the lymphocyte transformation test using 14C- or 3H-leucine and 3H-thymidine. As a positive control for T-cell stimulation phytohemagglutinin (PHA)-induced lymphocyte stimulation was measured. The following results were obtained: 1. Kinetics of the incorporation of 14C-leucine and 3H-thymidine in lymphocytes incubated with optimal and suboptimal PHA-doses respectively are quantitatively similar but different in time. 2. The results of the protein and DNA synthesis stimulation assays were correlated against influenza virus antigens. 3. The administration of influenza virus antigens in complete Freund's adjuvant induced a more intensive cell-mediated reaction than injections of antigens in aqueous suspensions, but the results of both methods of cell-mediated immune response (CMI) were correlated. 4. The optimal CMI under the experimental cinditions described is induced by an administration of 30 to 50 μg virus protein per animal and by a combined intramuscular - intraperitoneal immunization procedure. 5. The measurement of the early stimulation of protein synthesis in the protein synthesis stimulation test is substantially more rapid than for the classical lymphocyte transformation test. (author)

  1. Synthesis of a Hoechst 32258 analogue amino acid building block for direct incorporation of a fluorescent, high-affinity DNA binding motif into peptides

    DEFF Research Database (Denmark)

    Behrens, C; Harrit, N; Nielsen, P E

    2001-01-01

    The synthesis of a new versatile "Hoechst 33258-like" Boc-protected amino acid building block for peptide synthesis is described. It is demonstrated that this new ligand is an effective mimic of Hoechst 33258 in terms of DNA affinity and sequence specificity. Furthermore, this minor groove binder...

  2. Synthesis, spectroscopic characterization, biological screenings, DNA binding study and POM analyses of transition metal carboxylates

    Science.gov (United States)

    Uddin, Noor; Sirajuddin, Muhammad; Uddin, Nizam; Tariq, Muhammad; Ullah, Hameed; Ali, Saqib; Tirmizi, Syed Ahmed; Khan, Abdur Rehman

    2015-04-01

    This article contains the synthesis of a novel carboxylic acid derivative, its transition metal complexes and evaluation of biological applications. Six carboxylate complexes of transition metals, Zn(II) and Hg(II), have been successfully synthesized and characterized by FT-IR and NMR (1H, 13C). The ligand, HL, (4-[(2,6-Diethylphenyl)amino]-4-oxobutanoic acid) was also characterized by single crystal X-ray analysis. The complexation occurs via oxygen atoms of the carboxylate moiety. FT-IR date show the bidentate nature of the carboxylate moiety of the ligand as the Δν value in all complexes is less than that of the free ligand. The ligand and its complexes were screened for antifungal and antileishmanial activities. The results showed that the ligand and its complexes are active with few exceptions. UV-visible spectroscopy and viscometry results reveal that the ligand and its complexes interact with the DNA via intercalative mode of interaction. A new and efficient strategy to identify the pharmacophores and anti-pharmacophores sites in carboxylate derivatives for the antibacterial/antifungal activity using Petra, Osiris and Molinspiration (POM) analyses was also carried out.

  3. The DNA methylation inhibitor 5-azacytidine decreases melanin synthesis by inhibiting CREB phosphorylation.

    Science.gov (United States)

    Shin, Jun Seob; Jeong, Hyo-Soon; Kim, Myo-Kyoung; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Kim, Dong-Seok

    2015-10-01

    Here we examined the effects of a DNA methylation inhibitor, 5-azacytidine, on melanogenesis in Mel-Ab cells. We found that 5-azacytidine decreased the melanin content and tyrosinase activity in these cells in a dose-dependent manner; importantly, 5-azacytidine was not cytotoxic at the concentrations used in these experiments. On the other hand, 5-azacytidine did not affect tyrosinase activity in a cell-free system, indicating that 5-azacytidine is not a direct tyrosinase inhibitor. Instead, 5-azacytidine decreased the protein levels of microphthalmia-associated transcription factor (MITF) and tyrosinase. Thus, we investigated the effects of 5-azacytidine on signal transduction pathways related to melanogenesis. However, 5-azacytidine did not have any effect on either Akt or glycogen synthase kinase 3β (GSK3β) phosphorylation. The phosphorylation of cAMP response element-binding protein (CREB) is well known to regulate MITF expression, thereby also regulating tyrosinase expression. We found that 5-azacytidine decreased the phosphorylation of CREB. Therefore, we propose that 5-azacytidine may decrease melanin synthesis by downregulating MITF and tyrosinase via CREB inactivation. PMID:26601420

  4. Candida famata (Debaryomyces hansenii) DNA sequences containing genes involved in riboflavin synthesis.

    Science.gov (United States)

    Voronovsky, Andriy Y; Abbas, Charles A; Dmytruk, Kostyantyn V; Ishchuk, Olena P; Kshanovska, Barbara V; Sybirna, Kateryna A; Gaillardin, Claude; Sibirny, Andriy A

    2004-11-01

    Previously cloned Candida famata (Debaryomyces hansenii) strain VKM Y-9 genomic DNA fragments containing genes RIB1 (codes for GTP cyclohydrolase II), RIB2 (encodes specific reductase), RIB5 (codes for dimethylribityllumazine synthase), RIB6 (encodes dihydroxybutanone phosphate synthase) and RIB7 (codes for riboflavin synthase) were sequenced. The derived amino acid sequences of C. famata RIB genes showed extensive homology to the corresponding sequences of riboflavin synthesis enzymes of other yeast species. The highest identity was observed to homologues of D. hansenii CBS767, as C. famata is the anamorph of this hemiascomycetous yeast. The D. hansenii CBS767 RIB3 gene encoding specific deaminase was cloned. This gene successfully complemented riboflavin auxotrophy of the rib3 mutant of flavinogenic yeast, Pichia guilliermondii. Putative iron-responsive elements (potential sites for binding of the transcription factors Fep1p or Aft1p and Aft2p) were found in the upstream regions of some C. famata and D. hansenii RIB genes. The sequences of C. famata RIB genes have been submitted to the EMBL data library under Accession Nos AJ810169-AJ810173. PMID:15543522

  5. Harnessing DNA Synthesis to Develop Rapid Responses to Emerging and Pandemic Pathogens

    Directory of Open Access Journals (Sweden)

    Lisa M. Runco

    2011-01-01

    Full Text Available Given the interconnected nature of our world today, emerging pathogens and pandemic outbreaks are an ever-growing threat to the health and economic stability of the global community. This is evident by the recent 2009 Influenza A (H1N1 pandemic, the SARS outbreak, as well as the ever-present threat of global bioterrorism. Fortunately, the biomedical community has been able to rapidly generate sequence data so these pathogens can be readily identified. To date, however, the utilization of this sequence data to rapidly produce relevant experimental results or actionable treatments is lagging in spite of obtained sequence data. Thus, a pathogenic threat that has emerged and/or developed into a pandemic can be rapidly identified; however, translating this identification into a targeted therapeutic or treatment that is rapidly available has not yet materialized. This commentary suggests that the growing technology of DNA synthesis should be fully implemented as a means to rapidly generate in vivo data and possibly actionable therapeutics soon after sequence data becomes available.

  6. Candida famata (Debaryomyces hansenii) DNA sequences containing genes involved in riboflavin synthesis.

    Science.gov (United States)

    Voronovsky, Andriy Y; Abbas, Charles A; Dmytruk, Kostyantyn V; Ishchuk, Olena P; Kshanovska, Barbara V; Sybirna, Kateryna A; Gaillardin, Claude; Sibirny, Andriy A

    2004-11-01

    Previously cloned Candida famata (Debaryomyces hansenii) strain VKM Y-9 genomic DNA fragments containing genes RIB1 (codes for GTP cyclohydrolase II), RIB2 (encodes specific reductase), RIB5 (codes for dimethylribityllumazine synthase), RIB6 (encodes dihydroxybutanone phosphate synthase) and RIB7 (codes for riboflavin synthase) were sequenced. The derived amino acid sequences of C. famata RIB genes showed extensive homology to the corresponding sequences of riboflavin synthesis enzymes of other yeast species. The highest identity was observed to homologues of D. hansenii CBS767, as C. famata is the anamorph of this hemiascomycetous yeast. The D. hansenii CBS767 RIB3 gene encoding specific deaminase was cloned. This gene successfully complemented riboflavin auxotrophy of the rib3 mutant of flavinogenic yeast, Pichia guilliermondii. Putative iron-responsive elements (potential sites for binding of the transcription factors Fep1p or Aft1p and Aft2p) were found in the upstream regions of some C. famata and D. hansenii RIB genes. The sequences of C. famata RIB genes have been submitted to the EMBL data library under Accession Nos AJ810169-AJ810173.

  7. Establishment of a non-radioactive cleavage assay to assess the DNA repair capacity towards oxidatively damaged DNA in subcellular and cellular systems and the impact of copper

    International Nuclear Information System (INIS)

    Oxidative stress is involved in many diseases, and the search for appropriate biomarkers is one major focus in molecular epidemiology. 8-Oxoguanine (8-oxoG), a potentially mutagenic DNA lesion, is considered to be a sensitive biomarker for oxidative stress. Another approach consists in assessing the repair capacity towards 8-oxoG, mediated predominantly by the human 8-oxoguanine DNA glycosylase 1 (hOGG1). With respect to the latter, during the last few years so-called cleavage assays have been described, investigating the incision of 32P-labelled and 8-oxoG damaged oligonucleotides by cell extracts. Within the present study, a sensitive non-radioactive test system based on a Cy5-labelled oligonucleotide has been established. Sources of incision activity are isolated proteins or extracts prepared from cultured cells and peripheral blood mononuclear cells (PBMC). After comparing different oligonucleotide structures, a hairpin-like structure was selected which was not degraded by cell extracts. Applying this test system the impact of copper on the activity of isolated hOGG1 and on hOGG activity in A549 cells was examined, showing a distinct inhibition of the isolated protein at low copper concentration as compared to a modest inhibition of hOGG activity in cells at beginning cytotoxic concentrations. For investigating PBMC, all reaction conditions, including the amounts of oligonucleotide and cell extract as well as the reaction time have been optimized. The incision activities of PBMC protein extracts obtained from different donors have been investigated, and inter-individual differences have been observed. In summary, the established method is as sensitive and even faster than the radioactive technique, and additionally, offers the advantage of reduced costs and low health risk.

  8. Cellular response to DNA damage is enhanced by the pR plasmid in mouse cells and in Escherichia coli

    International Nuclear Information System (INIS)

    The pR plasmid, which enhances the survival of Escherichia coli C600 exposed to UV light by induction of the SOS regulatory mechanism, showed the same effect when it transformed mouse LTA cells (tk-, aprt-). With Tn5 insertion mutagenesis which inactivates UV functions in the pR plasmid, we recognized two different regions of the plasmid, uvp1 and uvp2. These pR UVR- mutants exhibited the same effect in LTA transformed cells, demonstrating that resistance to UV light, carried by the pR plasmid, was really due to the expression of these two regions, which were also in the mouse cells. Statistical analysis showed that the expression of the uvp1 and uvp2 regions significantly increased (P less than 0.01) the survival upon exposure to UV light in mouse cells and bacteria. These results might suggest the presence of an inducible repair response to DNA damage in mouse LTA cells

  9. Cellular ATP Synthesis Mediated by Type III Sodium-dependent Phosphate Transporter Pit-1 Is Critical to Chondrogenesis*

    OpenAIRE

    Sugita, Atsushi; Kawai, Shinji; Hayashibara, Tetsuyuki; Amano, Atsuo; Ooshima, Takashi; Michigami, Toshimi; Yoshikawa, Hideki; Yoneda, Toshiyuki

    2010-01-01

    Disturbed endochondral ossification in X-linked hypophosphatemia indicates an involvement of Pi in chondrogenesis. We studied the role of the sodium-dependent Pi cotransporters (NPT), which are a widely recognized regulator of cellular Pi homeostasis, and the downstream events in chondrogenesis using Hyp mice, the murine homolog of human X-linked hypophosphatemia. Hyp mice showed reduced apoptosis and mineralization in hypertrophic cartilage. Hyp chondrocytes in culture displayed decreased ap...

  10. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis.

    Science.gov (United States)

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia; Altmannova, Veronika; Sebesta, Marek; Pacesa, Martin; Fugger, Kasper; Sorensen, Claus Storgaard; Lee, Marietta Y W T; Haracska, Lajos; Krejci, Lumir

    2016-04-20

    Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HRin vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy. PMID:26792895

  11. Revisiting Plus-Strand DNA Synthesis in Retroviruses and Long Terminal Repeat Retrotransposons: Dynamics of Enzyme: Substrate Interactions

    Directory of Open Access Journals (Sweden)

    Stuart F. J. Le Grice

    2009-11-01

    Full Text Available Although polypurine tract (PPT-primed initiation of plus-strand DNA synthesis in retroviruses and LTR-containing retrotransposons can be accurately duplicated, the molecular details underlying this concerted series of events remain largely unknown. Importantly, the PPT 3’ terminus must be accommodated by ribonuclease H (RNase H and DNA polymerase catalytic centers situated at either terminus of the cognate reverse transcriptase (RT, and in the case of the HIV-1 enzyme, ~70Å apart. Communication between RT and the RNA/DNA hybrid therefore appears necessary to promote these events. The crystal structure of the HIV-1 RT/PPT complex, while informative, positions the RNase H active site several bases pairs from the PPT/U3 junction, and thus provides limited information on cleavage specificity. To fill the gap between biochemical and crystallographic approaches, we review a multidisciplinary approach combining chemical probing, mass spectrometry, NMR spectroscopy and single molecule spectroscopy. Our studies also indicate that nonnucleoside RT inhibitors affect enzyme orientation, suggesting initiation of plus-strand DNA synthesis as a potential therapeutic target.

  12. Effect of hydrophobic scaffold on the cellular uptake and gene transfection activities of DNA-encapsulating liposomal nanoparticles via intracerebroventricular administration.

    Science.gov (United States)

    Akita, Hidetaka; Nakatani, Taichi; Kuroki, Kimiko; Maenaka, Katsumi; Tange, Kota; Nakai, Yuta; Harashima, Hideyoshi

    2015-07-25

    Efficient DNA carriers are needed as a gene medication for curing brain disorders. In the present study, the function of a neutral lipid envelope-type nanoparticle (LNP) encapsulating pDNA was evaluated after intracerebroventricular administration. The lipid envelope was composed of a series of SS-cleavable and pH-activated lipid like materials (ssPalm) including myristic acid, vitamin A and vitamin E in the hydrophobic scaffold (LNPssPalmM, LNPssPalmA, LNPssPalmE, respectively). The LNPssPalmA and LNPssPalmE were extensively distributed in the corpus callosum, and then gene expression occurred mainly astrocytes in this region, while not in LNPssPalmM. The recombinant human ApoE3-dependent enhancement of the uptake into an astrocyte-derived cell line (KT-5) was observed in LNPssPalmA and LNPssPalmE. Thus, ApoE in the brain plays a key role in the cellular uptake of these particles by astrocytes, and this uptake is dependent on the structure of the hydrophobic scaffold.

  13. Distinct genetic loci control plasma HIV-RNA and cellular HIV-DNA levels in HIV-1 infection: the ANRS Genome Wide Association 01 study.

    Directory of Open Access Journals (Sweden)

    Cyril Dalmasso

    Full Text Available Previous studies of the HIV-1 disease have shown that HLA and Chemokine receptor genetic variants influence disease progression and early viral load. We performed a Genome Wide Association study in a cohort of 605 HIV-1-infected seroconverters for detection of novel genetic factors that influence plasma HIV-RNA and cellular HIV-DNA levels. Most of the SNPs strongly associated with HIV-RNA levels were localised in the 6p21 major histocompatibility complex (MHC region and were in the vicinity of class I and III genes. Moreover, protective alleles for four disease-associated SNPs in the MHC locus (rs2395029, rs13199524, rs12198173 and rs3093662 were strikingly over-represented among forty-five Long Term HIV controllers. Furthermore, we show that the HIV-DNA levels (reflecting the HIV reservoir are associated with the same four SNPs, but also with two additional SNPs on chromosome 17 (rs6503919; intergenic region flanked by the DDX40 and YPEL2 genes and chromosome 8 (rs2575735; within the Syndecan 2 gene. Our data provide evidence that the MHC controls both HIV replication and HIV reservoir. They also indicate that two additional genomic loci may influence the HIV reservoir.

  14. Elucidation of lead-induced oxidative stress in Talinum triangulare roots by analysis of antioxidant responses and DNA damage at cellular level.

    Science.gov (United States)

    Kumar, Abhay; Prasad, M N V; Mohan Murali Achary, V; Panda, Brahma B

    2013-07-01

    Hydroponic experiments were performed with Talinum triangulare (Jacq.) Willd. focusing the root cellular biochemistry with special emphasis on DNA damage, structural, and elemental analyses in Pb(NO3)2 exposed with 0, 0.25, 0.5, 0.75, 1.0, and 1.25 mM for 7 days. Lead (Pb) increased reactive oxygen species production, lipid peroxidation, protein oxidation, cell death, and DNA damage and decreased the protein content in a dose-dependent manner. Likewise, a dose-dependent induction of antioxidative enzymes superoxide dismutase and catalase by Pb was evident. Ascorbate peroxidase on the other hand responded biphasically to Pb treatments by showing induction at low (0.25 and 0.50) and repression at high (0.75-1.25 mM) concentrations. The estimation of proline content also indicated a similar biphasic trend. Scanning electron microscope and energy-dispersive X-ray spectroscopy analysis showed that 1.25 mM Pb treatment resulted in ultrastructural modifications in roots and stem tissue that was marked by the change in the elemental profile. The findings pointed to the role of oxidative stress in the underlying Pb phytotoxicity and genotoxicity in T. triangulare. PMID:23263755

  15. Comparisons of ape and human sequences that regulate mitochondrial DNA transcription and D-loop DNA synthesis.

    OpenAIRE

    Foran, D R; Hixson, J E; Brown, W. M.

    1988-01-01

    The mitochondrial DNA (mtDNA) control regions for common chimpanzee, pygmy chimpanzee and gorilla were sequenced and the lengths and termini of their D-loop DNA's characterized. In these and all other species for which there are data, 5' termini map to sequences that contain the trinucleotide YAY. 3' termini are 25-51 nucleotides downstream from a sequence that is moderately conserved among vertebrates. Substitutions were greater than 1.5 times more frequent in the control region than in regi...

  16. Protein accounting in the cellular economy

    Science.gov (United States)

    Vázquez-Laslop, Nora; Mankin, Alexander S.

    2014-01-01

    Knowing the copy number of cellular proteins is critical for understanding cell physiology. By being able to measure the absolute synthesis rates of the majority of cellular proteins, Li et al. (2014) gain insights into key aspects of translation regulation and fundamental principles of cellular strategies to adjust protein synthesis according to the needs. PMID:24766801

  17. The validity of the autoradiographic method for detecting DNA repair synthesis in rat hepatocytes in primary culture

    International Nuclear Information System (INIS)

    The autoradiographic detection of unscheduled DNA synthesis (UDS) in primary cultures of rat hepatocytes (HPC) was used to measure excision repair of DNA lesions induced by genotoxic agents. Both directly and indirectly acting agents were tested. The HPC/DNA repair test has been claimed to have advantages over screening tests based on non-metabolizing cells in combination with a system for bio-activation. The experiments reported here, however, show that its advantages are greatly reduced by the difficulty of obtaining cell preparations of reliable and reproducible quality. The reproducibility of the system is affected by the large variations in the functional state of the isolated cells and by other factors. For some of these variations a correction is possible. For instance, differences due to the size of the nuclei can be eliminated by reporting the grains counted above the nucleus in proportion to the size of the nuclear area scored. (orig./AJ)

  18. Synthesis, solubilization, and surface functionalization of highly fluorescent quantum dots for cellular targeting through a small molecule

    Science.gov (United States)

    Galloway, Justin F.

    To achieve long-term fluorescence imaging with quantum dots (QDs), a CdSe core/shell must first be synthesized. The synthesis of bright CdSe QDs is not trivial and as a consequence, the role of surfactant in nucleation and growth was investigated. It was found that the type of surfactant used, either phosphonic or fatty acid, played a pivotal role in the size of the CdSe core. The study of surfactant on CdSe synthesis, ultimately led to an electrical passivation method that utilized a short-chained phosphonic acid and highly reactive organometallic precursors to achieve high quantum yield (QY) as has been previously described. The synthesis of QDs using organometallic precursors and a phosphonic acid for passivation resulted in 4 out of 9 batches of QDs achieving QYs greater than 50% and 8 out of 9 batches with QYs greater than 35%. The synthesis of CdSe QDs was done in organic solutions rendering the surface of the particle hydrophobic. To perform cell-targeting experiments, QDs must be transferred to water. The transfer of QDs to water was successfully accomplished by using single acyl chain lipids. A systematic study of different lipid combinations and coatings demonstrated that 20-40 mol% single acyl chained lipids were able to transfer QDs to water resulting in monodispersed, stable QDs without adversely affecting the QY. The advantage to water solubilization using single acyl chain lipids is that the QD have a hydrodynamic radius less than 15 nm, QYs that can exceed 50% and additional surface functionalization can be down using the reactive sites incorporated into the lipid bilayer. QDs that are bright and stable in water were studied for the purpose of targeting G protein-coupled Receptors (GPCR). GPCRs are transmembrane receptors that internalize extracellular cues, and thus mediate signal transduction. The cyclic Adenosine Monophosphate Receptor 1 of the model organism Dictyostelium disodium was the receptor of interest. The Halo protein, a genetically

  19. Dithiocarbamate/piperazine bridged pyrrolobenzodiazepines as DNA-minor groove binders: synthesis, DNA-binding affinity and cytotoxic activity.

    Science.gov (United States)

    Kamal, Ahmed; Sreekanth, Kokkonda; Shankaraiah, Nagula; Sathish, Manda; Nekkanti, Shalini; Srinivasulu, Vunnam

    2015-04-01

    A new series of C8-linked dithiocarbamate/piperazine bridged pyrrolo[2,1-c][1,4]benzodiazepine conjugates (5a-c, 6a,b) have been synthesized and evaluated for their cytotoxic potential and DNA-binding ability. The representative conjugates 5a and 5b have been screened for their cytotoxicity against a panel of 60 human cancer cell lines. Compound 5a has shown promising cytotoxic activity on selected cancer cell lines that display melanoma, leukemia, CNS, ovarian, breast and renal cancer phenotypes. The consequence of further replacement of the 3-cyano-3,3-diphenylpropyl 1-piperazinecarbodithioate in 5b and 5c with 4-methylpiperazine-1-carbodithioate yielded new conjugates 6a and 6b respectively. In addition, the compounds 5c and 6a,b have been evaluated for their in vitro cytotoxicity on some of the selected human cancer cell lines and these conjugates have exhibited significant cytotoxic activity. Further, the DNA-binding ability of these new conjugates has been evaluated by using thermal denaturation (ΔTm) studies. The correlation between structure and DNA-binding ability has been investigated by molecular modeling studies which predicted that 6b exhibits superior DNA-binding ability and these are in agreement with the experimental DNA-binding studies. PMID:25665519

  20. Targeted Nanogel Conjugate for Improved Stability and Cellular Permeability of Curcumin: Synthesis, Pharmacokinetics, and Tumor Growth Inhibition

    Science.gov (United States)

    2015-01-01

    Curcumin (CUR) is a unique natural compound with promising anticancer and anti-inflammatory activities. However, the therapeutic efficacy of curcumin was challenged in clinical trials, mostly due to its low bioavailability, rapid metabolism, and elimination. We designed a nanodrug form of curcumin, which makes it stable and substantially enhances cellular permeability and anticancer activity at standard oral administration. Curcumin was conjugated as an ester to cholesteryl-hyaluronic acid (CHA) nanogel that is capable of targeted delivery to CD44-expressing drug-resistant cancer cells. CHA-CUR nanogels demonstrated excellent solubility and sustained drug release in physiological conditions. It induced apoptosis in cancer cells, suppressing the expression of NF-κB, TNF-α, and COX-2 cellular targets similar to free curcumin. Pharmacokinetic/pharmacodynamic (PK/PD) studies also revealed improved circulation parameters of CHA-CUR at oral, i.p. and i.v. administration routes. CHA-CUR showed targeted tumor accumulation and effective tumor growth inhibition in human pancreatic adenocarcinoma MiaPaCa-2 and aggressive orthotropic murine mammary carcinoma 4T1 animal models. CHA-CUR treatment was well-tolerated and resulted in up to 13-fold tumor suppression, making this nanodrug a potential candidate for cancer prevention and therapeutic treatment. PMID:25072100

  1. [Overgrowth and DNA synthesis of neuroepithelium in embryonic stages of induced Long-Evans rat myeloschisis].

    Science.gov (United States)

    Chono, Y

    1993-01-01

    Overgrowth of the myeloschisis, namely the excessive amount of the neural plate tissue, has been reported in the human myeloschisis. However, it is still debatable how the overgrowth develops and whether the overgrowth is the cause, or the secondary effect of spinal dysraphism. The author induced myeloschisis in the fetuses of Long-Evans rats by the administration of ethylenethiourea (ETU) to pregnant rats on day 10 of gestation. The fetuses were removed 1 hour after the treatment with bromodeoxyuridine (BrdU) to the dams on day 14 and 21. The fetuses were fixed in alcohol and embedded in paraffin. H-E staining and the immunohistologic examination were performed on the staining patterns to anti-neurofilament (NFP), anti-glial fibrillary acidic protein (GFAP) and anti-BrdU antibody by ABC method. On day 14, the lateral portion of everted neural plate showed a loose arrangement of cells and there was rosette formation in the mesoderm. On day 21, cell necrosis was observed at the dorsolateral portion of myeloschisis, although the ventral portion showed almost normal cytoarchitecture and was positive to NFP and GFAP. The cause of myeloschisis in this model is supposed to be the local and direct cytotoxic effect of ETU to neuro-ectodermal junction. On day 14, control animals contained few BrdU-incorporated cells at the basal plate of neural tube. In contrast, everted neural plate showed an active uptake of BrdU diffusely in the subependymal matrix layer cells. Overgrowth was not yet identified. On day 21, overgrowth of myeloschisis was found in spite of a few positive cells to BrdU which was identical to the control animals. These findings seem to suggest that cells in the myeloschisis retain their ability of DNA synthesis for longer periods of development and overgrowth found on day 21 is possibly a secondary effect of spinal dysraphism in this model.

  2. Facile synthesis of nitrogen-doped carbon dots for Fe{sup 3+} sensing and cellular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Xiaojuan; Lu, Wenjing [Institute of Environmental Science, and School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Paau, Man Chin; Hu, Qin [Partner State Key Laboratory of Environmental and Biological Analysis, and Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong SAR (China); Wu, Xin; Shuang, Shaomin [Institute of Environmental Science, and School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Dong, Chuan, E-mail: dc@sxu.edu.cn [Institute of Environmental Science, and School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Choi, Martin M.F., E-mail: mmfchoi@gmail.com [Partner State Key Laboratory of Environmental and Biological Analysis, and Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong SAR (China)

    2015-02-25

    Highlights: • Fast synthesis of nitrogen-doped carbon dots (N-CDs) by microwave method. • Optimization of synthesis of N-CDs. • Fluorescence sensing of Fe{sup 3+} by N-CDs. • Cell imaging and detecting Fe{sup 3+} in biosystem by N-CDs. - Abstract: A fast and facile approach to synthesize highly nitrogen (N)-doped carbon dots (N-CDs) by microwave-assisted pyrolysis of chitosan, acetic acid and 1,2-ethylenediamine as the carbon source, condensation agent and N-dopant, respectively, is reported. The obtained N-CDs are fully characterized by elemental analysis, transmission electron microscopy, high-resolution transmission electron microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray diffraction pattern, X-ray photoelectron spectroscopy, UV–vis absorption, and photoluminescence spectroscopy. Doping N heteroatoms benefits the generation of N-CDs with stronger fluorescence emission. As the emission of N-CDs is efficiently quenched by Fe{sup 3+}, the as-prepared N-CDs are employed as a highly sensitive and selective probe for Fe{sup 3+} detection. The detection limit can reach as low as 10 ppb, and the linear range is 0.010–1.8 ppm Fe{sup 3+}. The as-synthesized N-CDs have been successfully applied for cell imaging and detecting Fe{sup 3+} in biosystem.

  3. A Novel Cobalt(Ⅲ) Mixed-polypyridyl Complex: Synthesis,Characterization and DNA Binding

    Institute of Scientific and Technical Information of China (English)

    CHEN,Hui-Li(陈绘丽); YANG,Pin(杨频)

    2002-01-01

    A novel complex[Co(phen)2HPIP]Cl3[phen=phenanethroline,HPIP=2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanethroline]has been synthesized and structurally characterized by elemental analysis,UV,IR and 1H NMR spectroscopies. The interaction of the complex with calf thymus DNA(CT DNA)has been studied using absorption and emission spectroscopy, DNA melting techniques and cyclic voltammetry. The compound shows absorption hypochromicity, fluorescence enhancement and DNA melting temperature increment when binding to CT DNA. CV measurement shows a shift in reduction potential and a change in peak current with addition of DNA.These results prove that the compound inserts into DNA base pairs. The shift of peak potential indicates the ion interaction mode between the complex and DNA. The binding constant of the compound to DNA is 4.37×104. The complex also seems to be an efficient photocleavage reagent.

  4. Mechanism of Translesion Synthesis Past an Equine Estrogen-DNA Adduct by Y-Family DNA Polymerases

    OpenAIRE

    Yasui, Manabu; Suzuki, Naomi; Liu, Xiaoping; Kim, Yoshinori Okamoto Sung Yeon; Laxmi, Y. R. Santosh; Shibutani, Shinya

    2007-01-01

    4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) η, κ and ι in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol η, κ and ι. Pol η inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and ...

  5. Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis.

    OpenAIRE

    Ariga, H

    1984-01-01

    The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixtur...

  6. DNA binding, cytotoxicity and inhibitory effect on RNA synthesis of two new 1-nitro-9-aminoacridine dimers.

    Science.gov (United States)

    Markovits, J; Wilmańska, D; Lescot, E; Studzian, K; Szmigiero, L; Gniazdowski, M

    1989-01-01

    Two 1-nitro-9-aminoacridine dimers were prepared: one bearing a spermine flexible linking chain, compound 4, the other a rigid dipiperidine-type linker, compound 7. Both dimers elicited a higher affinity constant for DNA than the parent monomeric drug nitracrine 2. This affinity was several orders lower than what was found for other dimeric compounds having the same linkers and no nitro group on the acridine ring (3, 5, 6 and 8). Bisintercalation was evidenced for compound 4 by viscosimetric measurements. In the absence of dithiothreitol, an inhibitory effect of RNA synthesis in vitro was observed for all the tested compounds except 2 and 7. In the presence of dithiothreitol, 4 and 7 formed irreversible complexes with DNA of decreased template properties. The level of the dimers binding was lower than that of the parent compound 2. Cross-links were detected by means of hydroxylapatite chromatography in a complex of the dimer bearing a flexible linking chain, compound 4 with DNA, while the compound 7-DNA complex eluted in the single-stranded DNA region. The extent of cytotoxicity of the two 1-nitro-9-aminoacridine dimers against L1210 cultured cells was different. PMID:2472225

  7. Synthesis of titanium oxide nanoparticles using DNA-complex as template for solution-processable hybrid dielectric composites

    Energy Technology Data Exchange (ETDEWEB)

    Ramos, J.C. [Center for Sustainable Materials Chemistry, 153 Gilbert Hall, Oregon State University, Corvallis, OR (United States); Mejia, I.; Murphy, J.; Quevedo, M. [Department of Materials Science and Engineering, University of Texas at Dallas, Dallas, TX (United States); Garcia, P.; Martinez, C.A. [Engineering and Technology Institute, Autonomous University of Ciudad Juarez, Ciudad Juarez, Chihuahua (Mexico)

    2015-09-15

    Highlights: • We developed a synthesis method to produce TiO{sub 2} nanoparticles using a DNA complex. • The nanoparticles were anatase phase (~6 nm diameter), and stable in alcohols. • Composites showed a k of 13.4, 4.6 times larger than the k of polycarbonate. • Maximum processing temperature was 90 °C. • Low temperature enables their use in low-voltage, low-cost, flexible electronics. - Abstract: We report the synthesis of TiO{sub 2} nanoparticles prepared by the hydrolysis of titanium isopropoxide (TTIP) in the presence of a DNA complex for solution processable dielectric composites. The nanoparticles were incorporated as fillers in polycarbonate at low concentrations (1.5, 5 and 7 wt%) to produce hybrid dielectric films with dielectric constant higher than thermally grown silicon oxide. It was found that the DNA complex plays an important role as capping agent in the formation and suspension stability of nanocrystalline anatase phase TiO{sub 2} at room temperature with uniform size (∼6 nm) and narrow distribution. The effective dielectric constant of spin-cast polycarbonate thin-films increased from 2.84 to 13.43 with the incorporation of TiO{sub 2} nanoparticles into the polymer host. These composites can be solution processed with a maximum temperature of 90 °C and could be potential candidates for its application in low-cost macro-electronics.

  8. Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: Testing of 24 compounds

    International Nuclear Information System (INIS)

    The in vivo-in vitro hepatocyte DNA repair assay has been shown to be useful for studying genotoxic hepatocarcinogens. In addition, measurement of S-phase synthesis (SPS) provides an indirect indicator of hepatocellular proliferation, which may be an important mechanism in rodent carcinogenesis. This assay was used to examine 24 chemicals for their ability to induce unscheduled DNA synthesis (UDS) or SPS in Fischer-344 rats or B6C3F1 mice following in vivo treatment. Hepatocytes were isolated by liver perfusion and incubated with 3H-thymidine following in vivo treatment by gavage. Chemicals chosen for testing were from the National Toxicology Program (NTP) genetic toxicology testing program and most were also evaluated in long-term animal studies conducted by the NTP. Dinitrotoluene and Michler's Ketone induced positive UDS response in rat, while N-nitrosodiethanolamine and selenium sulfide induced equivocal UDS results in mouse and rat, respectively. BCMEE, bromoform, chloroform, PBB, 1,1,2-trichloroethane, and trichloroethylene were all potent inducers of SPS in mouse liver, while C.I. Solvent Yellow 14, and 1,1,2,2-tetrachloroethane yielded equivocal SPS results in rat and mouse, respectively. These results indicate that most of the test compounds do not induced UDS in the liver; however, the significant S-phase response induced by many of these compounds, especially the halogenated solvents, may be an important mechanism in their hepatocarinogenicity

  9. One-pot synthesis of FePt/CNTs nanocomposites for efficient cellular imaging and cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Weihong; Zheng, Xiuwen, E-mail: xwzheng1976@163.com [Linyi University, School of Chemistry & Chemical Engineering, Shandong Provincial Key Laboratory of Detection Technology for Tumor Makers (China); Li, Shulian [Linyi Tumor Hospital (China); Zhang, Wei; Wen, Xin [Linyi University, School of Chemistry & Chemical Engineering, Shandong Provincial Key Laboratory of Detection Technology for Tumor Makers (China); Yue, Ludan [Shandong Normal University (China); Wang, Jinlong [Shandong University of Technology (China)

    2015-11-15

    Here, we developed a facile route to synthesize carbon nanotube-based FePt nanocomposites (FePt/CNTs) as a potential theranostic platform in the cancer treatment. FePt/CNTs were firstly synthesized via one-pot polyol route, and then functionalized with 6-arm-polyethylene glycol-amine polymer. The average size of FePt nanoparticles (NPs) is 3–4 nm, which is dispersed on the CNT surface (ca.50–150 nm). The as-prepared FePt NPs display high cytotoxicity by highly reactive oxygen species in cancer cells. Folic acid and fluorescein isothiocyanate are assembled onto the surface of FePt/CNTs for effective targeting of folate receptor-positive cancer cells and simultaneously for the visualization of cellular uptake. Therefore, the FePt/CNTs NPs capability of simultaneously performing diagnosis, therapy, and targeting is, therefore, promising for future potential widespread application in biomedicine.

  10. Synthesis of a Neutral Mixed-Valence Diferrocenyl Carborane for Molecular Quantum-Dot Cellular Automata Applications.

    Science.gov (United States)

    Christie, John A; Forrest, Ryan P; Corcelli, Steven A; Wasio, Natalie A; Quardokus, Rebecca C; Brown, Ryan; Kandel, S Alex; Lu, Yuhui; Lent, Craig S; Henderson, Kenneth W

    2015-12-14

    The preparation of 7-Fc(+) -8-Fc-7,8-nido-[C2 B9 H10 ](-) (Fc(+) FcC2 B9 (-) ) demonstrates the successful incorporation of a carborane cage as an internal counteranion bridging between ferrocene and ferrocenium units. This neutral mixed-valence Fe(II) /Fe(III) complex overcomes the proximal electronic bias imposed by external counterions, a practical limitation in the use of molecular switches. A combination of UV/Vis-NIR spectroscopic and TD-DFT computational studies indicate that electron transfer within Fc(+) FcC2 B9 (-) is achieved through a bridge-mediated mechanism. This electronic framework therefore provides the possibility of an all-neutral null state, a key requirement for the implementation of quantum-dot cellular automata (QCA) molecular computing. The adhesion, ordering, and characterization of Fc(+) FcC2 B9 (-) on Au(111) has been observed by scanning tunneling microscopy. PMID:26516063

  11. Simple synthesis of carbon-11-labeled chromen-4-one derivatives as new potential PET agents for imaging of DNA-dependent protein kinase (DNA-PK) in cancer

    International Nuclear Information System (INIS)

    Carbon-11-labeled chromen-4-one derivatives were synthesized as new potential PET agents for imaging of DNA repair enzyme DNA-dependent protein kinase (DNA-PK) in cancer. The target tracers, X-[11C]methoxy-2-morpholino-4H-chromen-4-ones (X=8, 7, 6, 5; [11C]4a–d), were prepared from their corresponding precursors, X-hydroxy-2-morpholino-4H-chromen-4-ones (X=8, 7, 6, 5; 5a–d), with [11C]CH3OTf through O-[11C]methylation and isolated by a simplified solid-phase extraction (SPE) method using a C-18 Sep-Pak Plus cartridge. The radiochemical yields decay corrected to end of bombardment (EOB), from [11C]CO2, were 40–60%. The specific activity at end of synthesis (EOS) was 185–370 GBq/μmol. - Highlights: ► New chromen-4-one derivatives were synthesized. ► New carbon-11-labeled chromen-4-one derivatives were synthesized. ► Simple solid-phase extraction (SPE) method was employed in radiosynthesis.

  12. Studies on the repair of double strand break of DNA and cellular carcinogenesis, and consideration on the concept of extinction of nuclear power

    International Nuclear Information System (INIS)

    This paper describes the relationship between the repair of double strand break (DSB) of DNA and cellular carcinogenesis mainly on author's investigations, and his recent thought aiming at the extinction of nuclear power. The molecular repairing system is explained about DNA DSB induced by radiation and chemicals. When DSB occurs, nucleosome consisting from 4 core-histones participates to link the broken ends and then repair mechanisms of homologous recombination (HRR) and non-homologous end joining (NHEJ) begin to work. The latter is dominant in mammalians. Thus the genetic defect in these systems of DSB response and repair is a course of disorders such as ataxia telangiectasia (AT) (DSB sensor defect), genetic breast cancer (HRR defect), and radiosensitive-severe combined immunodeficiency (RS-SCID) (NHEJ defect), all of which result in cancer formation. NHEJ repair is known to be error-prone. Against multi-step carcinogenesis where accumulated gene mutations lead to the cancer formation, the author thinks chromosomal instability is one of important carcinogenic causes: the instability can be a trigger of producing cancer stem cells because the cells can be yielded from mouse embryonic stem cells where DSB is shown to participate in the process. Low dose radiation produces a small amount of DSB, to which the repair response is less sensitive at G2/M checkpoint, ultimately leading to genomic instability. Considering effects of the low dose radiation exposure above, and of the internal exposure to 3H-thymidine beta ray in cells, of indoor Rn participating 16% of lung cancer incidence (Canadian epidemiological data) and so on, together with moral and social responsibility of scientist and technologist, the author says to have attained to the concept of the ''Extinction of Nuclear Power''. (T.T)

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

    Science.gov (United States)

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

    2016-01-01

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

  14. DNA photobinding of 7-methylpyrido[3,4-c]psoralen and 8-methoxypsoralen. Effects on macromolecular synthesis, repair and survival in cultured human cells

    International Nuclear Information System (INIS)

    The photobinding to DNA of tritiated 7-methylpyrido[3,4-c]psoralen (MPP), a recently synthesized monofunctional compound of therapeutical interest, and of 8-methoxypsoralen (8-MOP) was determined in cultured normal human fibroblasts. Employing compounds at 10-6 M, MPP photobinds approximately 11 times more efficiently than 8-MOP. For equivalent photobinding MPP ad 8-MOP induce similar inhibitions of DNA synthesis. However, the recovery of DNA synthesis during post-treatment incubation is lower after photoaddition of MPP than after that of 8-MOP. MPP also exerts a much higher lethal effect than 8-MOP. Alkaline elution experiments confirmed the monofunctional nature of MPP and indicated that in MPP-damaged cells DNA breaks accumulate with time of post-treatment incubation. In 8-MOP-treated cells, DNA cross-links appear to be partially repaired. In conclusion, MPP monoadducts turn out to constitute more cytotoxic lesions than 8-MOP mono- and bi-adducts. (Auth.)

  15. Effect of exogenous surfactants on viability and DNA synthesis in A549, immortalized mouse type II and isolated rat alveolar type II cells

    Directory of Open Access Journals (Sweden)

    Haller Thomas

    2011-02-01

    Full Text Available Abstract Background In mechanically ventilated preterm infants with respiratory distress syndrome (RDS, exogenous surfactant application has been demonstrated both to decrease DNA-synthesis but also and paradoxically to increase epithelial cell proliferation. However, the effect of exogenous surfactant has not been studied directly on alveolar type II cells (ATII cells, a key cell type responsible for alveolar function and repair. Objective The aim of this study was to investigate the effects of two commercially available surfactant preparations on ATII cell viability and DNA synthesis. Methods Curosurf® and Alveofact® were applied to two ATII cell lines (human A549 and mouse iMATII cells and to primary rat ATII cells for periods of up to 24 h. Cell viability was measured using the redox indicator resazurin and DNA synthesis was measured using BrdU incorporation. Results Curosurf® resulted in slightly decreased cell viability in all cell culture models. However, DNA synthesis was increased in A549 and rat ATII cells but decreased in iMATII cells. Alveofact® exhibited the opposite effects on A549 cells and had very mild effects on the other two cell models. Conclusion This study showed that commercially available exogenous surfactants used to treat preterm infants with RDS can have profound effects on cell viability and DNA synthesis.

  16. Down-regulation of SMT3A gene expression in association with DNA synthesis induction after X-ray irradiation in nevoid basal cell carcinoma syndrome (NBCCS) cells

    Energy Technology Data Exchange (ETDEWEB)

    Sugaya, Shigeru [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Nakanishi, Hiroshi [Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Tanzawa, Hideki [Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Sugita, Katsuo [Department of Clinical Medicine, Faculty of Education, Chiba University, 1-33 Yayoi, Inage-ku, Chiba 263-8522 (Japan); Kita, Kazuko [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Suzuki, Nobuo [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan)]. E-mail: nobuo@faculty.chiba-u.jp

    2005-10-15

    Fibroblast cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation. Genes, whose expression is modulated in association with the DNA synthesis induction, were searched by using PCR-based mRNA differential display analysis in one of the NBCCS cell lines, NBCCS1 cells. Decreased levels of SMT3A gene expression were found in X-ray-irradiated NBCCS1 cells. This decrease was also shown by RT-PCR analysis in another cell line, NBCCS3 cells. In addition to NBCCS cells, normal fibroblast cells showed the DNA synthesis induction after X-ray irradiation when they were treated with antisense oligonucleotides (AO) for SMT3A. However, treatment of normal fibroblasts with the random oligonucleotides (RO) resulted in decreased levels of DNA synthesis after X-ray irradiation. Thus, down-regulation of SMT3A gene expression may be involved in the DNA synthesis induction after X-ray irradiation in the NBCCS cells at least tested.

  17. Facile synthesis of Graphene Oxide/Double-stranded DNA composite liquid crystals and Hydrogels

    Indian Academy of Sciences (India)

    Rajendra Kurapati; Ashok M Raichur; U Venkateswara Reddy; N Suryaprakash

    2016-03-01

    Investigation of the interactions between graphene oxide (GO) and biomolecules is very crucialfor the development of biomedical applications based on GO. This study reports the first observation of thespontaneous formation of self-assembled liquid crystals and three-dimensional hydrogels of graphene oxidewith double-stranded DNA by simple mixing in an aqueous buffer media without unwinding double-strandedDNA to single-stranded DNA. The GO/dsDNA hydrogels have shown controlled porosity by changing the concentration of the components. The strong binding between dsDNA and graphene is proved by Ramanspectroscopy

  18. Effects of Captan on DNA and DNA metabolic processes in human diploid fibroblasts.

    Science.gov (United States)

    Snyder, R D

    1992-01-01

    The fungicide Captan has been examined for its effects on DNA and DNA processing in order to better understand the genotoxicity associated with this agent. Captan treatment resulted in production of DNA single strand breaks and DNA-protein cross-links and elicited an excision repair response in human diploid fibroblasts. Captan was also shown to inhibit cellular DNA synthesis and to form stable adducts in herring sperm and human cellular DNA. Misincorporation of nucleotides into Captan-treated synthetic DNA templates was significantly elevated in an in vitro assay using E. coli DNA polymerase I, suggesting that DNA adduct formation by Captan could have mutagenic consequences. In sum, these studies demonstrate that Captan is capable of interacting with DNA at a number of levels and that these interactions could provide the basis for Captan's genotoxicity. The extreme cytotoxicity of this fungicide, however, could be due to other cellular effects since at the IC50 for cell killing, approximately 0.8 microM, none of the above genotoxic events could be detected by the methods employed.

  19. Mechanism of the clinical effects of uv-irradiated blood: stimulation of dna synthesis by human cells in culture

    International Nuclear Information System (INIS)

    This paper studies the DNA-synthetic activity of hyman embryonic cells (EC) cultured in the presence of supernatants from intact and irradiated cell fractions of blood or plasma. Human EC obtained from abortion material were incubated; after incubation, tritium-thymidine was added to the growth medium for 30 min. It is shown that stimulation of DNA synthesis in EC growing in the presence of supernatants from irradiated whole blood is not connected with photoactivation of growth factors in the blood plasma, but takes place as a result of their release from the cells. Donated blood, irradiated with UV light of the same wavelength and within the same dose range as are used under clinical conditions (up to 1200 J/m2), possesses growth-stimulating properties

  20. DNA microarray synthesis by using PDMS molecular stamp (II) -- Oligonucleotide on-chip synthesis using PDMS stamp

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5′-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleotide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the conventional synthesis methods.

  1. Sleep, Plasticity and the Pathophysiology of Neurodevelopmental Disorders: The Potential Roles of Protein Synthesis and Other Cellular Processes

    Directory of Open Access Journals (Sweden)

    Dante Picchioni

    2014-03-01

    Full Text Available Sleep is important for neural plasticity, and plasticity underlies sleep-dependent memory consolidation. It is widely appreciated that protein synthesis plays an essential role in neural plasticity. Studies of sleep-dependent memory and sleep-dependent plasticity have begun to examine alterations in these functions in populations with neurological and psychiatric disorders. Such an approach acknowledges that disordered sleep may have functional consequences during wakefulness. Although neurodevelopmental disorders are not considered to be sleep disorders per se, recent data has revealed that sleep abnormalities are among the most prevalent and common symptoms and may contribute to the progression of these disorders. The main goal of this review is to highlight the role of disordered sleep in the pathology of neurodevelopmental disorders and to examine some potential mechanisms by which sleep-dependent plasticity may be altered. We will also briefly attempt to extend the same logic to the other end of the developmental spectrum and describe a potential role of disordered sleep in the pathology of neurodegenerative diseases. We conclude by discussing ongoing studies that might provide a more integrative approach to the study of sleep, plasticity, and neurodevelopmental disorders.

  2. DNA replication at the single-molecule level

    NARCIS (Netherlands)

    Stratmann, S A; van Oijen, A M

    2014-01-01

    A cell can be thought of as a highly sophisticated micro factory: in a pool of billions of molecules - metabolites, structural proteins, enzymes, oligonucleotides - multi-subunit complexes assemble to perform a large number of basic cellular tasks, such as DNA replication, RNA/protein synthesis or i

  3. Synthesis of metal nanowires using nanocracks and DNA-templates and their characterization

    Science.gov (United States)

    Mani, Sathyanarayanan

    A major challenge in the field of nanotechnology is the synthesis and testing of nanostructures, such as nanowires, in a cost effective manner. Currently, there are two general approaches for fabricating nanowires, namely top-down and bottom-up. While the top-down approach that uses nano-lithography is controlled but expensive, the bottom-up approach that uses self-assembly is inexpensive but uncontrolled. This research, describes the mechanism behind an innovative and inexpensive method that combines both the top-down and bottom-up approaches to produce metal nanowires. The method uses cracks in PECVD silicon dioxide thin films on silicon substrate as molds to produce electroless deposited nickel nanowires. The cracks are initiated from voids formed due to nonconformal deposition of oxide on RIE etched trenches in silicon substrate. A model was developed to characterize void formation and the stress response of these oxide films during a thermal ramp, and identify the operating conditions such as film deposit thickness, trench dimensions and annealing temperature for controlled crack pattern formation. The nickel nanowires (50-250 nm wide) are polycrystalline with grain size ranging between 10 to 50 nm. Electrical characterization revealed that the resistivity of these wires decreased 10 times on sintering. However, this resistivity was still twice that of their bulk counterpart. This higher resistivity can be explained by the enhanced scattering of conductive electrons by grain boundaries and phosphorous impurities. An alternate nanowire fabrication technique that simplifies their testing by enabling their direct integration with MEMS test-beds has also been presented. In this approach, commercially available lambda DNA's (16.1 microns long and 2 nm wide) are used as templates to form nanowires of various metals through sputter deposition or evaporation. The test-bed is compatible with commercially available TEM stages for conducting in situ nanostructural

  4. In vitro expression levels of cell-cycle checkpoint proteins are associated with cellular DNA repair capacity in peripheral blood lymphocytes: a multivariate analysis

    OpenAIRE

    Fan, You-Hong; Hu, Zhibin; Li, Chunying; Wang, Li-E; Guo, Zhaozheng; Qiao, Yawei; Zhang, Li; Zhang, Wei; Mao, Li; Wei, Qingyi

    2007-01-01

    DNA repair should occur after cells sense DNA damage signals and undergo cell-cycle arrest to provide sufficient time for DNA repair, and suboptimal DNA repair capacity (DRC) in peripheral lymphocytes has been suggested as a cancer susceptibility marker. Numerous studies showed a functional link between DNA damage sensing, cell-cycle checkpoint and DNA repair. We hypothesized that in vitro cell-cycle checkpoint-related protein expression levels in stimulated lymphocytes predict DRC levels. To...

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

    International Nuclear Information System (INIS)

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

  6. SYNTHESIS OF THE FULLY PROTECTED PHOSPHORAMIDITE OF THE BENZENE-DNA ADDUCT, N2- (4-HYDROXYPHENYL)-2'-DEOXYGUANOSINE AND INCORPORATION OF THE LATER INTO DNA OLIGOMERS

    Energy Technology Data Exchange (ETDEWEB)

    Chenna, Ahmed; Gupta, Ramesh C.; Bonala, Radha R.; Johnson, Francis; Huang, Bo

    2008-06-09

    N2-(4-Hydroxyphenyl)-2'-deoxyguanosine-5'-O-DMT-3'-phosphoramidite has been synthesized and used to incorporate the N2-(4-hydroxyphenyl)-2'-dG (N2-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N2-arylation (Buchwald-Hartwig reaction) of a fully protected 2'-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5'-O-DMT-3'-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N2-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.

  7. Urinary tract infection drives genome instability in uropathogenic Escherichia coli and necessitates translesion synthesis DNA polymerase IV for virulence.

    Science.gov (United States)

    Gawel, Damian; Seed, Patrick C

    2011-01-01

    Uropathogenic Escherichia coli (UPEC) produces ~80% of community-acquired UTI, the second most common infection in humans. During UTI, UPEC has a complex life cycle, replicating and persisting in intracellular and extracellular niches. Host and environmental stresses may affect the integrity of the UPEC genome and threaten its viability. We determined how the host inflammatory response during UTI drives UPEC genome instability and evaluated the role of multiple factors of genome replication and repair for their roles in the maintenance of genome integrity and thus virulence during UTI. The urinary tract environment enhanced the mutation frequency of UPEC ~100-fold relative to in vitro levels. Abrogation of inflammation through a host TLR4-signaling defect significantly reduced the mutation frequency, demonstrating in the importance of the host response as a driver of UPEC genome instability. Inflammation induces the bacterial SOS response, leading to the hypothesis that the UPEC SOS-inducible translesion synthesis (TLS) DNA polymerases would be key factors in UPEC genome instability during UTI. However, while the TLS DNA polymerases enhanced in vitro, they did not increase in vivo mutagenesis. Although it is not a source of enhanced mutagenesis in vivo, the TLS DNA polymerase IV was critical for the survival of UPEC during UTI during an active inflammatory assault. Overall, this study provides the first evidence of a TLS DNA polymerase being critical for UPEC survival during urinary tract infection and points to independent mechanisms for genome instability and the maintenance of genome replication of UPEC under host inflammatory stress.

  8. Amplified and multiplexed detection of DNA using the dendritic rolling circle amplified synthesis of DNAzyme reporter units.

    Science.gov (United States)

    Wang, Fuan; Lu, Chun-Hua; Liu, Xiaoqing; Freage, Lina; Willner, Itamar

    2014-02-01

    The amplified, highly sensitive detection of DNA using the dendritic rolling circle amplification (RCA) is introduced. The analytical platform includes a circular DNA and a structurally tailored hairpin structure. The circular nucleic acid template includes a recognition sequence for the analyte DNA (the Tay-Sachs mutant gene), a complementary sequence to the Mg(2+)-dependent DNAzyme, and a sequence identical to the loop region of the coadded hairpin structure. The functional hairpin in the system consists of the analyte-sequence that is caged in the stem region and a single-stranded loop domain that communicates with the RCA product. The analyte activates the RCA process, leading to DNA chains consisting of the Mg(2+)-dependent DNAzyme and sequences that are complementary to the loop of the functional hairpin structure. Opening of the coadded hairpin releases the caged analyte sequence, resulting in the dendritic RCA-induced synthesis of the Mg(2+)-dependent DNAzyme units. The DNAzyme-catalyzed cleavage of a fluorophore/quencher-modified substrate leads to a fluorescence readout signal. The method enabled the analysis of the target DNA with a detection limit corresponding to 1 aM. By the design of two different circular DNAs that include recognition sites for two different target genes, complementary sequences for two different Mg(2+)-dependent DNAzyme sequences and two different functional hairpin structures, the dendritic RCA-stimulated multiplexed analysis of two different genes is demonstrated. The amplified dendritic RCA detection of DNA is further implemented to yield the hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme as catalytic labels that provide colorimetric or chemiluminescent readout signals. PMID:24377284

  9. Effects of chronic renal failure on protein synthesis and albumin messenger ribonucleic acid in rat liver.

    OpenAIRE

    Zern, M A; Yap, S.H.; Strair, R K; Kaysen, G A; Shafritz, D A

    1984-01-01

    Previously we reported that chronic renal failure in rats leads to preferential disaggregation of liver membrane-bound polysomes associated with a decrease in albumin synthesis. To determine whether reduced albumin synthesis results from reduced cellular levels of albumin messenger RNA (mRNA) or some other molecular mechanism, we have employed mRNA-DNA hybridization in conjunction with cell-free protein synthesis to determine albumin mRNA sequence content and biological activity in subcellula...

  10. Abnormal levels of UV-induced unscheduled DNA synthesis in ataxia telangiectasia cells after exposure to ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Jaspers, N.G.J. (Erasmus Universiteit, Rotterdam (Netherlands). Dept. of Cell Biology and Genetics; Nederlandse Centrale Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek, Rijswijk. Medical Biological Lab.); Bootsma, D. (Erasmus Universiteit, Rotterdam (Netherlands). Dept. of Cell Biology and Genetics)

    1982-01-01

    In cultured cells from normal individuals and from patients having ataxia telangiectasia (AT) the rate of unscheduled DNA synthesis (UDS) induced by UV light was investigated by autoradiography. The number of grains in 6 different AT cell strains was similar to that observed in normal cells. Exposure of normal cells to doses of X-rays up to 20 krad had no influence on the rate of UV-induced UDS. In contrast, the UV-induced UDS was significantly modified in AT cells by treatment with X-rays. In AT cell strains that were reported to have reduced levels of ..gamma..-ray-induced repair DNA synthesis ('excision-deficient' AT cells) the effect of X-rays on UV-induced UDS was inhibitory, whereas UV-induced UDS was stimulated by X-ray exposure in 'excision-proficient' AT cell strains. Different UV and X-ray dose-response relationships were seen in the two categories of AT cell strains.

  11. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

    OpenAIRE

    Sinara Mônica Vitalino de Almeida; Elizabeth Almeida Lafayette; Lúcia Patrícia Bezerra Gomes da Silva; Cézar Augusto da Cruz Amorim; Tiago Bento de Oliveira; Ana Lucia Tasca Gois Ruiz; João Ernesto de Carvalho; Ricardo Olímpio de Moura; Eduardo Isidoro Carneiro Beltrão; Maria do Carmo Alves de Lima; Luiz Bezerra de Carvalho Júnior

    2015-01-01

    In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as re...

  12. Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA.

    OpenAIRE

    Defais, M; Lesca, C; Monsarrat, B.; Hanawalt, P

    1989-01-01

    Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, e...

  13. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives.

    Science.gov (United States)

    de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; da Silva, Lúcia Patrícia Bezerra Gomes; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Ruiz, Ana Lucia Tasca Gois; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

    2015-01-01

    In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a-h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 10(4) to 1.0 × 10(6) M(-1) and quenching constants from -0.2 × 10(4) to 2.18 × 10(4) M(-1) indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N- (4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

  14. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

    Directory of Open Access Journals (Sweden)

    Sinara Mônica Vitalino de Almeida

    2015-06-01

    Full Text Available In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z-2-(acridin-9-ylmethylene-N-phenylhydrazinecarbothioamide derivatives (3a–h were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z-2-(acridin-9-ylmethylene-N- (4-chlorophenyl hydrazinecarbothioamide (3f, while the most active compound in antiproliferative assay was (Z-2-(acridin-9-ylmethylene-N-phenylhydrazinecarbothioamide (3a. There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.

  15. Cyclophilin A as a potential genetic adjuvant to improve HIV-1 Gag DNA vaccine immunogenicity by eliciting broad and long-term Gag-specific cellular immunity in mice

    Science.gov (United States)

    Hou, Jue; Zhang, Qicheng; Liu, Zheng; Wang, Shuhui; Li, Dan; Liu, Chang; Liu, Ying; Shao, Yiming

    2016-01-01

    Previous research has shown that host Cyclophilin A (CyPA) can promote dendritic cell maturation and the subsequent innate immune response when incorporated into an HIV-1 Gag protein to circumvent the resistance of dendritic cells to HIV-1 infection. This led us to hypothesize that CyPA may improve HIV-1 Gag-specific vaccine immunogenicity via binding with Gag antigen. The adjuvant effect of CyPA was evaluated using a DNA vaccine with single or dual expression cassettes. Mouse studies indicated that CyPA specifically and markedly promoted HIV-1 Gag-specific cellular immunity but not an HIV-1 Env-specific cellular response. The Gag/CyPA dual expression cassettes stimulated a greater Gag-specific cellular immune response, than Gag immunization alone. Furthermore, CyPA induced a broad Gag-specific T cell response and strong cellular immunity that lasted up to 5 months. In addition, CyPA skewed to cellular rather than humoral immunity. To investigate the mechanisms of the adjuvant effect, site-directed mutagenesis in CyPA, including active site residues H54Q and F60A resulted in mutants that were co-expressed with Gag in dual cassettes. The immune response to this vaccine was analyzed in vivo. Interestingly, the wild type CyPA markedly increased Gag cellular immunity, but the H54Q and F60A mutants drastically reduced CyPA adjuvant activation. Therefore, we suggest that the adjuvant effect of CyPA was based on Gag-CyPA-specific interactions. Herein, we report that Cyclophilin A can augment HIV-1 Gag-specific cellular immunity as a genetic adjuvant in multiplex DNA immunization strategies, and that activity of this adjuvant is specific, broad, long-term, and based on Gag-CyPA interaction. PMID:26305669

  16. A simple and accurate two-step long DNA sequences synthesis strategy to improve heterologous gene expression in pichia.

    Directory of Open Access Journals (Sweden)

    Jiang-Ke Yang

    Full Text Available In vitro gene chemical synthesis is a powerful tool to improve the expression of gene in heterologous system. In this study, a two-step gene synthesis strategy that combines an assembly PCR and an overlap extension PCR (AOE was developed. In this strategy, the chemically synthesized oligonucleotides were assembled into several 200-500 bp fragments with 20-25 bp overlap at each end by assembly PCR, and then an overlap extension PCR was conducted to assemble all these fragments into a full length DNA sequence. Using this method, we de novo designed and optimized the codon of Rhizopus oryzae lipase gene ROL (810 bp and Aspergillus niger phytase gene phyA (1404 bp. Compared with the original ROL gene and phyA gene, the codon-optimized genes expressed at a significantly higher level in yeasts after methanol induction. We believe this AOE method to be of special interest as it is simple, accurate and has no limitation with respect to the size of the gene to be synthesized. Combined with de novo design, this method allows the rapid synthesis of a gene optimized for expression in the system of choice and production of sufficient biological material for molecular characterization and biotechnological application.

  17. The Effects of Magnesium Ions on the Enzymatic Synthesis of Ligand-Bearing Artificial DNA by Template-Independent Polymerase

    Directory of Open Access Journals (Sweden)

    Yusuke Takezawa

    2016-06-01

    Full Text Available A metal-mediated base pair, composed of two ligand-bearing nucleotides and a bridging metal ion, is one of the most promising components for developing DNA-based functional molecules. We have recently reported an enzymatic method to synthesize hydroxypyridone (H-type ligand-bearing artificial DNA strands. Terminal deoxynucleotidyl transferase (TdT, a template-independent DNA polymerase, was found to oligomerize H nucleotides to afford ligand-bearing DNAs, which were subsequently hybridized through copper-mediated base pairing (H–CuII–H. In this study, we investigated the effects of a metal cofactor, MgII ion, on the TdT-catalyzed polymerization of H nucleotides. At a high MgII concentration (10 mM, the reaction was halted after several H nucleotides were appended. In contrast, at lower MgII concentrations, H nucleotides were further appended to the H-tailed product to afford longer ligand-bearing DNA strands. An electrophoresis mobility shift assay revealed that the binding affinity of TdT to the H-tailed DNAs depends on the MgII concentration. In the presence of excess MgII ions, TdT did not bind to the H-tailed strands; thus, further elongation was impeded. This is possibly because the interaction with MgII ions caused folding of the H-tailed strands into unfavorable secondary structures. This finding provides an insight into the enzymatic synthesis of longer ligand-bearing DNA strands.

  18. Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

    NARCIS (Netherlands)

    Hamdan, Samir M.; Loparo, Joseph J.; Takahashi, Masateru; Richardson, Charles C.; Oijen, Antoine M. van

    2009-01-01

    In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesiz

  19. Synthesis of Distamycin Analogs and Their Interactions withCalf Thymus DNA

    Institute of Scientific and Technical Information of China (English)

    肖军华; 袁谷; 黄伟强; 杜卫红; 王保怀; 李芝芬

    2001-01-01

    Two distamycin analogs (PyPyPy-γ-Dp and PyPyPyPy-γ- Dp)were synthesized by a halform reaction and the DCC/HOBT coupling reaction in a simple and fast way without amino protection.By using calf thymus DNA,the interaction between the analogs and DNA duplex was studied by CD, and ITC.

  20. DNA-surfactant complexes : preparation, self-assembly properties and applications in synthesis and bioelectronics

    NARCIS (Netherlands)

    Liu, Kai

    2015-01-01

    The powerful ionic self-assembly behavior of DNA-surfactant complexes make it a unique material for various applications from optoelectronics to biomedicine. Three types of DNA-surfactant assemblies, including bulk films, lyotropic liquid crystals (LCs) and hydrogels have been investigated extensive

  1. Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas microspores.

    Science.gov (United States)

    Dubas, Ewa; Custers, Jan; Kieft, Henk; Wędzony, Maria; van Lammeren, André A M

    2011-11-01

    In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A 'mild' heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3-8 celled. PMID:21779827

  2. Boron Clusters as a Platform for New Materials: Synthesis of Functionalized o-Carborane (C2 B10 H12 ) Derivatives Incorporating DNA Fragments.

    Science.gov (United States)

    Janczak, Slawomir; Olejniczak, Agnieszka; Balabańska, Sandra; Chmielewski, Marcin K; Lupu, Marius; Viñas, Clara; Lesnikowski, Zbigniew J

    2015-10-19

    A synthetic strategy for functionalization of the three vertices of o-carborane and the attachment of the obtained triped to the solid support was developed. Further functionalization of the triped with short DNA sequences by automated DNA synthesis was achieved. The proposed methodology is a first example of boron cluster chemistry on a solid support opening new perspectives in boron cluster functionalization. PMID:26346614

  3. Chitosan-DNA microparticles as mucosal delivery system:synthesis, characterization and release in vitro

    Institute of Scientific and Technical Information of China (English)

    LI Yu-hong; FAN Min-wen; BIAN Zhuan; CHEN Zhi; ZHANG Qi; YANG Hai-rui

    2005-01-01

    Background Mucosal immunity is important to defense against dental caries. To enhance mucosal immunity, a DNA vaccine mucosal delivery system was prepared by encapsulating anticaries DNA vaccine (plasmid pGJA-P/VAX) in chitosan under optimal conditions and the characteristics of the microparticles was investigated. Furthermore, the release properties and protective action of microparticles for plasmid were studied in vitro.Methods Plasmid loaded chitosan microparticles were prepared by complex coacervation. Three factors, concentration of DNA, sodium sulfate, and the chitosan/DNA ratios in complexes [better expressed as N/P ratio: the number of poly nitrogen (N) per DNA phosphate (P)] influencing preparation were optimized by orthogonal test. The characteristics of microparticles were evaluated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). DNA release rate of microparticles in similar gastro fluid (SGF) or similar intestinal fluid (SIF) at 37℃ was determined by ultraviolet spectrophotometry.Results High encapsulation efficiency (96.8%) was obtained with chitosan microparticles made under optimal conditions of 50 mmol/L Na2SO4, 200 μg/ml DNA and N/P ratio of 4. The size of particles was about 4 to 6 μm. The encapsulation process did not destroy the integrity of DNA. When incubated with SIL, after a release of about 10% in the first 60 minutes, no further DNA was released during the following 180 minutes. When incubated with SGL, the microparticles released a small burst (about 11%) in the first 60 minutes, and then slowly released at a constant, but different rate.Conclusions These chitosan microparticles showed suitable characteristics in vitro for mucosal vaccination and are therefore a promising carrier system for DNA vaccine mucosal delivery.

  4. DNA binding and cleavage activity by a mononuclear iron(II)Schiff base complex: Synthesis and structural characterization

    Indian Academy of Sciences (India)

    Abhijit Pal; Bhaskar Biswas; Merry Mitra; Subramaniyam Rajalakshmi; Chandra Shekhar Purohit; Soumitra Hazra; Gopinatha Suresh Kumar; Balachandran Unni Nair; Rajarshi Ghosh

    2013-09-01

    Synthesis and characterization of a mononuclear Fe(II) compound [Fe(L)](ClO4)2 (1) [L = N-(1-pyridin-2-yl-phenylidene)-N'-[2-({2-[(1-pyridin-2-ylphenylidene)amino]ethyl}amino)ethyl] ethane-1,2-diamine] (1) is reported. 1 crystallizes in P-1 space group with a = 11.9241(3) Å, b = 12.1994(3) Å and c = 13.0622(4) Å. The binding property of the complex with DNA has been investigated using absorption and emission studies, thermal melting, viscosity experiments and circular dichroism studies. The binding constant (b) and the linear Stern-Volmer quenching constant (sv) of the complex have been determined as 3.5 × 103M-1 and 2.73 × 104M-1, respectively. Spectroscopic and hydrodynamic investigations revealed intercalative mode of binding of 1 with DNA. 1 is also found to induce oxidative cleavage of the supercoiled pUC 18 DNA to its nicked circular form in a concentration dependent manner.

  5. Molecular and cellular analysis of the DNA repair defect in a patient in xeroderma pigmentosum complementation group D who has the clinical features of xeroderma pigmentosum and Cockayne syndrome.

    OpenAIRE

    Broughton, B.C.; Thompson, A F; Harcourt, S A; Vermeulen, Wim; Hoeijmakers, Jan; Botta, Elena; Stefanini, Miria; King, M.D.; Weber, Christine; Cole, J.; Arlett, C F; Lehmann, Alan

    1995-01-01

    textabstractXeroderma pigmentosum (XP) and Cockayne syndrome (CS) are quite distinct genetic disorders that are associated with defects in excision repair of UV-induced DNA damage. A few patients have been described previously with the clinical features of both disorders. In this paper we describe an individual in this category who has unusual cellular responses to UV light. We show that his cultured fibroblasts and lymphocytes are extremely sensitive to irradiation with UV-C, despite a level...

  6. Synthesis of modified oligonucleotides for repair and replication studies of single and double radio-induced DNA lesions

    International Nuclear Information System (INIS)

    Several oxidative processes induce the formation of DNA lesions. In order to evaluate the biological and structural significance of such damage, several DNA lesions were inserted into synthetic oligonucleotides at defined sites. The research work aimed at describing the preparation of oligonucleotides t hat contained DNA damage and the evaluation of the biological properties of the lesions. A first part described the incorporation of radiation-induced lesions, namely (5'S,6S)-5',6-cyclo-5,6-dihydro-2'-deoxyuridine and (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-desoxyuridine into oligonucleotides. The modified DNA fragments were characterised by several spectroscopic and biochemical analyses including ESI MS, MALDI-TOF MS, CLHP and enzymatic digestions. During in vitro DNA synthesis by Taq DNA polymerase and Klenow exo fragment, the pyrimidine cyclo-nucleosides were found to block the progression of the enzymes. Then, repair studies by ADN N glycosylases, operating in the base excision repair pathway, have shown that the anhydro-nucleoside lesions were not recognised nor excised by Fpg, endo III, endo VIII, yNtg1 yNtg2 and yOgg1. Interestingly, the Latococcus lactis Fpg protein recognises (formation of a non covalent complex) but do not excise the damage. The incorporation into oligonucleotides of the (5R*) and (5S*) diastereoisomers of 1-[2-deoxy-β-D-erythro-pentofuranosyl]-5-hydroxy-hydantoin, generated by several oxidative processes was then described. In vitro DNA replication assays using modified oligonucleotides matrix showed a lethal potential of the latter base damage. Repair studies by ADN N-glycosylases showed that the damage was substrate for Fpg, endo III, endo VIII, Ntg1, Ntg2 and Fpg-L1. The rates of excision as inferred from the determination of the Michaelis kinetics constants were found to be affected by the presence of the damage. MALDI-TOF MS was used in order to gain insights into mechanistic aspects of oligonucleotides cleavage by the

  7. 遗传物质DNA的复制合成%Synthesis and Replication of DNA

    Institute of Scientific and Technical Information of China (English)

    贺清兰; 索红军

    2011-01-01

    The articles described the nature of DNA replication, the four logical stages of DNA replication process and the repair after the abnormal situation and damage of DNA replication.It maybe help with the genetic information during cell division.%文章结合相关的酶,介绍了DNA复制的性质、DNA复制过程的4个逻辑阶段以及DNA复制过程出现异常或损伤后的修复,为熟悉细胞分裂过程中信息遗传提供帮助.

  8. Synthesis and properties of defined DNA oligomers containing base mispairs involving 2-aminopurine.

    OpenAIRE

    Eritja, R.; Kaplan, B E; Mhaskar, D; Sowers, L C; Petruska, J; Goodman, M F

    1986-01-01

    DNA heptamers containing the mutagenic base analogue 2-aminopurine (AP) have been chemically synthesized and physically characterized. We report on the relative stabilities of base pairs between AP and each of the common DNA bases, as determined from heptamer duplex melts at 275 and 330 nm. Base pairs are ranked in order of decreasing stability: AP.T greater than AP.A greater than AP.C greater than AP.G. It is of interest that AP.A is more stable than AP.C even though DNA polymerase strongly ...

  9. Structurally Diverse Polyamines: Solid-Phase Synthesis and Interaction with DNA.

    Science.gov (United States)

    Umezawa, Naoki; Horai, Yuhei; Imamura, Yuki; Kawakubo, Makoto; Nakahira, Mariko; Kato, Nobuki; Muramatsu, Akira; Yoshikawa, Yuko; Yoshikawa, Kenichi; Higuchi, Tsunehiko

    2015-08-17

    A versatile solid-phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher-order structures of DNA.

  10. Recent Developments in the Chemistry of Deoxyribonucleic Acid (DNA Intercalators: Principles, Design, Synthesis, Applications and Trends

    Directory of Open Access Journals (Sweden)

    Alexandre A. M. Lapis

    2009-05-01

    Full Text Available In the present overview, we describe the bases of intercalation of small molecules (cationic and polar neutral compounds in DNA. We briefly describe the importance of DNA structure and principles of intercalation. Selected syntheses, possibilities and applications are shown to exemplify the importance, drawbacks and challenges in this pertinent, new, and exciting research area. Additionally, some clinical applications (molecular processes, cancer therapy and others and trends are described.

  11. Synthesis, structure, DNA binding and cleavage activity of a new copper(Ⅱ) complex of bispyridylpyrrolide

    Institute of Scientific and Technical Information of China (English)

    MIN Rui; HU Xiao-hui; YI Xiao-yi; ZHANG Shou-chun

    2015-01-01

    A copper-bispyridylpyrrolide complex [Cu(PDPH)Cl] (PDPH = 2,5-bis(2′-pyridyl)pyrrole) was synthesized and characterized. The complex crystallizes in the orthorhombic system with space groupPccn,a = 0.9016(3) nm,b = 1.0931(4) nm,c = 2.5319(8) nm, andV = 2.4951(15) nm3. The copper center is situated in a square planar geometry. The interaction of the copper(Ⅱ) complexwith calf thymus DNA (CT-DNA) was investigated by electronic absorption, circular dichroism (CD) and fluorescence spectra. It is proposed that the complex binds to CT-DNA through groove binding mode. Nuclease activity of the complex was also studied by gel electrophoresis method. The complex can efficiently cleave supercoiled pBR322 DNA in the presence of ascorbate (H2A) via oxidative pathway. The preliminary mechanism of DNA cleavage by the complex with different inhibiting reagents indicates that the hydroxyl radicals were involved as the active species in the DNA cleavage process.

  12. Study of DNA light switch Ru(II) complexes: synthesis, characterization, photocleavage and antimicrobial activity.

    Science.gov (United States)

    Yata, Praveen Kumar; Shilpa, M; Nagababu, P; Reddy, M Rajender; Kotha, Laxma Reddy; Gabra, Nazar Md; Satyanarayana, S

    2012-05-01

    The three Ru(II) complexes of [Ru(phen)(2)dppca](2+) (1) [Ru(bpy)(2)dppca](2+) (2) and [Ru(dmb)(2)dppca](2+) (3) (where phen = 1,10 phenanthroline, bpy = 2,2-bipyridine, dmb = 2 ,2-dimethyl 2',2'-bipyridine and polypyridyl ligand containing a single carboxylate functionality dppca ligand (dipyridophenazine-11-carboxylic acid) have been synthesized and characterized. These complexes have been shown to act as promising calf thymus DNA intercalators and a new class of DNA light switches, as evidenced by UV-visible and luminescence titrations with Co(2+) and EDTA, steady-state emission quenching by [Fe(CN)(6)](4-) and KI, DNA competitive binding with ethidium bromide, viscosity measurements, and DNA melting experiments. The results suggest that 1, 2, and 3 complexes bind to CT-DNA through intercalation and follows the order 1 > 2 > 3. Under irradiation at 365 nm, the three complexes have also been found to promote the photocleavage of plasmid pBR322 DNA. PMID:22194001

  13. Jak1/STAT3 pathway mediates the inhibition of lipoxin A4 on TNF-α-induced DNA synthesis of glomerular mesangial cells in rats

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective: To examine whether lipoxin A4 (LXA4) has an inhibitory effect on tumor necrosis factor-α(TNF-α)-induced DNA synthesis of glomerular mesangial cells of rat, and explore the molecular mechanisms of LXA4 action. Methods: Glomerular mesangial cells of rat were cultured and preincubated with LXA4 at different concentrations, and then treated with TNF-α( 10 ng/ml). DNA synthesis was assessed by the incorporation of [3H]-thymidine in mesangial cells. Expression of cyclin E protein was determined by Western blotting analysis. Activities of signal transducers and activators of transcription-3 (STAT3) were analyzed by electrophoretic mobility shift assay (EMSA). Results: TNF-α-stimulated DNA synthesis of mesangial cells, upregulation of cyclin E protein and STAT3 activities were inhibited by LXA4 in a dose-dependent manner. Conclusion: TNF-α-induced DNA synthesis of mesangial cells can be inhibited by TXA4probably through the mechanism of Jak1/STAT3 pathway-dependent signal transduction.

  14. Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups.

    NARCIS (Netherlands)

    A.J.R. de Jonge; W. Vermeulen (Wim); W. Keijzer; J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1985-01-01

    textabstractThe UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of

  15. DNA Synthesis during Endomitosis Is Stimulated by Insulin via the PI3K/Akt and TOR Signaling Pathways in the Silk Gland Cells of Bombyx mori

    Directory of Open Access Journals (Sweden)

    Yaofeng Li

    2015-03-01

    Full Text Available Silk gland cells undergo multiple endomitotic cell cycles during silkworm larval ontogeny. Our previous study demonstrated that feeding is required for continued endomitosis in the silk gland cells of silkworm larvae. Furthermore, the insulin signaling pathway is closely related to nutritional signals. To investigate whether the insulin signaling pathway is involved in endomitosis in silk gland cells, in this study, we initially analyzed the effects of bovine insulin on DNA synthesis in endomitotic silk gland cells using 5-bromo-2'-deoxyuridine (BrdU labeling technology, and found that bovine insulin can stimulate DNA synthesis. Insulin signal transduction is mainly mediated via phosphoinositide 3-kinase (PI3K/Akt, the target of rapamycin (TOR and the extracellular signal-regulated kinase (ERK pathways in vertebrates. We ascertained that these three pathways are involved in DNA synthesis in endomitotic silk gland cells using specific inhibitors against each pathway. Moreover, we investigated whether these three pathways are involved in insulin-stimulated DNA synthesis in endomitotic silk gland cells, and found that the PI3K/Akt and TOR pathways, but not the ERK pathway, are involved in this process. These results provide an important theoretical foundation for the further investigations of the mechanism underlying efficient endomitosis in silk gland cells.

  16. 8-Methoxypsoralen DNA interstrand cross-linking of the ribosomal RNA genes in Tetrahymena thermophila. Distribution, repair and effect on rRNA synthesis

    DEFF Research Database (Denmark)

    Fengquin, X; Nielsen, Henrik; Zhen, W;

    1993-01-01

    between three domains (terminal spacer, transcribed region and central spacer) as defined by restriction enzyme analysis (BamHI and ClaI). It is furthermore shown that a dosage resulting in approximately one cross-link per rDNA molecule (21 kbp, two genes) is sufficient to block RNA synthesis. Finally...

  17. Design, synthesis, physicochemical studies, solvation, and DNA damage of quinoline-appended chalcone derivative: comprehensive spectroscopic approach toward drug discovery.

    Science.gov (United States)

    Kumar, Himank; Chattopadhyay, Anjan; Prasath, R; Devaraji, Vinod; Joshi, Ritika; Bhavana, P; Saini, Praveen; Ghosh, Sujit Kumar

    2014-07-01

    The present study epitomizes the design, synthesis, photophysics, solvation, and interaction with calf-thymus DNA of a potential antitumor, anticancer quinoline-appended chalcone derivative, (E)-3-(anthracen-10-yl)-1-(6,8-dibromo-2-methylquinolin-3-yl)prop-2-en-1-one (ADMQ) using steady state absorption and fluorescence spectroscopy, molecular modeling, molecular docking, Fourier-transform infrared spectroscopy (FTIR), molecular dynamics (MD) simulation, and gel electrophoresis studies. ADMQ shows an unusual photophysical behavior in a variety of solvents of different polarity. The dual emission has been observed along with the formation of twisted intramolecular charge transfer (TICT) excited state. The radiationless deactivation of the TICT state is found to be promoted strongly by hydrogen bonding. Quantum mechanical (DFT, TDDFT, and ZINDO-CI) calculations show that the ADMQ is sort of molecular rotor which undergoes intramolecular twist followed by a complete charge transfer in the optimized excited state. FTIR studies reveals that ADMQ undergoes important structural change from its native structure to a β-hydroxy keto form in water at physiological pH. The concentration-dependent DNA cleavage has been identified in agarose gel DNA electrophoresis experiment and has been further supported by MD simulation. ADMQ forms hydrogen bond with the deoxyribose sugar attached with the nucleobase adenine DA-17 (chain A) and result in significant structural changes which potentially cleave DNA double helix. The compound does not exhibit any deleterious effect or toxicity to the E. coli strain in cytotoxicity studies. The consolidated spectroscopic research described herein can provide enormous information to open up new avenues for designing and synthesizing chalcone derivatives with low systematic toxicity for medicinal chemistry research.

  18. 一种高保真单向DNA合成方法初步探索%A high-fidelity DNA unidirection synthesis method:preliminary exploration

    Institute of Scientific and Technical Information of China (English)

    吴逊; 陈惠鹏; 李玉霞; 李北平; 李炳娟; 白东梅; 张昕; 凌焱; 周围; 刘刚

    2014-01-01

    Objective To establish a simple but quick method to improve the high fidelity of the synthesis of DNA frag -ments.Methods High fidelity DNA unidirection synthesis method (HFUS) was presented and used that involved Phusion DNA polymerase, BsrDⅠrestriction enzymes and λexonuclease.Using the same system at different temperatures , HFUS method synthesized one positive single-strand DNA and several reverse single -stranded DNA one by one into the target DNA fragment.Results Two random sequences DNA fragments of 340 bp and 450 bp were synthesized using HFUS meth-od.Conclusion This article explores a new method for the synthesis of genes .Through the harvest of 450 bp DNA, HFUS may promise to be a new approach to the synthesis of DNA .%目的:建立一种高保真、简单快速的DNA片段合成方法。方法提出一种高保真DNA单向合成方法( high fidelity DNA unidirection synthesis method , HFUS),主要在Phusion DNA聚合酶、BsrDⅠ限制性内切酶和λ外切酶3种酶协同作用下,将一条正向DNA单链和数条反向DNA单链通过逐个顺序扩增的方式,最终合成出目的DNA片段。该方法采用37℃、50℃和72℃3个温度的快速转换实现片段扩增的分步有序化合成。结果通过HFUS法,初步合成出2条长度分别为340 bp、450 bp的DNA随机序列片段。结论该研究探索了一种新的合成DNA片段的方法,初步实现了长达450 bp DNA片段的快速合成,为DNA合成方法提供了新的思路。

  19. UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Kelley N. Newton

    2012-01-01

    Full Text Available UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structures in vitro and is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair.

  20. UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli.

    Science.gov (United States)

    Newton, Kelley N; Courcelle, Charmain T; Courcelle, Justin

    2012-01-01

    UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structures in vitro and is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair. PMID:23056919