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Sample records for ccc dna synthesis

  1. Complete Spectrum of CRISPR/Cas9-induced Mutations on HBV cccDNA.

    Science.gov (United States)

    Seeger, Christoph; Sohn, Ji A

    2016-08-01

    Hepatitis B virus (HBV) causes chronic infections that cannot yet be cured. The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in nondividing cells. Antiviral therapies with nucleoside analogues inhibit HBV DNA synthesis in capsids in the cytoplasm of infected hepatocytes, but do not destroy nuclear cccDNA. Because over 200 million people are still infected, a cure for chronic hepatitis B (CHB) has become one of the major challenges in antiviral therapy. As a first step toward the development of curative therapies, we previously demonstrated that the CRISPR/Cas9 system can be used to functionally inactivate cccDNA derived from infectious HBV. Moreover, some evidence suggests that certain cytokines might induce an APOBEC-mediated cascade leading to the destruction of cccDNA. In this report we investigated whether a combination of the two mechanisms could act synergistically to inactivate cccDNA. Using next generation sequencing (NGS), we determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by nonhomologous end joining (NHEJ). We found that over 90% of HBV DNA was cleaved by Cas9. In addition our results showed that editing of HBV DNA after Cas9 cleavage is at least 15,000 times more efficient that APOBEC-mediated cytosine deamination following treatment of infected cells with interferon alpha (IFNα). We also found that a previously used method to detect cytosine deaminated DNA, termed 3D-PCR, overestimates the amount and frequency of edited HBV DNA. Taken together, our results demonstrated that the CRISPR/Cas9 system is so far the best method to functionally inactivate HBV cccDNA and provide a cure for CHB. PMID:27203444

  2. Attacking hepatitis B virus cccDNA--The holy grail to hepatitis B cure.

    Science.gov (United States)

    Lucifora, Julie; Protzer, Ulrike

    2016-04-01

    HBV deposits a covalently closed circular DNA form, called cccDNA, in the nucleus of infected cells. As the central transcription template, the cccDNA minichromosome is a key intermediate in the HBV life cycle. Its location in the nucleus makes cccDNA a difficult target for antivirals and immune response, and therefore it is responsible for chronicity of HBV infection. While little is known about the mechanisms involved in cccDNA formation, current research is accumulating data on the mechanisms regulating transcription from cccDNA, and the first potential targeting approaches have been reported. This review will summarize our knowledge about cccDNA biology and the latest advances in cccDNA targeting strategies in order to finally achieve an HBV cure. PMID:27084036

  3. The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely

    OpenAIRE

    Xiaoyan Guo; Ping Chen; Xiaohu Hou; Wenjuan Xu; Dan Wang; Tian-yan Wang; Liping Zhang; Gang Zheng; Zhi-liang Gao; Cheng-Yi He; Boping Zhou; Zhi-Ying Chen

    2016-01-01

    HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C g...

  4. Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status.

    Science.gov (United States)

    Koumbi, Lemonica; Pollicino, Teresa; Raimondo, Giovanni; Stampoulis, Dimitrios; Khakoo, Salim; Karayiannis, Peter

    2016-07-15

    In chronic hepatitis B virus (HBV) infection, variants with mutations in the basal core promoter (BCP) and precore region predominate and associate with more severe disease forms. Studies on their effect on viral replication remain controversial. Increasing evidence shows that epigenetic modifications of cccDNA regulate HBV replication and disease outcome. Here we determined the transcription and viral replication efficiency of well-defined BCP and precore mutations and their effect on cccDNA epigenetic control. HBV monomers bearing BCP mutations A1762T/G1764A and A1762T/G1764A/C1766T, and precore mutations G1896A, G1899A and G1896A/G1899A, were transfected into HepG2 cells using a plasmid-free approach. Viral RNA transcripts were detected by Northern blot hybridization and RT PCR, DNA replicative intermediates by Southern blotting and RT PCR, and viral release was measured by ELISA. Acetylation of cccDNA-bound histones was assessed by Chromatin ImmunoPrecipitation (ChIP) assay and methylation of cccDNA by bisulfite sequencing. BCP mutations resulted in low viral release, mRNA transcription and pgRNA/cccDNA ratios that paralleled the acetylation of cccDNA-bound H4 histone and inversely correlated with the HDAC1 recruitment onto cccDNA. Independently of the mutations, cccDNA was a target for methylation, accompanied by the upregulation of DNMT1 expression and DNMT1 recruitment onto cccDNA. Our results suggest that BCP mutations decrease viral replication capacity possibly by modulating the acetylation and deacetylation of cccDNA-bound histones while precore mutations do not have a significant effect on viral replication. These data provide evidence that epigenetic factors contribute to the regulation of HBV viral replication. PMID:27132039

  5. The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.

    Science.gov (United States)

    Guo, Xiaoyan; Chen, Ping; Hou, Xiaohu; Xu, Wenjuan; Wang, Dan; Wang, Tian-Yan; Zhang, Liping; Zheng, Gang; Gao, Zhi-Liang; He, Cheng-Yi; Zhou, Boping; Chen, Zhi-Ying

    2016-01-01

    HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus. PMID:27174254

  6. The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely

    Science.gov (United States)

    Guo, Xiaoyan; Chen, Ping; Hou, Xiaohu; Xu, Wenjuan; Wang, Dan; Wang, Tian-yan; Zhang, Liping; Zheng, Gang; Gao, Zhi-liang; He, Cheng-Yi; Zhou, Boping; Chen, Zhi-Ying

    2016-01-01

    HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus. PMID:27174254

  7. Targeting hepatitis B virus cccDNA using CRISPR/Cas9.

    Science.gov (United States)

    Kennedy, Edward M; Kornepati, Anand V R; Cullen, Bryan R

    2015-11-01

    Despite the existence of an excellent prophylactic vaccine and the development of highly effective inhibitors of the viral polymerase, chronic hepatitis B virus (HBV) infection remains a major source of morbidity and mortality, especially in Africa and Asia. A significant problem is that, while polymerase inhibitors can effectively prevent the production of viral genomic DNA from pre-genomic RNA transcripts, they do not prevent the transcription and translation of viral mRNAs from the covalently closed circular DNA (cccDNA) templates present in the nuclei of infected cells. Moreover, because these cccDNAs are highly stable, chronic HBV infections are only very rarely cured by the use of polymerase inhibitors and these drugs clearly cannot entirely prevent the subsequent development of HBV-related morbidities such as cirrhosis and hepatocellular carcinoma. As a result, there has been considerable interest in the possibility of developing treatment approaches that directly target cccDNA for elimination. Here, we discuss recent publications that analyze the ability of the bacterial CRISPR/Cas DNA editing machinery to be repurposed as a tool for the specific cleavage and destruction of HBV cccDNAs in the nuclei of infected cells and consider which steps will be necessary to make CRISPR/Cas targeting of HBV DNA a clinically feasible approach to the treatment of chronic infections in humans. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B." PMID:26476375

  8. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli.

    Science.gov (United States)

    Kieser, T

    1984-07-01

    Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed. The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature. Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform. The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis. Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S. lividans protoplasts or competent E. coli cells in about 2 h. All steps of the procedure are explained in detail with information about the effects of changing parameters. This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids. PMID:6387733

  9. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

    Energy Technology Data Exchange (ETDEWEB)

    Reaiche-Miller, Georget Y.; Thorpe, Michael; Low, Huey Chi; Qiao, Qiao; Scougall, Catherine A. [School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005 (Australia); Mason, William S.; Litwin, Samuel [Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111 (United States); Jilbert, Allison R., E-mail: allison.jilbert@adelaide.edu.au [School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005 (Australia)

    2013-11-15

    Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10{sup 5}-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. - Highlights: • The hepatitis B virus nuclear template is covalently closed circular DNA (cccDNA). • cccDNA was studied during liver growth in duck hepatitis B virus infected ducks. • Virus DNA replication and new cccDNA synthesis were inhibited with Entecavir. • At least 49% of cccDNA appeared to survive hepatocyte mitosis. • Low level virus DNA synthesis may contribute to survival of cccDNA through mitosis.

  10. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

    International Nuclear Information System (INIS)

    Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 105-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. - Highlights: • The hepatitis B virus nuclear template is covalently closed circular DNA (cccDNA). • cccDNA was studied during liver growth in duck hepatitis B virus infected ducks. • Virus DNA replication and new cccDNA synthesis were inhibited with Entecavir. • At least 49% of cccDNA appeared to survive hepatocyte mitosis. • Low level virus DNA synthesis may contribute to survival of cccDNA through mitosis

  11. 乙型肝炎病毒与cccDNA在血液中的清除动力学比较研究%Comparative study on clearance dynamics of hepatitis B virion and cccDNA in blood

    Institute of Scientific and Technical Information of China (English)

    赵克开; 王青; 缪晓辉; 徐文胜

    2009-01-01

    Objective To confirm whether it is feasible to detect HBV cecDNA based on an analysis of its stability and clearance dynamics in the blood of ducks.Methods Twelve 1-week-old ducklings were randomized into group A and group B,and each duckling was intravenously injected with 1 milliliter of serum containing 1.2×109 copies of HBV virion(group A)or plasmid pBS HBV 3.6 Ⅱ(group B).At 0.25,2,4,6,8,12,24 and 32 h post-injection,serum HBV DNA levelin each duckling was quantified by Real-time fluorescent PCR.Results Both of HBV virion and plasmid pBS HBV 3.6Ⅱ could be detected from 0.25 h to 24 h after intravenous administration,and declined approximately by 1 log10 every 2 hours within 8 h and was more slowly cleared at 8 h.and became undetectable at 32 h.Both of them are characteristic of one-phase exponential decay-as indicated by the relative index of the one-phase exponential curve ranging from 0.9593 to 0.9976.The mean half-life of HBV virion and plasmid(cecDNA)was 4.536±0.769 h(range:3.5-5.4 h)and 4.5±0.8 h(range:3.9-6.1 h)respectively.No significant difference was observed in the half-life between HBV virion and plasmid/cccDNA(t=0.0523.P=0.9593).Conclusions HBV cceDNA is similar to HBV virion in its characteristics of clearance dynamics in blood.Thus it is feasible to detect serum HBV cccDNA.The clearance half-life of Hepatitis B virus in serum is not as long as previously believed at 1.0-1.2 d%目的 以HBV为对照观察HBV cccDNA在外周血中的血液清除动力学特征,探讨在外周血中检测cccDNA的可行性.方法 将12只1周龄上海麻鸭随机分为A、B两组,A组和B组每只鸭分别以1.2×109拷贝/ml的HBV携带者血清和含1.2×109拷贝/mi pBS HBV 3.6Ⅱ质粒(模拟cccDNA)的人血清1ml颈静脉注射.在注射后0.25、2、4、6、8、12、24和32 h,分别采静脉血进行HBVDNA定量检测.结果 在注射后0.25~24 h内血液中均可检测到HBV颗粒和质粒(cccDNA).在注射后0.25~8 h时

  12. Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells

    Institute of Scientific and Technical Information of China (English)

    Xiao-Min Xin; Gui-Qiu U; Ying-Yu Jin; Min Zhuang; Di Li

    2008-01-01

    AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression in HepG2.2.15 cells by combination of small interfering RNAs (siRNAs).METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells.At 48 h,72 h and 96 h after transfection,culture media were collected and cells were harvested for HBV replication assay.HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay (ELISA).Intracellular viral DNA and covalently closed circular DNA (cccDNA)were quantified by real-time polymerase chain reaction (PCR).HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR (RT-PCR).RESULTS: siRNAs showed marked anti-HBV effects.siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dosedependent manner.Furthermore,combination of siRNAs,compared with individual use of each siRNA,exerted a stronger inhibition on antigen expression and viral replication.More importantly,combination of siRNAs significantly suppressed HBV cccDNA amplification.CONCLUSION: Combination of siRNAs mediates a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells,especially on cccDNA amplification.

  13. Pulse time and agarose concentration affect the electrophoretic mobility of cccDNA during PFGE and FIGE [corrected].

    OpenAIRE

    Sobral, B W; Atherly, A G

    1989-01-01

    Circular DNAs have been shown to migrate in an unusual manner during field inversion gel electrophoresis (FIGE) and orthogonal field alternating gel electrophoresis (OFAGE). We studied the effect of varying pulse time and agarose concentration on the electrophoretic mobility of supercoiled (ccc) DNAs ranging from 2 kbp to 16 kbp during FIGE and contoured homogeneous electric fields (CHEF). Both supercoiled and linear molecules display a minimum mobility as a function of pulse time in a CHEF a...

  14. The effect of the plant growth retardants AMO-1618 and CCC on the synthesis of ribonucleic acids and proteins in triticale embryos during the initial phase of germination

    Directory of Open Access Journals (Sweden)

    Stanisław Weidner

    2014-02-01

    Full Text Available Triticale var. Grado caryopses were subjected to imbibition and germination in the presence of the growth retardants, AMO-1618 (2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride and CCC (2-chloroethyl-trimethylammonium chloride at the following concentrations, 3 x 10-4 M and 10-3 M. These compounds exerted a very strong inhibitory effect on the initiation of germination processes, growth of embryos and the germination capacity of the caryopses. At the concentration of 10-3 M, AMO-1618 showed an especially strong effect, lowering the germination capacity of the caryopses to about 50%. It was also shown that both retardants are decidedly more effective on the germination of whole, intact caryopses than on that of isolated embryos. During the very earliest hours of germination, these retardants already inhibited RNA synthesis. The participation of the polyribosome fraction in the total ribosome fraction of embryos in the control sample after 24 hrs of germination of caryopses equalled about 70%, while in the samples treated with CCC (10-3 M- about 57%, in the samples treated with AMO-1618 (10-3 M about 35%,. The inhibition of incorporation of 14C-amino acids into ribosomal proteins in the polyribosome fraction was in the case of CCC about 13%, while in the samples treated with AMO-1618, about 55%. In the monosome fraction (80S, the inhibition by CCC was about 23%, whereas in the samples treated with AMO-1618 it reached around 73%. From this data it is evident that the studied retardants have a significant influence on the synthesis of ribonucleic acids as well as on ribosome proteins. These results also suggest the existance of another mechanism, aside from that of inhibition of gibberellin biosynthesis, inhibiting the growth and development of cells. The high percentage of ribosome subunits in the samples treated with CCC, in comparison with controls and samples treated with AMO-1618, points to different mechanisms

  15. 甲泼尼龙和他克莫司在体外对HepG2.2.15细胞中乙型肝炎病毒cccDNA合成的影响%Effects of methylprednisolone and tacrolimus on cccDNA replication of hepatitis B virus in HepG 2.2.15 cells

    Institute of Scientific and Technical Information of China (English)

    沈中阳; 郑卫平; 宋红丽; 王建

    2009-01-01

    Objective To investigate the effect of methylprednisolone(MP) and tacrolimus (Tac) on hepatitis B virus(HBV)cccDNA in vitro.Methods The HepG 2.2.15 cells were the line of HBV genome permanently transfected to human liver cancer cells.MTT was used to determine the nontoxic concentrations of MP and Tac.HepG 2.2.15 cells were treated with safe concentrations of MP and Tac for 72 h. Intracellular HBV cccDNA and supernatant HBV DNA were analyzed by real time polymerase chain reaction(RT-PCR).Results MTT revealed that the nontoxic concentrations of MP and Tac were 0-250 ng/ml and 0-500 ng/ml,Respectively.After treatment with MP at the concentrations of 10,50,100 and 250 ng/ml,the HBV DNA replication levels were 5.7823±0.1 861,5.1337±0.1364,4.7865±0.0398 and 4.1468±0.1016 log_(10) copy/ml,respectively(P0.05).结论 在体外,MP能抑制HepG2.2.15细胞中HBV DNA的表达,同时可以减少细胞内HBV cccDNA的合成,该作用呈剂量依赖性;Tac无此作用.

  16. 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.

  17. 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

  18. 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

  19. CCC and the Fermi paradox

    CERN Document Server

    Gurzadyan, V G

    2016-01-01

    Within the scheme of conformal cyclic cosmology (CCC), information can be transmitted from aeon to aeon. Accordingly, the "Fermi paradox" and the SETI programme - of communication by remote civilizations - may be examined from a novel perspective: such information could, in principle, be encoded in the cosmic microwave background. The current empirical status of CCC is also discussed.

  20. CCC and the Fermi paradox

    Science.gov (United States)

    Gurzadyan, V. G.; Penrose, R.

    2016-01-01

    Within the scheme of conformal cyclic cosmology (CCC), information can be transmitted from aeon to aeon. Accordingly, the "Fermi paradox" and the SETI programme --of communication by remote civilizations-- may be examined from a novel perspective: such information could, in principle, be encoded in the cosmic microwave background. The current empirical status of CCC is also discussed.

  1. 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...

  2. 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

  3. Spontaneous unscheduled DNA synthesis in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Forell, B.; Myers, L.S. Jr.; Norman, A.

    1979-01-01

    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.

  4. 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...

  5. 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...

  6. Polymerase synthesis of new photocaged DNA

    Czech Academy of Sciences Publication Activity Database

    Vaníková, Zuzana; Hocek, Michal

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i, 2014 - (Hocek, M.), s. 392-393 ISBN 978-80-86241-50-0. - (Collection Symposium Series. 14). [Symposium on Chemistry of Nucleic Acid Components /16./. Český Krumlov (CZ), 08.06.2014-13.06.2014] R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : DNA * polymerase synthesis Subject RIV: CC - Organic Chemistry

  7. DNA synthesis in the nuclei of Pinus silvestris embryos during

    OpenAIRE

    P. Brodzki

    2015-01-01

    DNA synthesis starts earliest in the apical meristem of the shoot, and latest in the cotyledons. Mitoses appear simultaneously in the apical meristem and in the hypocotyl cortex. Synthesis continues in the mother cells of vascular elements and cotyledon parenchyma when mitosis ceases. In the cotyledons DNA synthesis is rather synchronous and leads to the elimination of 2 C nuclei.

  8. DNA synthesis in the nuclei of Pinus silvestris embryos during

    Directory of Open Access Journals (Sweden)

    P. Brodzki

    2015-05-01

    Full Text Available DNA synthesis starts earliest in the apical meristem of the shoot, and latest in the cotyledons. Mitoses appear simultaneously in the apical meristem and in the hypocotyl cortex. Synthesis continues in the mother cells of vascular elements and cotyledon parenchyma when mitosis ceases. In the cotyledons DNA synthesis is rather synchronous and leads to the elimination of 2 C nuclei.

  9. 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)

  10. 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.

  11. 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...

  12. 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...

  13. Fresh Water Cyanobacteria Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 as an Anticancer Drug Resource

    OpenAIRE

    Akanksha Srivastava; Ratnakar Tiwari; Vikas Srivastava; Tej Bali Singh; Ravi Kumar Asthana

    2015-01-01

    An increasing number of cancer patients worldwide, especially in third world countries, have raised concern to explore natural drug resources, such as the less explored fresh water filamentous cyanobacteria. Six strains of cyanobacteria (Phormidium sp. CCC727, Geitlerinema sp. CCC728, Arthrospira sp. CCC729, Phormidium sp. CCC731, Phormidium sp. CCC730, and Leptolyngbya sp. CCC732) were isolated (paddy fields and ponds in the Banaras Hindu University, campus) and five strains screened for ant...

  14. DNA polymerase activity and radiation-induced unscheduled synthesis of DNA at the nuclear matrix

    International Nuclear Information System (INIS)

    It is shown that both DNA polymerase α and β are involved in DNA synthesis at the nuclear matrix. DNA polymerase β is more firmly attached to the nuclear matrix of normal than of regenerating liver cells. In the nuclear matrix of UV- and gamma-irradiated cells of Zajdela hepatoma a higher level of hydroxyurea-resistant DNA synthesis has been observed in the initial 1.5-5 min of postradiation incubation if compared to that of total nuclear DNA. However 1-β-D-arabinofuranosylcytosine-resistant radiation-induced synthesis of DNA is similar in both the nuclear matrix and the whole nuclei of these cells. Poly(ADP-ribose)synthetase activity is shown to be associated with the nuclear matrix. Inhibition of this activity results in increase of the hydroxyurea-resistant synthesis of DNA at nuclear matrix. (author)

  15. CCC/WPA study : Des Lacs NWR

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — Summary of the Civilian Conservation Corps (CCC) camp at Des Lacs National Wildlife Refuge from July 1935-May 1942 to carry on restoration and development of Des...

  16. 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 that...

  17. Amiloride inhibits rat mucosal ornithine decarboxylase activity and DNA synthesis

    International Nuclear Information System (INIS)

    Refeeding fasted rats induces a dramatic trophic response in gastrointestinal mucosa and is associated with elevations in both rate of DNA synthesis and ornithine decarboxylase (ODC) activity. The signal for these increases is unknown. Amiloride prevents cell alkalinization by blocking Na+-H+ exchange at apical epithelial cell membranes. In study 1, rats were fasted 48 h, treated with amiloride (0.5 to 500 mg/kg), and refed for 4 h. Refeeding increased ODC activities in the jejunal mucosa (X8) and liver (X19) but not in the oxyntic gland mucosa. In the jejunum, but not the liver, the activation of ODC was completely abolished by 100 mg/kg amiloride. In study 2, the rate of DNA synthesis was determine by measuring the rate of [3H]thymidine incorporation 16 h after refeeding. Refeeding resulted in significantly increased rates of DNA synthesis over fasted levels, and amiloride at 100 mg/kg significantly reduced the elevations in the jejenum and liver. In conclusion, amiloride inhibits the postprandial increases in jejunal ODC activity and DNA synthesis in the jejunum and liver. The results indicate that (1) the Na+-H+ antiport is essential to the increased ODC activity in the jejunum and liver after a meal and (2) increases in DNA synthesis and their suppression by amiloride are not necessary linked to ODC activity

  18. 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. PMID:26530671

  19. 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

  20. 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.

  1. 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)

  2. DNA-membrane complex restoration in Micrococcus radiodurans after X-irradiation: relation to repair, DNA synthesis and DNA degradation

    International Nuclear Information System (INIS)

    The DNA-membrane complex in Micrococcus radiodurans was shown to be essentially constituted of proteins, lipids and DNA. The complex was dissociated immediately after X-irradiation of cells and restored during post-incubation in complete medium. In X-irradiated protoplasts some DNA remained associated with the complex. Restoration of the complex during post-incubation was only seen in a medium favouring DNA polymerase and ligase activities. Under this condition no DNA synthesis occurred, suggesting that complex restoration may involve ligase activity. The complex restoration in the wild type and the X-ray sensitive mutant UV17 of M. radiodurans was strictly dependent on the X-ray dose. It was correlated with survival and DNA degradation but always preceded the onset of DNA synthesis after X-irradiation. At the same dose the complex restoration was about 2 fold lower in mutant than in wild type cells indicating that the restoration of the complex is related to repair capacity. The results are consistent with the idea that the complex protects X-irradiated DNA of M. radiodurans from further breakdown and, subsequently, permits DNA synthesis and repair to occur. (author)

  3. Synthesis of Palladium Conductive DNA-based Nanowires

    Science.gov (United States)

    Nguyen, Khoa; Streiff, Stéphane; Lyonnais, Sébastien; Goux-Capes, Laurence; Filoramo, Arianna; Goffman, Marcelo; Bourgoin, Jean Philippe

    2006-09-01

    We present here a simple method to metallize DNA by Electroless Plating of palladium, a trusty metal for contacting SWNT devices. Indeed, DNA is a promising scaffolding candidate for molecular electronic bottom-up self-assembly approaches of SWNT devices. We report in this work the synthesis and characterization of individual Pd nanowires as thin as 30 nm showing ohmic behavior at room temperature.

  4. 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)

  5. 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

  6. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    International Nuclear Information System (INIS)

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling

  7. 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

  8. 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...

  9. Design and synthesis of DNA four-helix bundles

    Energy Technology Data Exchange (ETDEWEB)

    Rangnekar, Abhijit; Gothelf, Kurt V [Department of Chemistry, Centre for DNA Nanotechnology (CDNA) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C (Denmark); LaBean, Thomas H, E-mail: kvg@chem.au.dk, E-mail: thl@cs.duke.edu [Department of Chemistry, Duke University, Durham, NC 27708 (United States)

    2011-06-10

    The field of DNA nanotechnology has evolved significantly in the past decade. Researchers have succeeded in synthesizing tile-based structures and using them to form periodic lattices in one, two and three dimensions. Origami-based structures have also been used to create nanoscale structures in two and three dimensions. Design and construction of DNA bundles with fixed circumference has added a new dimension to the field. Here we report the design and synthesis of a DNA four-helix bundle. It was found to be extremely rigid and stable. When several such bundles were assembled using appropriate sticky-ends, they formed micrometre-long filaments. However, when creation of two-dimensional sheet-like arrays of the four-helix bundles was attempted, nanoscale rings were observed instead. The exact reason behind the nanoring formation is yet to be ascertained, but it provides an exciting prospect for making programmable circular nanostructures using DNA.

  10. 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

  11. 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

  12. 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.

  13. 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)

  14. Synthesis and AFM visualization of DNA nanostructures

    International Nuclear Information System (INIS)

    We propose a novel bottom-up approach for the fabrication of various desired nanostructures, based on self-assembly of oligonucleotides governed by Watson-Crick base pairing. Using this approach, we designed Y-shaped, closed Y-shaped, H-shaped, and hexagonal structures with oligonucleotides. These structures were autonomously fabricated simply by mixing equimolar solutions of oligonucleotides and performing hybridization. After synthesis of the nanostructures, we confirmed their validity by agarose gel electrophoresis and atomic force microscope (AFM) visualization. We detected bands of the desired molecular sizes in the gel electrophoresis and observed the desired structures by AFM analysis. We concluded that the synthesized structures were consistent with our intended design and that AFM visualization is a very useful tool for the observation of nanostructures

  15. 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,...

  16. 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

  17. 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

  18. Association of DNA sequence variation in mitochondrial DNA polymerase with mitochondrial DNA synthesis and risk of oral cancer.

    Science.gov (United States)

    Datta, Sayantan; Ray, Anindita; Roy, Roshni; Roy, Bidyut

    2016-01-10

    Enzymes responsible for mitochondrial (mt) DNA synthesis and transcription are encoded by nuclear genome and inherited mutations in these genes may play important roles in enhancing risk of precancer and cancer. Here, genetic variations in 23 functionally relevant tagSNPs in 6 genes responsible for mtDNA synthesis and transcription were studied in 522 cancer and 241 precancer (i.e. leukoplakia) patients and 525 healthy controls using Illumina Golden Gate assay to explore association with risk of oral precancer and cancer. Two SNPs, rs41553913 at POLRMT and rs9905016 at POLG2, significantly increased risk of oral leukoplakia and cancer, respectively, at both genotypic and allelic levels. Gene-environment interaction models also revealed that tobacco habits and SNPs at POLG2 and TFAM may modulate risk of both leukoplakia and cancer. In silico analysis of published data-set also revealed that variant heterozygote (TC) significantly increased transcription of POLG2 compared to wild genotype (p=0.03). Cancer tissues having variant allele genotypes (TC+CC) at POLG2 contained 1.6 times (pcancer tissues having wild genotype (TT). In conclusion, polymorphisms at POLG2 and POLRMT increased risk of oral cancer and leukoplakia, respectively, probably modulating synthesis and activity of the enzymes. Enhanced synthesis of mtDNA in cancer tissues may have implication in carcinogenesis, but the mechanism is yet to be explored. PMID:26403317

  19. 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.)

  20. A computer simulation of the new Control Centre (CCC)

    CERN Multimedia

    2004-01-01

    In a development crucial for the success of the LHC, CERN will build a Control Centre (CCC) for the operation of all its beams and accelerators. The CCC will be an extension of the existing PCR building at Prévessin and is due to be operational by 1 February 2006.

  1. 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

  2. Radiosensitive Down syndrome lymphoblastoid lines have normal ionizing-radiation-induced inhibition of DNA synthesis

    International Nuclear Information System (INIS)

    The extent of X-ray-induced inhibition of DNA synthesis was determined in radiosensitive lymphoblastoid lines from 3 patients with Down syndrome and 3 patients with ataxia telangiectasia (AT). Compared to 6 normal control lines, the 3 AT lines were abnormally resistant to X-ray-induced inhibition of DNA synthesis, while the 3 Down syndrome lines had normal inhibition. These results demonstrate that radiosensitive human cells can have normal X-ray-induced inhibition of DNA synthesis and provide new evidence for the dissociation of radioresistant DNA synthesis. (author). 27 refs.; 1 fig.; 1 tab

  3. Analysis of plasmid DNA synthesis by double tracer method

    International Nuclear Information System (INIS)

    An Escherichia coli strain, CR34, harboring both pSC101 and ColEl-amp plasmids was exposed to media containing rifampicin (100 μg/ml) and/or chloramphenicol (180 μg/ml) and the cells were labeled for 20 min with 3H-thymine at 3,25 and 50 min after exposure to drug(s). The plasmid DNA synthesis was assayed by DNA-DNA hybridization with 14C-labeled pSC134 DNA as internal marker. In the presence of rifampicin, the replication of pSC 101 was from 57 to 104% that in its absence, and that of ColEl-amp was from 17 to 26%. The DNA replication of pSC101 after addition of chloramphenicol was reduced to 35 to 75%, and that of ColEl-amp was reduced to 39% and then restored to 92%. This restoration was not observed in the presence of rifampicin. (author)

  4. 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.

  5. 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

  6. Co-opting the Fanconi Anemia Genomic Stability Pathway Enables Herpesvirus DNA Synthesis and Productive Growth

    OpenAIRE

    Karttunen, Heidi; Savas, Jeffrey N; McKinney, Caleb; Chen, Yu-Hung; Yates, John R.; Hukkanen, Veijo; Huang, Tony T.; Mohr, Ian

    2014-01-01

    DNA damage associated with viral DNA synthesis can result in double strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi Anemia (FA) genomic stability pathway is exploited by HSV1 to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV1-infected cells resulted in monoubiquitination of FA effector proteins, FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized...

  7. 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.

  8. DNA (DEOXYRIBONUCLEIC ACID) SYNTHESIS FOLLOWING MICROINJECTION OF HETEROLOGOUS SPERM AND SOMATIC CELL NUCLEI INTO HAMSTER OOCYTES

    Science.gov (United States)

    The authors have investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in 3H-thymidine after being parthenogenetically activated by sha...

  9. 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

  10. Radioresistant DNA synthesis in fibroblasts of a patient with Down's syndrome

    International Nuclear Information System (INIS)

    Ionizing radiation effect on DNA replication on fibroblasts of a healthy donor and a patient with Down's syndrome either by direct 3H-thymidine inclusion into DNA, or by analysis of the sizes of daughter DNA moleculs at the state of stable distribution in acid saccharose, gradients was studied. Gamma-radiation doses (5-10 Gy) suppressing DNA synthesis in normal fibroblasts practically had no effect on DNA synthesisin fibroblasts of a patient with Down's syndrome. Radioresistant DNA synthesis in Down's syndrome is conditioned by a far less supression of replicon initiation as compared with the one in normal cells. So, it is stated that in Down's disease there is no delay in DNA synthesis by ionizing radiation that enables the normal cells to repair DNA damages before replication renewal

  11. 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.

  12. 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.

  13. 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.

  14. Ωccc production in high energy nuclear collisions

    Directory of Open Access Journals (Sweden)

    Hang He

    2015-06-01

    Full Text Available We investigate the production of Ωccc baryon in high energy nuclear collisions via quark coalescence mechanism. The wave function of Ωccc is solved from the Schrödinger equation for the bound state of three charm quarks by using the hyperspherical method. The production cross section of Ωccc per binary collision in a central Pb+Pb collision at sNN=2.76 TeV reaches 9 nb, which is at least two orders of magnitude larger than that in a p+p collision at the same energy. Therefore, it is most probable to discover Ωccc in heavy ion collisions at LHC, and the observation will be a clear signature of the quark–gluon plasma formation.

  15. 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)

  16. Automation of cDNA Synthesis and Labelling Improves Reproducibility

    Directory of Open Access Journals (Sweden)

    Daniel Klevebring

    2009-01-01

    Full Text Available Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

  17. Study of stimulators of DNA synthesis in nerve tissue cells

    International Nuclear Information System (INIS)

    Changes in proliferative activity in different regions of the brain during ontogenesis are connected with changes in the composition and properties of regulators of cell proliferation. Extracts of regions of the brain in which active cell division takes place in a given stage of development (cortex of 15- to 17-day-old embryos or cerebellum of 8- to 10-day-old rats) can stimulate the incorporation of labeled precursors into the brain cell DNA of both newborn and adult rats. Salting out at increasing ammonium sulfate concentrations, gel filtration on Sephadex, and isoelectric focusing led to the isolation of three fractions of stimulators of DNA synthesis: in acid, neutral, and alkaline pH regions. A method is described for obtaining purified preparations and for determining some physicochemical properties of the acid activator, which is a low-molecular-weight peptide capable of noticeably stimulating the incorporation of labeled precursors into the DNA of nerve tissue cells when added to an in vitro system in a concentration of the order of 1 μg/ml

  18. 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)

  19. 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.)

  20. 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

  1. The effect of bleomycin on DNA synthesis in ataxia telangiectasia lymphoid cells

    International Nuclear Information System (INIS)

    Bleomycin, a radiomimetic glycopeptide, inhibits de novo DNA synthesis in ataxia telangiectasia lymphoblastoid B cells to a markedly lesser extent than in normal and xeroderma pigmentosum lymphoid cells. This observation is similar to that following ionizing radiation; however, the effect is slower following the chemical treatment. Recovery of the normal cells occurs 15-18 hours after treatment, whereas the ataxia telangiectasia lines do not attain normal levels of DNA synthesis during the entire 24-hour observation period. Similar differences were not observed following treatment with mitomycin C, a bifunctional alkylating agent, indicating a specific effect of bleomycin on DNA synthesis in ataxia telangiectasia cells. Following bleomycin treatment and preincubation with hydroxyurea, residual DNA synthesis in ataxia telangiectasia cells was similar to that in both normal and xeroderma pigmentosum lymphoid lines, suggesting that the capacity to repair the induced DNA lesion is present

  2. In vivo measurement of DNA synthesis rates of colon epithelial cells in carcinogenesis

    International Nuclear Information System (INIS)

    We describe here a highly sensitive technique for measuring DNA synthesis rates of colon epithelial cells in vivo. Male SD rats were given 2H2O (heavy water). Colon epithelial cells were isolated, DNA was extracted, hydrolyzed to deoxyribonucleosides, and the deuterium enrichment of the deoxyribose moiety was determined by gas chromatographic/mass spectrometry. Turnover time of colon crypts and the time for migration of cells from basal to top fraction of the crypts were measured. These data were consistent with cell cycle analysis and bromodeoxyuridine labeling. By giving different concentrations of a promoter, dose-dependent increases in DNA synthesis rates were detected, demonstrating the sensitivity of the method. Administration of a carcinogen increased DNA synthesis rates cell proliferation in all fractions of the crypt. In conclusion, DNA synthesis rates of colon epithelial cells can be measured directly in vivo using stable-isotope labeling. Potential applications in humans include use as a biomarker for cancer chemoprevention studies

  3. 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 in...... isolated peripheral lymphocytes from the patients by measuring the rejoining of strand breaks following alkylation damage to the lymphocytes or by measuring unscheduled DNA synthesis. Day-to-day variability in the assays was considerable, but findings were that 5 out of 7 SL patients had repair...... deficiencies as measured by their ability to rejoin strand breaks, and 5 out of 7 had increased unscheduled DNA synthesis compared to treated and normal controls. All patients with SL and 4 out of 8 treated controls had inherent strand breaks in their DNA as compared to the normal controls when measured by...

  4. Radiation-induced depression of DNA synthesis in cultured mammalian cells

    International Nuclear Information System (INIS)

    A 313-nm light source was constructed in order to study the mechanisms by which ultraviolet and ionizing radiations inhibit DNA synthesis. It was found that in CHO, MDBK and HeLa cells, grown for one generation in the DNA sensitizer bromodeoxyuridine (BrdUrd), 313-nm light inhibited DNA synthesis with a pattern similar to that of the effect of x-rays on normal cells. A biphasic dose response curve for inhibition of total synthesis was observed, with a sensitive component representing depression of initiation of new replicons and a resistant component representing interference with elongation of replicons already growing at the time of irradiation. Since the BrdUrd plus 313-nm light treatment produces DNA lesions similar to those produced by x-rays (base damage, strand breaks, crosslinks) these results suggest that the effect of x-rays on DNA synthesis is mediated by DNA damage. In experiments with synchronized cells, it was found that in cells in which about half the chromosomes had incorporated BrdUrd, 313-nm light inhibited replication of the BrdUrd-containing DNA, but had no effect on the replication of the unsubstituted DNA in the same cell. Thus the information that DNA is damaged appears to be propagated along the DNA molecule from the sites of damage to the replication initiation sites as some kind of conformational change, possibly a relaxation of superhelical tension. Target theory calculations suggest that a single DNA lesion prevents the initiation of several adjacent replicons

  5. 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...

  6. Ornithine decarboxylase as an early indicator of in vitro hepatocyte DNA synthesis

    International Nuclear Information System (INIS)

    The enzyme ornithine decarboxylase, one of the key enzymes involved in polyamine biosynthesis, catalyzes the decarboxylation of ornithine to give putrescine. The activity of this enzyme in an in vitro hepatocyte culture assay system was measured because it is known that ornithine decarboxylase levels increase in instances where active protein synthesis, DNA synthesis, and cell growth is initiated. A good correlation was found between ornithine decarboxylase activity and the rate of tritiated thymidine incorporation into hepatocyte DNA. The increase in enzyme activity precedes the incorporation of tritiated thymidine into DNA (enzyme activity increases 2-3 hr following stimulation of cell growth; whereas the tritiated thymidine uptake increases at about 14-18 hr). Experimental results obtained with this assay system, suggest that hepatocytes from the regenerating liver remnant, grown in vitro, secrete a factor(s) into the culture medium which stimulates DNA synthesis of normal hepatocytes. Use of the increase in ornithine decarboxylase activity in this hepatocyte monolayer culture system confirmed the observation made by several investigators: that the serum of rats which underwent partial hepatectomy contains a factor(s) which stimulates hepatocyte DNA synthesis in vitro. In conclusion, these results suggest that ornithine decarboxylase activity can be used as a sensitive, early indicator of the degree of stimulation of hepatocyte DNA synthesis and thus be of use in determining the effect of various trophic factors on hepatocyte DNA synthesis in vitro

  7. 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

  8. 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

  9. Unscheduled DNA synthesis induced by low energy N+ heavy ion of seed embryos in triticum aestivum

    International Nuclear Information System (INIS)

    Using the method of 3H-TdR incorporation, the authors studied the change of DNA synthesis during the early period of germination of wheat weeds irradiated by low energy N+ heavy ion. The results showed: 1) the low energy N+ heavy ion was also able to induce a unscheduled DNA synthesis (UDS) in seed embryos of wheat: 2) the peak of the UDS occurred at the 6 h after seeds soaking, being 4 h earlier than the peak of the normal DNA synthesis of seed embryos: 3) the rate of UDS was roughly in positive proportion to irradiation doses: 4) the level of the normal DNA synthesis decreased with the increase of the UDS in the wheat seed embryos irradiated

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

    International Nuclear Information System (INIS)

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

  11. 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. PMID:27019227

  12. 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

  13. piggyBac can bypass DNA synthesis during cut and paste transposition

    OpenAIRE

    Mitra, Rupak; Fain-Thornton, Jennifer; Craig, Nancy L.

    2008-01-01

    DNA synthesis is considered a defining feature in the movement of transposable elements. In determining the mechanism of piggyBac transposition, an insect transposon that is being increasingly used for genome manipulation in a variety of systems including mammalian cells, we have found that DNA synthesis can be avoided during piggyBac transposition, both at the donor site following transposon excision and at the insertion site following transposon integration. We demonstrate that piggyBac tra...

  14. Histone gene expression remains coupled to DNA synthesis during in vitro cellular senescence

    International Nuclear Information System (INIS)

    Despite a decrease in the extent to which confluent monolayers of late compared to early passage CF3 human diploid fibroblasts can be stimulated to proliferate, the time course of DNA synthesis onset is similar regardless of the in vitro age of the cells. A parallel and stoichiometric relationship is maintained between the rate of DNA synthesis and the cellular levels of histone mRNA independent of the age of the cell cultures. Furthermore, DNA synthesis and cellular histone mRNA levels decline in a coordinate manner after inhibition of DNA replication by hydroxyurea treatment. These results indicate that while the proliferative activity of human diploid fibroblasts decreases with passage in culture, those cells that retain the ability to proliferate continue to exhibit a tight coupling of DNA replication and histone gene expression

  15. 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

  16. DNA synthesis is initiated at two positions within the origin of replication of plasmid R1162.

    OpenAIRE

    Lin, L S; Meyer, R J

    1987-01-01

    DNA synthesis of broad host-range plasmid R1162 is initiated from two positions, flanking a large (40 bp stem, 40 bp loop) inverted repeat. Each start-point is located within a highly conserved, but oppositely oriented, 10 base-pair sequence. Synthesis from the two positions converges within the intervening inverted repeat. An analysis of deletions suggests that both start positions must be present for synthesis. A model describing possible early events in replication of plasmid R1162 is pres...

  17. DNA replication initiation, doubling of rate of phospholipid synthesis, and cell division in Escherichia coli.

    OpenAIRE

    Joseleau-Petit, D; Képès, F; Peutat, L; D'Ari, R; Képès, A

    1987-01-01

    In synchronized culture of Escherichia coli, the specific arrest of phospholipid synthesis (brought about by glycerol starvation in an appropriate mutant) did not affect the rate of ongoing DNA synthesis but prevented the initiation of new rounds. The initiation block did not depend on cell age at the time of glycerol removal, which could be before, during, or after the doubling in the rate of phospholipid synthesis (DROPS) and as little as 10 min before the expected initiation. We conclude t...

  18. 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

  19. Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [3H]DNA in alkaline sucrose gradients or by direct assay of the amount of [3H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations. (orig.)

  20. 76 FR 44836 - CCC Export Credit Guarantee (GSM-102) Program

    Science.gov (United States)

    2011-07-27

    ... regulatory requirements and U.S. anti-money laundering and terrorist financing statutes. The purpose of these... notice of proposed rulemaking (ANPR) in the Federal Register (73 FR 76568). This notice was intended to... this timeframe CCC hopes to mitigate the impact of any defaults, as the primary purpose of the NOD...

  1. 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.

  2. CCC Method: The Rules Of Professionals As A Building Certifier

    Directory of Open Access Journals (Sweden)

    Zakaria R.

    2014-01-01

    Full Text Available In order developers handing over a building to the client, the building must comply with the various rules and procedures set by the respective local authorities. Before submission for CCC can be carried out by the developer, it is necessary to get a certificate of completion for the building construction. For this purpose, a more effective system introduced by the Malaysian government namely the Certificate Completion and Compliance (CCC. This system used respective professional services to recommend the issuance of building certificates for the building to be occupied. Under this approach, professional architects and engineer have been identified by government as a profession who is responsible for overseeing the entire project’s development and building that resulting in the issuance of CCC. Appointed architect or engineer called the Principle Submitting Person (PSP.This research uses qualitative method involving seven local authorities as respondents based on the “Snowball Sampling”. This approach asked the last respondents’ opinions on who are the next respondent that are appropriate for interview. The selection of respondents in a row expected to acquire direct information for the subject issue. PBT of Selangor and Malacca only (developed states was selected and all types of buildings involved in this study. Reliability and normality tests of the variables showed normal distribution and a high level of reliability (above 0.8. Research results obtained through interview revealed that all respondents agreed that the implementation of the new system CCC is rather effective than the old system. However, to achieved the CCC goal comprehensively the PSP appointed needs to maintain their work ethic and a best practice in ensuring the service provided could be able to satisfy all stakeholders.

  3. Effects of a dwarfing compound, CCC, on the production and export of gibberellin-like substances by root systems.

    Science.gov (United States)

    Reid, D M; Carr, D J

    1967-03-01

    (2-Chloroethyl)trimethylammonium chloride (Cycocel or CCC), an inhibitor of gibberellin biosynthesis, when repeatedly supplied to the root systems of balsam (Impatiens glandulifera Royle) plants reduces growth in height and the level of gibberellin-like substances in the bleeding sap that exudes from the stumps of detopped plants. Within twelve hours after a single application of the inhibitor to decapitated field peas (Pisum arvense), there are quantitative and qualitative changes in the gibberellins of the sap compared with those in sap collected over the same period of time from untreated plants. These changes are interpreted in terms of the possible blockage by CCC of normal gibberellin production and diversion of precursors into synthesis of "abnormal" gibberellins. PMID:24554364

  4. 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...

  5. How GSH level changes DNA-fragmentation of plasmid DNA and DNA Synthesis of tumor cells treated by Cu (TAAB)Cl2

    International Nuclear Information System (INIS)

    Rate of DNA synthesis of leukemic cell lines K562 and L1210 was monitored by incorporation of radiolabelled 3H thymidine to unsoluble TSA-cell fraction. Obtained results indicate that three days treatment with Cu(TAAB)Cl2 in various concentrations had no effect on inhibition of DNA synthesis. Changes of thiols concentrations in the presence of H2O2 and Cu(TAAB)Cl2 was determined. Oxidation of thiols in the dependence on Cu(II) ions or Cu(TAAB)Cl2 concentration was evaluated. Analysis of GSH products was performed using HPLC after pre-colon derivatization

  6. 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

  7. 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...

  8. Activities of a lagging DNA strand synthesis of nucleoprotein complexes harboring an extrachromosomal DNA closely related to avian myeloblastosis virus core-bound DNA

    International Nuclear Information System (INIS)

    Nucleoprotein (NP) complexes constituting the material of the post-microsomal sediment (POMS) and its three basic components (A, B, C), harboring an extrachromosomal DNA closely related to AMV DNA were found to.possess DNA- and RNA-synthesizing activities (SAs) reflecting the ability of this material to be intensely labelled for DNA and RNA, respectively. The types of these NA-SAs were compatible with those significant for a lagging DNA strand synthesis (LSS). The use of selective inhibitors and of the proliferating cell nuclear antigen (PCNA) disclosed a successive involvement of alpha DNA polymerase (pol) and PCNA-insensitive delta DNA pol in LSS. In this respect, we show gradual changes in the representation of activities (As) of both mentioned DNA pols in the NP complexes of the individual POMS components. Those of POMS component C contained alpha DNA pol As only, while a distinct portion of DNA SAs of POMS component B was represented on expense of alpha DNA pol As by PCNA-insensitive delta DNA pol (epsilon DNA pol), As which represented practically all the DNA SAs of POMS component A. The type of RNA SAs of this material represented mostly by primase (Pr) As corresponded well with the nature of LSS. An exception was represented by a minor portion of RNA-SAs of POMS component A which was alpha amanitine-sensitive like RNA pol II. Moreover, analyzing this natural model replication system, we found that the carbonyl diphosphonate (COMDP), a selective inhibitor of the PCNA-insensitive delta DNA pol, was a strong activator of Pr-As and/or Pr-alpha DNA pol As of NP complexes of POMS component C. (authors)

  9. The DNA intercalating alkaloid cryptolepine interferes with topoisomerase II and inhibits primarily DNA synthesis in B16 melanoma cells.

    Science.gov (United States)

    Bonjean, K; De Pauw-Gillet, M C; Defresne, M P; Colson, P; Houssier, C; Dassonneville, L; Bailly, C; Greimers, R; Wright, C; Quetin-Leclercq, J; Tits, M; Angenot, L

    1998-04-14

    Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated from the roots of Cryptolepis sanguinolenta. It is characterized by a multiplicity of host-mediated biological activities, including antibacterial, antiviral, and antimalarial properties. To date, the molecular basis for its diverse biological effects remains largely uncertain. Several lines of evidence strongly suggest that DNA might correspond to its principal cellular target. Consequently, we studied the strength and mode of binding to DNA of cryptolepine by means of absorption, fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to reveal that the alkaloid binds tightly to DNA and behaves as a typical intercalating agent. In DNAase I footprinting experiments it was found that the drug interacts preferentially with GC-rich sequences and discriminates against homo-oligomeric runs of A and T. This study has also led to the discovery that cryptolepine is a potent topoisomerase II inhibitor and a promising antitumor agent. It stabilizes topoisomerase II-DNA covalent complexes and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. Taking advantage of the fluorescence of the indoloquinoline chromophore, fluorescence microscopy was used to map cellular uptake of the drug. Cryptolepine easily crosses the cell membranes and accumulates selectively into the nuclei rather than in the cytoplasm of B16 melanoma cells. Quantitative analyses of DNA in cells after Feulgen reaction and image cytometry reveal that the drug blocks the cell cycle in G2/M phases. It is also shown that the alkaloid is more potent at inhibiting DNA synthesis rather than RNA and protein synthesis. Altogether, the results provide direct evidence that DNA is the primary target of cryptolepine and suggest that this alkaloid is a valid candidate for the development of tumor

  10. Ataxia-telangiectasia cell extracts confer radioresistant DNA synthesis on control cells

    International Nuclear Information System (INIS)

    We have investigated in greater detail the radioresistant DNA synthesis universally observed in cells from patients with ataxia-telangiectasia (A-T). The approach employed in this study was to permeabilize cells with lysolecithin after gamma-irradiation and thus facilitate the introduction of cell extract into these cells. This permeabilization can be reversed by diluting the cells in growth medium. Cells treated in this way show the characteristic inhibition (control cells) or lack of it (A-T cells) after exposure to ionizing radiation. Introduction of A-T cells extracts into control cells prevented the radiation-induced inhibition of DNA synthesis normally observed in these cells. A-T cell extracts did not change the level of radioresistant DNA synthesis in A-T cells. Control cell extracts on the other hand did not influence the pattern of inhibition of DNA synthesis in either cell type. It seems likely that the agent involved is a protein because of its heat lability and sensitivity to trypsin digestion. It has a molecular weight (MW) in the range 20-30 000 D. The development of this assay system for a factor conferring radioresistant DNA synthesis on control cells provides a means of purifying this factor, and ultimately an approach to identifying the gene responsible

  11. 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)

  12. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    International Nuclear Information System (INIS)

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of [3H]thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity

  13. 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)

  14. 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

  15. 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.

  16. 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.

  17. The influence of some prostaglandins on DNA synthesis and DNA excision repair in mouse spleen cells ''in vitro''

    International Nuclear Information System (INIS)

    ''In vitro'' experiments were performed on mouse spleen cells to establish possible influences of some naturally occurring prostaglandins on DNA synthesis and DNA excision repair. The prostaglandins A1, B1, E1, E2 and Fsub(2α) were tested in concentrations of 10 pg, 5 ng and 2,5μg per ml cell suspension. DNA synthesis was significantly increased by PgFsub(2α) in all the three concentrations tested, while the other tested prostaglandins were essentially ineffective. DNA excision repair was significantly inhibited by PgE1 and PgE2 at 5 ng/ml and at 2,5 μg/ml but increased by PgFsub(2α) in the two lower concentrations. The rejoining of DNA-strand breaks after gamma-irradiation was slightly reduced by PgE1, PgE2 and PgF2 at 2,5 μg/ml. (author)

  18. 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

  19. 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

  20. Relationship of avian retrovirus DNA synthesis to integration in vitro.

    OpenAIRE

    Lee, Y.M.; Coffin, J M

    1991-01-01

    An in vitro integration system derived from avian leukosis virus-infected cells supports both intra- and intermolecular integration of the viral DNA. In the absence of polyethylene glycol, intramolecular integration of viral DNA molecules into themselves (autointegration) was preferred. In the presence of polyethylene glycol, integration into an exogenously supplied DNA target was greatly promoted. Analysis of integration intermediates revealed that the strand transfer mechanisms of both reac...

  1. 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)

  2. Cyclin D1 repression of nuclear respiratory factor 1 integrates nuclear DNA synthesis and mitochondrial function

    OpenAIRE

    Wang, Chenguang; Li, Zhiping; Lu, Yinan; Du, Runlei; Katiyar, Sanjay; Yang, Jianguo; Fu, Maofu; Leader, Jennifer E.; Quong, Andrew; Novikoff, Phyllis M.; Pestell, Richard G

    2006-01-01

    Cyclin D1 promotes nuclear DNA synthesis through phosphorylation and inactivation of the pRb tumor suppressor. Herein, cyclin D1 deficiency increased mitochondrial size and activity that was rescued by cyclin D1 in a Cdk-dependent manner. Nuclear respiratory factor 1 (NRF-1), which induces nuclear-encoded mitochondrial genes, was repressed in expression and activity by cyclin D1. Cyclin D1-dependent kinase phosphorylates NRF-1 at S47. Cyclin D1 abundance thus coordinates nuclear DNA synthesis...

  3. 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)

  4. 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.

  5. 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...

  6. 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.

  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. 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

  10. Human Cytomegalovirus UL84 Insertion Mutant Defective for Viral DNA Synthesis and Growth

    OpenAIRE

    Xu, Yiyang; Cei, Sylvia A.; Huete, Alicia Rodriguez; Pari, Gregory S.

    2004-01-01

    Human cytomegalovirus (HCMV) UL84 is required for oriLyt-dependent DNA replication, and evidence from transient transfection assays suggests that UL84 directly participates in DNA synthesis. In addition, because of its apparent interaction with IE2, UL84 is implicated as a possible regulatory protein. To address the role of UL84 in the context of the viral genome, we generated a recombinant HCMV bacterial artificial chromosome (BAC) construct that did not express the UL84 gene product. This c...

  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. X-ray dose-effect relationship on unscheduled DNA synthesis and spontaneous unscheduled DNA synthesis in mouse brain cells studied in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Korr, H.; Koeser, K.; Oldenkott, S.; Schmidt, H.; Schultze, B.

    1989-01-01

    X-irradiation of the head of adult mice leads to DNA repair synthesis (unscheduled DNA synthesis, UDS) in non-proliferating cells of the brain as shown autoradiographically after injection of /sup 3/H-thymidine and subsequent irradiation. The extent of UDS induced by one and the same X-ray dose varies between different cell types and also between different brain areas. Within the range of X-ray doses studied (2 to 100 Gy) a linear dose effect relationship was observed. No evidence of a saturation effect was found. The slopes of the regression lines for the dose effect relationship differ considerably for the different cell types. Two interesting correlations were found, if the present results were compared with other data in the literature: (i) There seems to be a correlation between the extent of UDS and radiosensitivity of different cell types, the cells with the low DNA repair synthetic rates being more radiosensitive. (ii) The extent of UDS of the different cell types correlates well with the extent of protein synthesis of the corresponding cell types. Apart from radiation induced UDS, spontaneous UDS was found to occur in sham-irradiated animals. The extent of spontaneous UDS also differs considerably between different cell types as well as between different brain areas. The increase of spontaneous UDS with increasing duration of immobilization of the animals during sham irradiation suggests a relationship between spontaneous UDS and stress.

  13. 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.

  14. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    Science.gov (United States)

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. PMID:27053724

  15. 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

  16. 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...

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

    International Nuclear Information System (INIS)

    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

  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. Templated synthesis of DNA nanotubes with controlled, predetermined lengths.

    Science.gov (United States)

    Lo, Pik Kwan; Altvater, Florian; Sleiman, Hanadi F

    2010-08-01

    We report a DNA-templated approach to construct nanotubes with controlled lengths and narrow molecular weight distribution, allowing the deliberate variation of this length. This approach relies on the facile and modular assembly of a DNA guide strand of precise length that contains single-stranded gaps repeating at every 50 nm. This is followed by positioning triangular DNA "rungs" on each of these single-stranded gaps and adding identical linking strands to the two other sides of the triangles to close the DNA nanotubes. The length of the guide strand can be deliberately changed. We show the use of this approach to produce nanotubes with lengths of 1 microm or 500 nm and narrow length distributions. This is in contrast to nontemplated approaches, which lead to long and polydisperse nanotubes. We also demonstrate the encapsulation of 20 nm gold nanoparticles within these well-defined nanotubes to form finite lines of gold nanoparticles with longitudinal plasmon coupling, with a number of potential nanophotonic applications. This guiding strand approach is a useful tool in the creation of DNA nanostructures, in this case allowing the use of a simple template generated by a minimal number of DNA strands to program the length and molecular weight distribution of assemblies, as well as to organize any number of DNA-labeled nano-objects into finite structures. PMID:20662492

  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. 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...

  2. 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 the...

  3. NOREPINEPHRINE AND EPIDERMAL GROWTH FACTOR: DYNAMICS OF THEIR INTERACTION IN THE STIMULATION OF HEPATOCYTE DNA SYNTHESIS

    Science.gov (United States)

    Primary cultures of adult rat hepatocytes are stimulated to enter DNA synthesis by norepinephrine (NE). This stimulation is maximal if the hepatocytes are incubated with NE for more than 12 hr, beginning no later than 2-4 hr after the cells are first plated. After 24 hr in cultur...

  4. Effect of hypertonicity and X radiation on DNA synthesis in normal and ataxia-telangiectasia cells

    International Nuclear Information System (INIS)

    Normal human cells and cells from patients with ataxia-telangiectasia (A-T) were exposed to culture medium made hypertonic by raising the NaCl concentration. The rate of DNA synthesis in both types of cells was depressed as a function of increasing hypertonicity. When cells of both types were exposed to X radiation and incubated in hypertonic medium, DNA synthesis appeared to be more radioresistant than in cells incubated in normal medium. Velocity sedimentation analysis showed that this was due to a hypertonicity-induced inhibition of replicon initiation, which is the same process affected by X radiation, indicating that the two treatments were not additive. After a 5-hr incubation in hypertonic medium, there was a new steady state of replicon initiation and elongation similar to that existing in cells grown in normal medium, except that fewer replicons were participating. At this time DNA synthesis in each type of cell had a characteristic response to radiation, i.e., radiosenstivie in normal cells and radioresistant in A-T cells. These results suggest that radioresistant DNA synthesis in A-T cells is not due to increased condensation of chromatin

  5. 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.

  6. 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-...

  7. 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

  8. 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

  9. 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.

  10. El Centro de Cardioestimuladores del Uruguay. CCC Medical Devices

    OpenAIRE

    Pablo Darscht

    2011-01-01

    Estudio de caso del Centro de Cardioestimuladores del Uruguay - CCC Medical Devices preparado a solicitud de Ingenio en el marco del proyecto financiado por la Iniciativa para Incubadoras de InfoDev - Grupo Banco Mundial. Este estudio detalla los pasos seguidos por una empresa nacional con un fuerte factor de innovación y los cambios producidos en el entorno de los negocios de la empresa. El comienzo de una pequeña empresa de marcapasos que tras pasar por diferentes etapas hoy gana mercados e...

  11. Purification, characterization and biological activity of tulipin, a novel inhibitor of DNA synthesis of plant origin.

    Science.gov (United States)

    Gasperi-Campani, A; Lorenzoni, E; Abbondanza, A; Perocco, P; Falasca, A I

    1987-01-01

    A DNA synthesis-inhibiting protein (for which the term tulipin is proposed) was isolated from the bulbs of Tulipa sp. The yield ranged from 3.4 to 4.1 per cent of total protein content of the crude extract. Mr, isoelectric point, neutral and amino sugar and amino acid composition were determined. Inhibition of DNA synthesis varied in intact cells according to the cellular types studied, with a minimum ID 50% (concentration giving 50% inhibition) of 400 ng/ml in neuroblastoma cells. The effect was reversible. No effect was obtained in cell-lysate. RNA and protein synthesis were unaffected. The acute toxicity, evaluated in Swiss mice, gave an LD of 6.1 mg/kg body wt. Results of electron microscopy are also given. A second protein, called tulipin 2, has been isolated and partially characterized. PMID:3592627

  12. 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.

  13. Site Specific Synthesis and in-situ Immobilization of Fluorescent Silver Nanoclusters on DNA Nanoscaffolds Using Tollens Reaction

    Energy Technology Data Exchange (ETDEWEB)

    Pal, Suchetan [Arizona State Univ., Tempe, AZ (United States); Varghese, R. [Arizona State Univ., Tempe, AZ (United States); Deng, Z. [Arizona State Univ., Tempe, AZ (United States); Zhao, Z. [Arizona State Univ., Tempe, AZ (United States); Kumar, A. [Arizona State Univ., Tempe, AZ (United States); Yan, Hao [Arizona State Univ., Tempe, AZ (United States); Liu, Yan [Arizona State Univ., Tempe, AZ (United States)

    2011-04-06

    DNA strands with specific sequences and covalently attached sugar moieties were used for the site-specific incorporation of the sugar units on a DNA origami scaffold. This approach enabled the subsequent site-specific synthesis and in situ immobilization of fluorescent Ag clusters at predefined positions on the DNA nanoscaffold by treatment with the Tollens reagent.

  14. DNA (deoxyribonucleic acid) synthesis following microinjection of heterologous sperm and somatic cell nuclei into hamster oocytes

    International Nuclear Information System (INIS)

    The authors investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in 3H-thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. The authors conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is present

  15. 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

  16. DNA synthesis in toluene-treated bacteriophage-infected minicells of Bacillus subtilis

    International Nuclear Information System (INIS)

    Bateriophage (phi29, SPP1, or SP01)-infected, toluene-treated minicells of Bacillus subtilis are capable of limited amounts of non-replicative DNA synthesis as measured by incorporation of [3H]dTTP into a trichloroacetic acid-precipitable form. The [3H]dTTP is covalently incorporated into small DNA fragments which result from the degradation of a small percentage of the infecting phage genomes (molecular weights in the range of 2.105). Short exposure of the DNA molecules containing the incorporated [3H]dTMP to Escherichia coli exonuclease III results in over 90% of the [3H]dTMP being converted to a trichloroacetic acid-soluble form. The synthesis is totally dependent on host-cell enzymes and is not inhibited by the addition of chloramphenicol, rifampicin, nalidixic acid and mitomycin C and only slightly (approx. 20%) inhibited by the addition of 6-(p-hydroxyphenylazo)-uracil. (Auth.)

  17. 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.

  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. Action of cytochalasin D on DNA synthesis in cells in culture

    International Nuclear Information System (INIS)

    To solve the problem of the effect of changes in the actin cytoskeleton on DNA replication during the action of cytochalasins, the effect of long-term incubation of normal cells with cytochalasin D (CCD), which selectively destroys the microfilament system but does not affect transport of sugars, was investigated. Incorporation of labeled thymidine into mononuclear and binuclear cells in the presence of CCD and after its removal by rinsing also was studied separately. To investigate DNA synthesis the method of autoradiography with 3H-thymidine was used. A culture of mouse fibroblasts of the BALB/3T3 line and a secondary culture of fibroblasts obtained by trypsinization of mouse embryos (MEF) were used. On incubation of MEF and 3T3 cells, gradual inhibition of DNA synthesis is observed. The results obtained indicate that structural changes in the active cytoskeleton can abruptly and reversibly disturb passage of the normal cell through the cycle

  20. 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)

  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. PMID:26883285

  2. Synthesis, antioxidant and DNA cleavage activities of novel indole derivatives.

    Science.gov (United States)

    Biradar, J S; Sasidhar, B S; Parveen, R

    2010-09-01

    A new series of novel indole derivatives containing barbitone moiety (5a-i) are synthesized by simple and efficient condensation of chalcones (3a-i) with barbituric acid (4). The synthesized compounds are screened for their antioxidant (free radical scavenging, total antioxidant capacity and ferric reducing antioxidant power) and DNA cleavage activities were evaluated. Among the synthesized compounds (5a), (5d) and (5g) exhibited excellent antioxidant activity and all the tested compounds in the series have exhibited promising DNA cleavage activities. The structures of the synthesized compounds are assigned on the basis of elemental analysis, IR, (1)H NMR, (13)C NMR and mass spectral data. PMID:20594623

  3. Synthesis, crystal structures, photoluminescence properties and DNA binding of triazine-nickel(II) complexes for DNA detection.

    Science.gov (United States)

    Duan, Ran-Ran; Ou, Zhi-Bin; Wang, Wei; Chen, Shi; Zhou, Xiao-Hua

    2015-12-01

    We report here the synthesis of three new nickel(II) complexes: [Ni(PzTA)2CO3]·5H2O (PzTA=2,4-diamino-6-(2'-pyrazin)-1,3,5-triazine) in 1, [NiQ(PyTA)(H2O)2]Cl·H2O (HQ=8-hydroxyquinoline, PyTA=2,4-diamino-6-(2'-pyridyl)-1,3,5-triazine) in 2, [NiQ(PzTA)(H2O)2]Cl·H2O in 3, and they were characterized by UV spectroscopy, elemental analysis, molar conductivity and X-ray single crystal diffraction. Binding of the complexes to ct-DNA was investigated with electronic spectroscopy, ethidium bromide displacement from DNA, viscometry and cyclic voltammetry. The results depicted the DNA binding mode of the three complexes was intercalation, and complex 1 together with external static-electricity. Moreover, the three complexes also presented potential anti-oxidant activity. Interestingly, we found 1 was sensitive to oxygen and to the polarity of nonaqueous solvents in fluorescence spectroscopy. Fluorescence of 2 and 3 is weak in neutral aqueous solvents, but is greatly enhanced by addition of ct-DNA. Thus, 2 and 3 can be used to DNA detection as DNA fluorescence probes with a LOD of 1.61 ng mL(-1), 4.90 ng mL(-1) for the relative wide linear range of 0.01-20 μg mL(-1), 0.02-30 μg mL(-1), respectively. These findings indicate that 1 may be a potential optical probe for oxygen-free environments in nonaqueous form, while 2 and 3 were DNA-targeted probes. PMID:26125984

  4. Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA

    International Nuclear Information System (INIS)

    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, error-prone replication through template lesions. Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode

  5. Passive AC network supplying the integration of CCC-HVDC and VSC-HVDC systems

    OpenAIRE

    BIDADFAR, Ali; Abedi, Mehrdad; KARRARI, Mehdi

    2014-01-01

    The integration of a capacitor-commutated converter (CCC) high-voltage direct current (HVDC) (CCC-HVDC) and voltage source converter (VSC) HVDC (VSC-HVDC) is proposed in this paper to supply entirely passive AC networks. The key point of this integration is the flat characteristic of the DC voltage of the CCC-HVDC, which provides the condition for the VSC to connect to the CCC DC link via a current regulator. The advantages of the proposed combined infeeding system are the requirement o...

  6. Thermodynamic Impact of Abasic Sites on Simulated Translesion DNA Synthesis

    Czech Academy of Sciences Publication Activity Database

    Malina, Jaroslav; Brabec, Viktor

    2014-01-01

    Roč. 20, č. 25 (2014), s. 7566-7570. ISSN 0947-6539 R&D Projects: GA ČR(CZ) GAP205/11/0856 Institutional support: RVO:68081707 Keywords : abasic sites * differential scanning calorimetry * DNA Subject RIV: BO - Biophysics Impact factor: 5.731, year: 2014

  7. 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.)

  8. Synthesis of streptavidin-conjugated magnetic nanoparticles for DNA detection

    Energy Technology Data Exchange (ETDEWEB)

    Gong Peijun, E-mail: skygpj@zjnu.cn; Peng Zheyang; Wang Yao; Qiao Ru; Mao Weixing; Qian Haisheng; Zhang Mengya; Li Congcong; Shi Shenyuan [College of Chemistry and Life Sciences, Zhejiang Normal University (China)

    2013-04-15

    In this paper, we report a fabrication of streptavidin-coated magnetic nanoparticles used for DNA detection. Initially, amino-functionalized Fe{sub 3}O{sub 4} nanoparticles with high saturation magnetization are prepared by a photopolymerization method using allylamine as monomer. It is followed by covalent immobilization of streptavidin onto the particle surface via a two-step reaction using glutaraldehyde as coupling agent. Streptavidin-coated magnetic nanoparticles are characterized and further tested for their ability to capture DNA target after binding biotinylated oligonucleotide probes. The results show that the products ({approx}27.2 nm) have a maximum biotin-binding capacity of 0.71 nmol mg{sup -1} when the immobilization reaction is conducted with a mass ratio of streptavidin to magnetic carriers above 0.2 in phosphate buffered saline (pH 7.4) for 24 h. In addition, highly negative {zeta}-potential and good magnetic susceptibility of the nanocomposites make them applicable for DNA collection and detection, which is verified by the results from the preliminary application of streptavidin-coated magnetic nanoparticles in DNA detection. Therefore, the magnetic nanoparticles provide a promising approach for rapid collection and detection of gene.

  9. PCR synthesis of base-modified DNA templates for transcription

    Czech Academy of Sciences Publication Activity Database

    Raindlová, Veronika; Hocek, Michal

    Praha : Czech Chemical Society, 2015. s. 132. [Liblice 2015. Advances in Organic , Bioorganic and Pharmaceutical Chemistry /50./. 06.11.2015-08.11.2015, Olomouc] R&D Projects: GA ČR GA14-04289S Institutional support: RVO:61388963 Keywords : base-modified DNA * polymerase chain reaction * RNA polymerase Subject RIV: CC - Organic Chemistry

  10. 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.

  11. 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

  12. 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

  13. 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

  14. Recovery of subchromosomal DNA synthesis in synchronous V-79 Chinese hamster cells after ultraviolet light exposure

    International Nuclear Information System (INIS)

    Previous work obtained from Chinese hamster V-79 cells indicated that, immediately following exposure, UV-induced lesions acted as blocks to elongation of nascent strands, but gradually lost that ability over a 10 h period after exposure to 10 J/m2. The work reported herein attempted to examine possible cell cycle mediated alterations in the recovery of DNA synthesis. Kinetic incorporation of radiolabeled thymidine studies indicated that there may have been a more rapid recover of DNA synthesis in cells irradiated in G1 or G2 vs cells irradiated in S phase. DNA fiber autoradiograms prepared from synchronous cells indicated that after irradiation in any phase of the cell cycle, the length of newly synthesized DNA was equal to control lengths 1 h after exposure to 5.0Jm2 (or 1 h after entering S phase for cells irradiated in G1 or G2). This observed recovery was not solely due to an excision process. No cell cycle mediated difference in the number of dimers induced or removed as a function of cell cycle position was observed. These results appear to be consistent with a continuum of effects, with initiation effects dominating the response at low fluences, gapped synthesis at intermediate fluences and elongation inhibition at high fluences. The fluences at which each event dominates may be cell-line specific. (author)

  15. Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors

    International Nuclear Information System (INIS)

    Cytoplasmic extracts from spontaneously proliferating and mitogen-activated lymphoid cells contain a protein factor called ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. ADR-containing preparations have proteolytic activity, as indicated by their ability to degrade fibrin in a plasminogen-independent and plasminogen-dependent manner. In addition, aprotinin, a nonspecific protease inhibitor, abrogates ADR-induced DNA synthesis in a dose-dependent fashion. Preincubation studies demonstrated that the effect of aprotinin is not due to its suppressive effects on the nuclei themselves. Other protease inhibitors such as leupeptin, p-aminobenzamidine, and N-α-tosyllysine chloromethyl ketone are also inhibitory, but soybean trypsin inhibitor is without effect. ADR activity can be removed from active extracts by adsorption with aprotinin-conjugated agarose beads and can be recovered by elution with an acetate buffer (pH 5). These finding are consistent with the interpretation that the initiation of DNA synthesis in resting nuclei may be protease dependent and, further, that the cytoplasmic stimulatory factor the authors have called ADR may be a protease itself

  16. 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

  17. In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174.

    OpenAIRE

    Zipursky, S. L.; Reinberg, D.; Hurwitz, J

    1980-01-01

    The origin of phage phi X174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for phi X174 RFI DNA as template in phi X174-specific reactions in vitro. We have shown that the recombinant plasmids: (i) are cleaved by the phi X174 A protein; (ii) support net synthesis of unit-length single-stranded circular DNA in the presence of the phi X174 A protein and E...

  18. Histoautoradiographic and liquid scintillometric studies on DNA synthesis in the liver, kidneys, spleen and tongue after bilateral adrenalectomy in rats

    International Nuclear Information System (INIS)

    Historadiographies and liquid scintillometries were carried out in 163 male Wistar rats in order to determine the effects of bilateral adrenalectomy on DNA synthesis in the liver, kidneys, spleen, and tongue. Both DNA synthesis and mitotic index are significantly increased from the 1st day p.o. onwards, with broad synthesis peaks between the 2nd and the 4th day. The intensity of DNA synthesis shows a gradual decrease with increasing duration of the experiment. In contrast to the adrenalectonized animals, the synthesis rate and mitotic index in the organs of sham-operated animals were significantly lower, although enhanced proliferation was observed after surgery. The enhanced DNA synthesis after bilateral adrenalectomy is interpreted in terms of a disinhibition; corticosteroids are assumed to play a key role. The effects of bilateral adrenalectromy on untreated organs are not organ-specific. The highest synthesis rate was observed in the tubular epithelia of the convoluted main parts, while the DNA synthesis in the tongue. The findings of autoradiography and liquid scintillometry are well correlated. (orig./MG)

  19. Synthesis, characterization and chemoprotective activity of polyoxovanadates against DNA alkylation.

    Science.gov (United States)

    Nunes, Giovana G; Bonatto, Ana C; de Albuquerque, Carla G; Barison, Andersson; Ribeiro, Ronny R; Back, Davi F; Andrade, André Vitor C; de Sá, Eduardo L; Pedrosa, Fábio de O; Soares, Jaísa F; de Souza, Emanuel M

    2012-03-01

    The alkylation of pUC19 plasmid DNA has been employed as a model reaction for the first studies on chemoprotective action by a mixed-valence (+IV/+V) polyoxovanadate. A new, non-hydrothermal route for the high yield preparation of the test compound is described. The deep green, microcrystalline solid A was isolated after a three-day reaction in water at 80°C and 1 atm, while the reaction at 100°C gave green crystals of B. Both solids were structurally characterized by X-ray diffractometry and FTIR, EPR, NMR and Raman spectroscopies. Product A was identified as (NH(4))(2)V(3)O(8), while B corresponds to the spherical polyoxoanion [V(15)O(36)(Cl)](6-), isolated as the NMe(4)(+) salt. The lack of solubility of A in water and buffers prevented its use in DNA interaction studies, which were then carried out with B. Complex B was also tested for its ability to react with DNA alkylating agents by incubation with diethylsulphate (DES) and dimethylsulphate (DMS) in both the absence and presence of pUC19. For DMS, the best results were obtained with 10 mM of B (48% protection); with DES, this percentage increased to 70%. The direct reaction of B with increasing amounts of DMS in both buffered (PIPES 50 mM) and non-buffered aqueous solutions revealed the sequential formation of several vanadium(IV), vanadium(V) and mixed-valence aggregates of different nuclearities, whose relevance to the DNA-protecting activity is discussed. PMID:22265837

  20. 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).

  1. Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome.

    Science.gov (United States)

    Baranovskiy, Andrey G; Babayeva, Nigar D; Zhang, Yinbo; Gu, Jianyou; Suwa, Yoshiaki; Pavlov, Youri I; Tahirov, Tahir H

    2016-05-01

    The human primosome, a 340-kilodalton complex of primase and DNA polymerase α (Polα), synthesizes chimeric RNA-DNA primers to be extended by replicative DNA polymerases δ and ϵ. The intricate mechanism of concerted primer synthesis by two catalytic centers was an enigma for over three decades. Here we report the crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58C) with bound DNA/RNA duplex. These structures, along with analysis of primase/polymerase activities, provide a plausible mechanism for all transactions of the primosome including initiation, elongation, accurate counting of RNA primer length, primer transfer to Polα, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs. PMID:26975377

  2. 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.

  3. 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.

  4. 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.

  5. Self-assembled catalytic DNA nanostructures for synthesis of para-directed polyaniline.

    Science.gov (United States)

    Wang, Zhen-Gang; Zhan, Pengfei; Ding, Baoquan

    2013-02-26

    Templated synthesis has been considered as an efficient approach to produce polyaniline (PANI) nanostructures. The features of DNA molecules enable a DNA template to be an intriguing template for fabrication of emeraldine PANI. In this work, we assembled HRP-mimicking DNAzyme with different artificial DNA nanostructures, aiming to manipulate the molecular structures and morphologies of PANI nanostructures through the controlled DNA self-assembly. UV-vis absorption spectra were used to investigate the molecular structures of PANI and monitor kinetic growth of PANI. It was found that PANI was well-doped at neutral pH and the redox behaviors of the resultant PANI were dependent on the charge density of the template, which was controlled by the template configurations. CD spectra indicated that the PANI threaded tightly around the helical DNA backbone, resulting in the right handedness of PANI. These reveal the formation of the emeraldine form of PANI that was doped by the DNA. The morphologies of the resultant PANI were studied by AFM and SEM. It was concluded from the imaging and spectroscopic kinetic results that PANI grew preferably from the DNAzyme sites and then expanded over the template to form 1D PANI nanostructures. The strategy of the DNAzyme-DNA template assembly brings several advantages in the synthesis of para-coupling PANI, including the region-selective growth of PANI, facilitating the formation of a para-coupling structure and facile regulation. We believe this study contributes significantly to the fabrication of doped PANI nanopatterns with controlled complexity, and the development of DNA nanotechnology. PMID:23272944

  6. 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

  7. STRUCTURE-ACTIVITY STUDY OF PARACETAMOL ANALOGUES: INHIBITION OF REPLICATIVE DNA SYNTHESIS IN V79 CHINESE HAMSTER CELLS

    Science.gov (United States)

    Experimental and theoretical evidence pertaining to cytotoxic and genotoxic activity of paracetamol in biological systems was used to formulate a simple mechanistic hypothesis to explain the relative inhibition of replicative DNA synthesis by a series of 19 structurally similar p...

  8. 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)

  9. Sister chromatid exchanges in X-ray irradiated blood lymphocytes from patients with hereditary diseases with radioresistant DNA synthesis

    International Nuclear Information System (INIS)

    X-ray irradiation induced sister chromatid exchanges (SCE) in blood lymphocytes from patient with Down's syndrome and adult progeria (in both the cases radioresistant DNA synthesis takes place). In normal lymphocytes (in which ionizing radiation inhibits the replicative synthesis of DNA) the rate of SCE rises with the rise of radiation dose. Thus, the rate of SCE in X-ray irradiated lymphocytes is in reverse dependence with radioresistance of replicative synthesis of DNA. The data obtained are explained in accordance with the replicative hypothesis of the SCE nature (Painter, 1980a): in cells of patients with Down's syndrome, xeroderma pigmentosum from 2 and progeria of adults the time of existence of partly replicated clusters of replicons is decreased due to radioresistant replicative synthesis of DNA, but the presence of partly replicated clusters of replicons in necessary for SCE formation. Therefore the rate of SCF in X-irradiated cells of these patients decreases

  10. RRR-α-tocopheryl succinate inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

    OpenAIRE

    Wu, Kun; ZHAO Yan; Liu, Bai-He; Li, Yao; Liu, Fang; Guo, Jian; Yu, Wei-Ping

    2002-01-01

    AIM: To investigate the effects of growth inhibition of human gastric cancer SGC-7901 cell with RRR-α-tocopheryl succinate (VES), a derivative of natural Vitamin E, via inducing apoptosis and DNA synthesis arrest.

  11. CCC-based muon telescope for examination of natural caves

    Directory of Open Access Journals (Sweden)

    L. Oláh

    2012-12-01

    Full Text Available A portable cosmic muon detector has been developed for geophysical applications: searching for large scale underground rock/soil inhomogeneities and underground cavities. The designed muon telescope called a muon tomograph is based on the recently developed closed cathode chamber (CCC technology, which provides a cheap, easy handling, portable, and power efficient detector system able to work even in extreme conditions (e.g. high humidity, low/high temperature. The muon telescope has a detection surface of approximately 0.1 m2 with a 10 mrad angular resolution. Tests have been performed in natural caves and artificial tunnel systems as well. In this paper a summary of the first results on tomographic cavities are presented and the geophysical and possible industrial use of the cosmic muon tomographic technology is indicated.

  12. CCC-based muon telescope for examination of natural caves

    Directory of Open Access Journals (Sweden)

    L. Oláh

    2012-09-01

    Full Text Available A portable cosmic muon detector has been developed for geophysical application: searching for large scale underground rock/soil inhomogeneities and underground cavities. The designed muon telescope called Muontomograph is based on the recently developed Closed Cathode Chamber (CCC technology, which provides a cheap, easy handling, portable, and power efficient detector system, able to work even at extreme conditions (e.g. high humidity, low/high temperature. The muon telescope has about 0.1 m2 detection surface with 10 mrad angular resolution. Tests have been performed in natural caves and artificial tunnel systems as well. In the paper a summary of the first results on tomographed cavities are presented and the geophysical and possible industrial use of the cosmic muon tomograph technology is indicated.

  13. Proliferative activity, DNA synthesis and reproductive death of near and distant descendants of irradiated cells

    International Nuclear Information System (INIS)

    In experiments on HeLa cells a study was made of a change in the rate of DNA synthesis, proliferative activity and reproductive death of exposed cells and their descendants throughout a number of generations. The rate of DNA synthesis decreased in 4 postirradiation generations, and a maximum inhibition (by 50%) was registered 48 h following irradiation. The proliferative activity of the irradiated cell descendants markedly decreased throughout 18-20 generations resulting in an increased death rate and a loss of cells from a generation. It is suggested that even the distant desendants (18-20 generations) of expose cells exhibited some lesions which may, in time, become fatal events leading to cell death

  14. 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

  15. 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 ...

  16. 78 FR 79253 - CCC Export Credit Guarantee (GSM-102) Program and Facility Guarantee Program (FGP)

    Science.gov (United States)

    2013-12-27

    ... smaller U.S. exporters. Changes are also intended to improve CCC's financial management of the program... the Uniform Customs and Practice for Documentary Credits (UCP 600). CCC agrees that this term should... obligation must be documented using one of the methods described in Sec. 1493.90. System for Award...

  17. 7 CFR 1435.504 - Timing of distribution of CCC-owned sugar.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Timing of distribution of CCC-owned sugar. 1435.504... CORPORATION, DEPARTMENT OF AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS SUGAR PROGRAM Processor Sugar Payment-In-Kind (PIK) Program § 1435.504 Timing of distribution of CCC-owned sugar. Distribution of...

  18. 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

  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. 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

  4. Lethality and the depression on DNA synthesis in UV-irradiated normal human and xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    Ultraviolet radiation suppresses the semiconservative DNA replication in mammalian cells. The rate of DNA synthesis is initially depressed and later recovers after low doses of UV radiation in human cells. Such a response is more sensitive to UV radiation in cells derived from patients with xeroderma pigmentosum (XP) than that in normal human cells. The relative rate of DNA synthesis is not always correlated with cell survival because, unlike cell survival, the dose-response curve of the relative rate of DNA synthesis shows the biphasic nature of the sensitivity. In the experiments reported herein, the total amount (not the rate) of DNA synthesized during a long interval of incubation which covers the period of inhibition and recovery (but not longer than one generation time) after irradiation with various doses of UV radiation was examined in normal human and XP cells, and was found to be well correlated with cell survival in all the cells tested. (Auth.)

  5. Inhibition of hydrogenase synthesis by DNA gyrase inhibitors in Bradyrhizobium japonicum

    International Nuclear Information System (INIS)

    Derepression of an uptake hydrogenase in Bradyrhizobium japonicum is dependent on a microaerophilic environment. Addition of DNA gyrase inhibitors during derepression of hydrogenase specifically prevented expression of the hydrogenase enzyme. Antibodies to individual hydrogenase subunits failed to detect the protein after derepression in the presence of inhibitors, although there was no general inhibition of protein synthesis. The general pattern of proteins synthesized from 14C-labeled amino acids during derepression was no significantly different whether proteins were labeled in the presence or in the absence of gyrase inhibitors. In contrast, if transcription or translation was inhibited by addition of inhibitors of those functions, virtually no proteins were labeled during derepression. This indicated that most of the 14C-labeled proteins were synthesized de novo during derepression, synthesis of most proteins was unaffected by gyrase inhibitors, and the dependence of hydrogenase synthesis on gyrase activity was a specific one

  6. Inhibition of DNA synthesis and radiosensitization effects of thalidomide on esophageal carcinoma TE1 cells

    International Nuclear Information System (INIS)

    Objective: To explore the radiosensitization effect of thalidomide combined with X-ray on esophageal carcinoma TE1 cells. Methods: Cell scratch assay was used to detect the inhibition ability of different concentration of Thalidomide on cell invasion and metastasis. H3-TdR incorporation assay was used to investigate the inhibition of DNA synthesis in TE1 cells by treated with Thalidomide singly or combination with X-rays. The colony formation assay was used to analyze the radiosensitization of Thalidomide effect on TE1 cells. Results: Thalidomide had obvious inhibition effect on TE1 cell metastasis, DNA synthesis and colony formation, which were correlated with drug concentration. The values D0, Dq and SF2 in TE1 cells were gradually decreased with thalidomide concentration increased. When the concentration of thalidomide was 100μg/ml, the SERD0 and SERD0 and SERDq were (1.4±0.2) and (1.5±0.1), respectively, While the concentration of thalidomide was 150 μg/ml, the SERD0 and SERDq were (1.5±0.2) and (1.8±0.2), respectively. Conclusions: Thalidomide could inhibit TE1 cell invasion, metastasis, DNA synthesis, and significantly enhance the radiosensitizing effect on esophageal carcinoma TE1 cells. (authors)

  7. 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

  8. Stimulation of DNA synthesis in human epidermis by UVB radiation and its inhibition by difluoromethylornithine

    International Nuclear Information System (INIS)

    The purpose of this study was to determine whether the rate of DNA synthesis in human skin could be increased by UVB radiation and to determine the potential for reversing the stimulatory effects of UVB radiation by alpha-difluoromethylornithine (DFMO). Split-thickness facial skin was grafted onto athymic CD-1 Nu/Nu mice on the anterolateral dorsal surface. Following graft healing for 6 weeks, grafts were treated with 0%, 2%, or 5% DFMO (a potent inhibitor of polyamine biosynthesis) and subsequently irradiated with 0.15 J/cm2 of UVB light. Two days after UVB exposure, [3H]thymidine was injected and the grafts were dissected and counted. Ultraviolet radiation significantly increased thymidine incorporation, indicating increased DNA synthesis. The stimulatory effects of UV radiation were significantly reduced by topical application of 5% DFMO. Thus administration of DFMO most likely decreased the polyamine level and decreased the rate of DNA synthesis, which may have caused a decreased rate of epidermal proliferation. Thus the topical application of DFMO may prove beneficial for UVB exposure and other hyperproliferative states where a decrease in the rate of cell turnover might be desirable

  9. 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

  10. 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)

  11. 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

  12. 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.)

  13. Prostaglandin and DNA synthesis in human skin: possible relationship to ultraviolet light effects

    International Nuclear Information System (INIS)

    The effect of prostaglandin E2 (PGE2) on DNA synthesis in human skin was evaluated. PGE2 (1 μg) was injected intradermally into normal buttock skin of 15 volunteers followed by tritiated thymidine for autoradiographic quantitation of DNA synthesizing cells. Controls of normal saline, histamine (50 μg), and lower doses of PGE2 were also injected into 8 of the volunteers. Forty-eight hours after injection of 1 μg and 0.1 μg PGE2 there was a 264 percent and 62 percent increase, respectively, in the number of DNA synthesizing epidermal cells/high-power field as compared to saline controls. These differences were statistically significant (p is less than 0.01). Histamine (50 μg) produced a statistically significant 36 percent higher labeling index compared to its saline controls (p is less than 0.05). Many types of skin injury, including ultraviolet light (UVL) irradiation, produce an increase in the number of DNA synthesizing cells about 48 hr after the stimulus. Our findings suggest that PGE, a putative mediator of UVL-induced inflammation, may be one of the chemical mediators for the UVL-induced increase in DNA synthesizing cells. Histamine may also contribute to the increase in DNA synthesizing cells following UVL-induced inflammation

  14. 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

  15. A versatile biosensing system for DNA-related enzyme activity assay via the synthesis of silver nanoclusters using enzymatically-generated DNA as template.

    Science.gov (United States)

    Yuan, Yijia; Li, Wenhua; Liu, Zhuoliang; Nie, Zhou; Huang, Yan; Yao, Shouzhuo

    2014-11-15

    In the present day, oligonucleotide-encapsulated silver clusters (DNA-AgNCs) have been widely applied into bio-analysis as a signal producer. Herein, we developed a novel method to synthesize DNA-AgNCs encapsulated by long-chain cytosine (C)-rich DNA. Such DNA was polymerized in a template-free way by terminal deoxynucleotidyl transferase (TdT). We demonstrated that TdT-polymerized long chain C-rich DNA can serve as an excellent template for AgNCs synthesis. Based on this novel synthesis strategy, we developed a label-free and turn-on fluorescence assay to detect TdT activity with ultralow limit of detection (LOD) of 0.0318 U and ultrahigh signal to background (S/B) of 46.7. Furthermore, our proposed method was extended to a versatile biosensing strategy for turn-on nucleases activity assay based on the enzyme-activated TdT polymerization. Two nucleases, EcoRI and ExoIII as model of endonuclease and exonuclease, respectively, have been detected with high selectivity and competitive low LOD of 0.0629 U and 0.00867 U, respectively. Our work demonstrates the feasibility of TdT polymerization-based DNA-AgNCs synthesis strategy as a versatile and potent biosensing platform to detect the activity of DNA-related enzymes. PMID:24907540

  16. 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 (DMS......) and N-acetoxy-N-acetylaminofluorene in vitro. Also, the binding of DMS to DNA was measured. An increased DNA repair synthesis was measured in monocyte/B-lymphocytes after induction of the two different types of DNA lesions, whereas no induction of unscheduled DNA synthesis was observed in T-lymphocytes....... A significantly higher DMS-DNA binding was also observed in monocyte/B-lymphocytes when compared with T-lymphocytes. Specific characterization of mononuclear blood cell populations used in biomonitoring of DNA adducts and repair is recommended....

  17. The Necessary Maximality Principle for c.c.c. forcing is equiconsistent with a weakly compact cardinal

    OpenAIRE

    Hamkins, Joel David; Woodin, W. Hugh

    2004-01-01

    The Necessary Maximality Principle for c.c.c. forcing asserts that any statement about a real in a c.c.c. extension that could become true in a further c.c.c. extension and remain true in all subsequent c.c.c. extensions, is already true in the minimal extension containing the real. We show that this principle is equiconsistent with the existence of a weakly compact cardinal.

  18. 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.

  19. Specific inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha, 3-deoxyaphidicolin and aphidicolin-17-monoacetate.

    OpenAIRE

    Haraguchi, T; Oguro, M; Nagano, H; Ichihara, A; Sakamura, S

    1983-01-01

    Of several phytotoxins isolated from culture filtrates of Phoma betae Frank PS-13, an incitant of leaf spot disease of sugar beet, three have been identified as aphidicolin, 3-deoxyaphidicolin and aphidicolin-17-monoacetate. Aphidicolin is a selective inhibitor of eukaryotic DNA polymerase alpha (Ikegami et al. (1978) Nature 275, 458-460). Consequently, we studied the action mechanism of 3-deoxyaphidicolin and aphidicolin-17-monoacetate. These aphidicolin analogues markedly inhibited the in v...

  20. 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.

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

    International Nuclear Information System (INIS)

    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 [3H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [3H] 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, [3H] 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

  2. 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

  3. Recovery of DNA synthesis from inhibition by ultraviolet light in mammalian cells

    International Nuclear Information System (INIS)

    In general mammalian cells recover from DNA synthesis inhibition by ultraviolet light (u.v.) before most of the pyrimidine dimers have been removed from the genome. Using metabolic inhibitors, it has been shown that (1) even the low repair rate exhibited by V79 cells is important for recovery; although most of the dimers remain in the V79 genome after recovery of DNA synthesis, either the removal of lesions from some important region of chromatin or the activity of the repair process itself is important for the recovery; (2) the recovery mechanism is induced and depends on RNA synthesis and the production of specific factors. Finally, we have observed that cells previously treated with fluorodeoxyuridine become more resistant to inhibition by u.v. Since it has been shown that this drug activates unused origins of replication in Chinese hamster cells, reducing the average replicon size, we assume that the acquired resistance has to do with the operation of a larger number of small replicons. (author)

  4. 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)

  5. Temporal and topographic changes in DNA synthesis after induced follicular atresia

    International Nuclear Information System (INIS)

    Hamsters were hypophysectomized on the morning of estrus (Day 1) and injected immediately with 30 IU pregnant mare's serum (PMS). This was followed on Day 4 by the injection of an antiserum to PMS (PMS-AS) that initiated follicular atresia (Time zero). From 0 to 72 h after PMS-AS, the animals were injected with [3H]thymidine and killed 4 h later. One ovary was saved for autoradiography and histology; from the other ovary, 5-10 large antral follicles were dissected and pooled, and incorporation into DNA was determined by scintillation counting. DNA synthesis dropped sharply between 12 and 18 h, coinciding with a fall in labeling index of the cumulus oophorus and thecal endothelial cells and a sharp fall in thecal vascularity. In contrast, for the mural granulosa cells bordering on the antral cavity, labeling index dropped sharply between 8 and 12 h when thecal vascularity was still high. The earliest sign of atresia was evident by 4 h in cumulus cells when, paradoxically, DNA synthesis was still high. It took 3 days for atresia of the antral follicles to progress to advanced stages, as evidenced by pseudo-pronuclei in the free floating ovum, further erosion of the mural granulosa, and minimal DNA/follicle. However, the theca still retained its histological integrity and contained no pyknotic cells. Although by 48 h the granulosal compartment was in disarray (DNA/follicle significantly different from earlier values), the egg was still viable, as judged by maximal fluorescence after the addition of fluoroscein diacetate

  6. 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.

  7. 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

  8. Host DNA synthesis-suppressing factor in culture fluid of tissue cultures infected with measles virus

    International Nuclear Information System (INIS)

    Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated componentnent which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither uv-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus. (auth)

  9. Effect of cisplatin alone and in combination with γ-radiation on the initiation of DNA synthesis in Friend leukemia cells

    International Nuclear Information System (INIS)

    The effect of the anticancer drug cisplatin (alone and in combination with γ-radiation) on the initiation of DNA synthesis in Friend leukemia cells was studied. A method for isolation of DNA fractions containing the origins of replication was used. It was found that cisplatin decreased the rate of the initiation of DNA synthesis. The mild γ-radiation has previously been observed to inhibit the initiation of DNA synthesis. In the present investigation the combination of cisplatin and γ-radiation showed additive effects without synergism on the initiation of DNA biosynthesis. (orig.)

  10. 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

  11. 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.

  12. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

    International Nuclear Information System (INIS)

    Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of [3H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [3H]hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of [3H]thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone

  13. Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells.

    OpenAIRE

    Smith, J. B.

    1983-01-01

    Vanadyl sulfate and sodium orthovanadate in the concentration range between 5 and 50 microM are shown to be mitogenic for quiescent cultures of Swiss mouse 3T3 and 3T6 cells. The compounds caused a striking shift in the dose-response for the effect of serum on [3H]thymidine incorporation and DNA synthesis. In the absence of serum the effect of vanadium was greatly potentiated by insulin. Vanadium ions produced no more than additive increases in [3H]thymidine incorporation when combined with e...

  14. 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

  15. 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)

  16. Malheur National Wildlife Refuge: Narrative Report: 1936: CCC Camps: 2: November

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — This narrative report from the Civilian Conservation Corps summarizes CCC activies on Malheur Migratory Waterfowl Refuge. Topics include construction, land...

  17. Malheur National Wildlife Refuge: Narrative Report: 1936: CCC Camps: 1: October

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — This narrative report from the Civilian Conservation Corps summarizes CCC activies on Malheur Migratory Waterfowl Refuge. Topics include construction, land...

  18. Narrative report : June - 1939 - July : Camp BS02 Co.2749 CCC

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — This CCC narrative report for June and July of 1939 for Sand Lake Migratory Waterfowl Refuge outlines the number of hours spent working on various construction...

  19. 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.

  20. 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...

  1. 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)

  2. 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

  3. 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....

  4. Induction of unscheduled DNA synthesis in suspensions of rat hepatocytes by an environmental toxicant, 3,3'4,4'-tetrachloroazobenzene.

    Science.gov (United States)

    Hsia, M T; Kreamer, B L

    1979-04-01

    Unscheduled DNA synthesis was induced by 3,3'4,4'-tetrachloroazobenzene (TCAB)) in freshly isolated suspensions of rat hepatocytes. A dose-dependent response was demonstrated. Hepatocellular DNA was obtained after the chloroform-isoamyl alchohol-phenol extraction of the isolated nuclei. The induction of unscheduled DNA synthesis was measured by the incorporation of [3H]-thymidine in the presence of hydroxyurea as determined by the scintillation counting assay. DNA repair data obtained in this study on benzo[a]pyrene and methyl methanesulfonate are comparable to a previous report using primary cultures of hepatocytes and cesium chloride gradients. Hence, the present method offers promise as a rapid and sensitive screen for chemical carcinogens. PMID:436117

  5. 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

  6. 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

  7. 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.

  8. 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)

  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. Action of caffeine on x-irradiated HeLa cells. I. Delayed inhibition of DNA synthesis

    International Nuclear Information System (INIS)

    Treatment of HeLa S3 cells with 1 mM caffeine delays progression through G1 by 1.5 hours but causes no other detectable inhibition of cell progression; it sometimes results in a large stimulation of thymidine incorporation. When this concentration is applied to cells that have been irradiated with 1-krad doses of 220-kV x rays, there is a marked suppression of both the inhibition of DNA synthesis and G2 arrest induced by the radiation. Larger doses require higher concentrations of caffeine to suppress the inhibition of DNA synthesis. Delaying addition until the rate of synthesis is at its minimum (1.5 hours after irradiation with 1 krad) results in a slightly accelerated recovery of the rate. Treatment before or during irradiation is without effect on the inhibition. Removal of the caffeine as late as 6 hours after its addition at the time of irradiation results in a prompt inhibition in DNA synthesis that mimics that observed immediately after irradiation in the absence of caffeine. These findings raise the possibility that the depression in rate of DNA systhesis might not result from radiation damage introduced into the replicon initiation system, but rather may be an indirect consequence of damage residing elsewhere in the irradiated cell

  11. PROLINE IS REQUIRED FOR THE STIMULATION OF DNA SYNTHESIS IN HEPATOCYTE CULTURES BY EGF (EPIDERMAL GROWTH FACTOR)

    Science.gov (United States)

    Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis in rat parenchymal hepatocytes both in vivo and in vitro (4,9). The authors report here that this response in vitro is dependent on the amino acids present in the media. Of all the amino acids, proline has th...

  12. 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.

  13. 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

  14. 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

  15. 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)

  16. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: Evidence for differential gene expression

    International Nuclear Information System (INIS)

    The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. The authors have established a single-cycle growth condition for HIV in H9 cells, a human CD4+ lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes

  17. 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.

  18. Depression of DNA synthesis rate following hyperthermia, gamma irradiation, cyclotron neutrons and mixed modalities

    International Nuclear Information System (INIS)

    The incorporation of the thymidine analogue I-UdR is proportional to the activity of DNA synthesis. The maximum depression of 125-I-UdR incorporation occurs approximately 4 hours after all kinds of treatment. The increase which follow reflects cell processes like reoxygeneration, recovery, recycling and recruitment (although a direct relation is not yet demonstrable). The degree of depression 4 hours after treatment and the time required needs to reach control level is dependent on dose and radiation quaility but no such dependence could be clearly seen for the times of hyperthermia treatment we used. Neutron irradiation and the combination gamma irradiation + hyperthermia show a higher depression and a slower return to normal than gamma irradiation at the same dose. (orig.)

  19. 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.

  20. 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

  1. Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

    International Nuclear Information System (INIS)

    Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125I-insulin specific binding was not affected. The decrease in 125I-epidermal growth factor specific binding was explained by the decrease in the number or available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/105 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immuno-detection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125I-epidermal growth factor binding. (au)

  2. 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.

  3. 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

  4. 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

  5. Glucocorticoid suppression of human lymphocyte DNA synthesis. Influence of phytohemagglutinin concentration

    International Nuclear Information System (INIS)

    Glucocorticoids have been shown to suppress lectin-stimulated lymphocyte DNA synthesis in some studies, whereas in other studies, the hormones have had little effect. We have found that the position on the PHA dose-response curve that is studied is the most important determinant of whether cortisol inhibits 3H-thymidine incorporation into lymphocyte DNA. The proportion of monocytes in culture also influenced the cortisol effect, but it was quantitatively less important than PHA concentration. Cortisol (5 nM to 100 μM) had little effect on blastogenesis or thymidine incorporation into DNA in cultures that contained both a high concentration (14% +- 2 (S.E.)) of monocytes and a concentration of PHA (0.6 to 1.2 μg/ml) that produced maximal stimulation of mitogenesis. When monocytes were reduced from 14 to 1.4%, cortisol (5 μM) caused a 30% reduction in thymidine incorporation in cultures stimulated by 0.6 to 1.2 μg/ml PHA. Much greater cortisol suppression of thymidine incorporation occurred if the concentration of PHA was reduced. For example, reduction of the PHA concentration from 1.2 to 0.075 μg/ml resulted in an increase in suppression by 5 μM cortisol from 5 to 90% even in the presence of 14% monocytes. These data indicate that the suppressive effects of glucocorticoids on blastogenesis and thymidine incorporation in vitro depend principally on the concentration of PHA used to stimulate blastogenesis and secondarily on the proportion of monocytes in the culture system

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.)

  12. 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

  13. 78 FR 38286 - Notice of Change to the CCC Sugar Purchase and Exchange To Include Certificates of Quota...

    Science.gov (United States)

    2013-06-26

    ... Refined Sugar Re-Export Program (78 FR 36508- 36510). This notice announces CCC's intent to also purchase... achieve the greatest cost reduction relative to the costs of later acquiring the domestic sugar through... Commodity Credit Corporation Notice of Change to the CCC Sugar Purchase and Exchange To Include...

  14. Influence of some radioprotective and radiosensitizing compounds on the replicative and repair induced DNA synthesis of rats spleen cells in vitro

    International Nuclear Information System (INIS)

    The effect of cysteine, dithiothreitol, N-ethylmaleimide, cytosinearabinoside, ethidiumbromide, bleomycine and diethyldithiocarbamate on the replicative and repair induced DNA synthesis in vitro was tested by using rats spleen cells. Besides the incorporation of a labeled DNA precursor (TdR-3H) the sedimentation of DNA in sucrose gradients was inquired. With respect to the DNA synthesis an uniform mechanism of action for the radioprotective substances can't be seen. Thymocytes and spleen cells seem to possess different systems of repair; this may be an explanation for their different sensibility against ionizing radiation. (orig./MG)

  15. Modulation of radiation-induced mutation frequencies and DNA synthesis by deoxycytidine in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    The goal of this project is to elucidate and model the impact of perturbations in cellular metabolism on the cellular response to DNA damage. Previously reported experiments established roles for DNA synthesis and the inhibition of DNA synthesis in the expression of radiation-induced mutations. This year, these experiments were continued and the study was expanded. Two different Chinese hamster ovary (CHO) cell lines were examined. Experimental measurements were obtained for cells in one of two growth media: (1) medium A, which consisted of Ham's F-12 containing 10% fetal calf serum; or (2) medium B, which consisted of medium A containing 2 mm deoxycytidine (CdR). The presence of CdR differentially affected the nature of DNA replication, the inhibition of replication by radiation, and the corresponding mutation frequency responses. Results suggest that the effect of CdR on mutation-induction is not locus-specific. Moreover, the phenomenal reduction in the mutation response in the presence of CdR is also seen following exposure to ionizing radiation, which implies that the effect of CdR is not mutagen-specific. Time-dependent changes in profiles of pulse-labeled DNA on alkaline sucrose gradients were employed to measure and compare nascent DNA chain growth processes in the two media. Results suggest that a more rapid DNA chain elongation and/or replicon mutation rate occurs in cells in medium B. In contrast, the sedimentation of one pulse-labeled CHO cell line DNA is always greater when cells are grown in medium A. UV radiation appears to inhibit postirradiation replicon initiation events and results in reduced mutation frequencies when cells are grown in the medium that promotes the more rapid rate of polymerization

  16. Characterizing Frothers through Critical Coalescence Concentration (CCC95-Hydrophile-Lipophile Balance (HLB Relationship

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    2012-08-01

    Full Text Available Frothers are surfactants commonly used to reduce bubble size in mineral flotation. This paper describes a methodology to characterize frothers by relating impact on bubble size reduction represented by CCC (critical coalescence concentration to frother structure represented by HLB (hydrophile-lipophile balance. Thirty-six surfactants were tested from three frother families: Aliphatic Alcohols, Polypropylene Glycol Alkyl Ethers and Polypropylene Glycols, covering a range in alkyl groups (represented by n, the number of carbon atoms and number of Propylene Oxide groups (represented by m. The Sauter mean size (D32 was derived from bubble size distribution measured in a 0.8 m3 mechanical flotation cell. The D32 vs. concentration data were fitted to a 3-parameter model to determine CCC95, the concentration giving 95% reduction in bubble size compared to water only. It was shown that each family exhibits a unique CCC95-HLB relationship dependent on n and m. Empirical models were developed to predict CCC95 either from HLB or directly from n and m. Commercial frothers of known family were shown to fit the relationships. Use of the model to predict D32 is illustrated.

  17. CCC effect on the 14CO2 assimilation and the growth of some crops

    International Nuclear Information System (INIS)

    Pot experiments with white mustard (Sinapis alba L.), common buckwheat (Fagopyrum esculentum Mnch.) and spring wheat (Triticum vulgare L.) of the Ostka Popularna variety were carried out. The aim of the experiments was to determine the effect of foliar and root application of chlorocholine chloride (CCC) in the photosynthetic fixation of 14CO2 and on the growth of the above crops. (author)

  18. Effects of CCC on dynamic changes in phosphate nutrition and yield of potato

    International Nuclear Information System (INIS)

    Effects of chlorocholine chloride (CCC) (0,1.5,2.0 and 2.5g/L) sprayed at early growth period, then the absorption of phosphate nutrition and the average yield per hill of potato cv. Zhongshu 3 were studied with 32P tracer technology. The results were showed that CCC increased the phosphate absorption during the first several days after emergence, and the absorbed phosphate was mainly distributed in stems and leaves, and the relative concentration of phosphate in organs was stem > leaf > root > tuber in turn. The total amount of phosphate absorped by plants increased with the growth of plants, but decreased at the late growth period. The total amount of phosphate absorped by plants was the highest when sprayed 2.0g/L CCC at different days after emergence. The average yield per hill had improved by 6.83%, 10.01% and 3.08% after sprayed 1.5, 2.0 and 2.5g/L CCC, respectively, and was positively correlated at 1% level with the phosphate content of leaves at 36d after emergence. (authors)

  19. Effects of inhibitors of DNA, RNA, and protein synthesis on frequencies and types of premature chromosome condensation from x-ray induced micronuclei. [Cytosine arabinoside, azathioprine, thymidine, trenimon

    Energy Technology Data Exchange (ETDEWEB)

    Madle, S.; Nowak, J.; Obe, G.

    1976-10-28

    Cells containing x-ray induced micronuclei were treated for a few hours before fixation with inhibitors of DNA synthesis (cytosine arabinoside; azathioprine; thymidine; trenimon), of RNA synthesis (actinomycin D; ethidium bromide), and of protein synthesis (puromycin). Only the inhibitors of DNA synthesis lead to a significant suppression of the frequencies of mitoses with micronucleus derived premature chromosome condensation (PCC). We tend to interpret the result as follows: Micronuclei that are in the G1 phase of their cell cycles are accumulated at the G1/S border or in the early S phase of their cell cycles under the influence of the inhibitors of the DNA synthesis. Micronuclei blocked in this way cannot be induced to undergo PCC and seem to disappear from the cells.

  20. 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...

  1. Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies.

    OpenAIRE

    Masse, J.E.; Bortmann, P; Dieckmann, T.; Feigon, J

    1998-01-01

    The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,...

  2. 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.

  3. 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)

  4. 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.

  5. 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, ...

  6. Changes in the synthesis of DNA, RNA and protein during somatic embryogenesis in wheat (triticum aestivum L.)

    International Nuclear Information System (INIS)

    Embryogenic and non-embryogenic callus formed from immature embryo of wheat (Triticum aestivum L.) in N6B5MS medium I supplemented with 2,4-D 2 mg/L, KT 0.5 mg/L, LH300 mg/L, sucrose 3% were sub-cultured and transferred respectively to N6B5MS medium II (2,4-D was decreased to 0.5 mg/L and 4 mol/L proline was added). Somatic embryos obtained from embryogenic callus, and plantlet formed from non-embryogenic callus through organogenesis respectively. By incorporation of 3H-thymidine, 3H-uridine and 3H-leucine into DNA, RNA and protein respectively, the rate of synthesis of DNA, RNA and protein during somatic embryogenesis were measured. A large amount of RNA and protein synthesized during the early somatic embryogenesis. The activities of RNA and protein synthesis reached the peak on the 4th and the 8th day respectively, then decreased a little, but kept a high level. The synthesis of DNA increased apparently during the early stage. No apparent change occurred when the embryogenic cell masses formed. The synthesis rate of RNA and protein in non-embryogenic callus were much less than that in embryogenic callus. Actinomycin and cycloheximide inhibited not only the synthesis of nucleic acid and protein, but also the growth of embryogenic callus and somatic embryogenesis. The earlier the inhibitors were added, the greater the influence was caused. The results indicate that the active expression of corresponding genes of wheat is the molecular base of somatic embryogenesis

  7. 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)

  8. Remote Activation of Host Cell DNA Synthesis in Uninfected Cells Signaled by Infected Cells in Advance of Virus Transmission

    OpenAIRE

    Schmidt, Nora; Hennig, Thomas; Remigiusz A Serwa; Marchetti, Magda; O'Hare, Peter

    2015-01-01

    ABSTRACT Viruses modulate cellular processes and metabolism in diverse ways, but these are almost universally studied in the infected cell itself. Here, we study spatial organization of DNA synthesis during multiround transmission of herpes simplex virus (HSV) using pulse-labeling with ethynyl nucleotides and cycloaddition of azide fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with the current understanding of the single-step growth cy...

  9. Effect of X-radiation on DNA and histone synthesis in ataxia telangiectasia and normal lymphoblastoid cells

    International Nuclear Information System (INIS)

    The possibility that the radiosensitivity of lymphoblastoid cell lines from patients with ataxia telangiectasia (A-T) is due to an aberrant content of histones has been examined. The histone pattern of lymphoblastoid cell lines derived from A-T patients was found to be indistinguishable from that obtained from normal individuals. X-ray irradiation led to a greater decrease in cell growth rate in the A-T cells than in the normal cells but was accompanied by a greater decrease of DNA synthesis rate in the normal cells. This difference in radiosensitivity was not reflected in differences in the content or rates of synthesis of histones or of major non-histone proteins in these cells. The authors conclude that the hypersensitivity to ionizing radiation in A-T cells is not due to fundamental differences in the composition or synthesis of the major chromosomal proteins. (Auth.)

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

    Czech Academy of Sciences Publication Activity Database

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

    2016-01-01

    Roč. 24, č. 6 (2016), s. 1268-1276. ISSN 0968-0896 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : nucleosides * nucleotides * pyrimidines * DNA methyltransferases * DNA polymerases Subject RIV: CC - Organic Chemistry Impact factor: 2.793, year: 2014

  11. 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...

  12. NAA-modified DNA oligonucleotides with zwitterionic backbones: stereoselective synthesis of A-T phosphoramidite building blocks.

    Science.gov (United States)

    Schmidtgall, Boris; Höbartner, Claudia; Ducho, Christian

    2015-01-01

    Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive charges into the otherwise polyanionic backbone of DNA oligonucleotides. Following initial studies with the introduction of the NAA-linkage at T-T sites, it is now envisioned to prepare NAA-modified oligonucleotides bearing the modification at X-T motifs (X = A, C, G). We have therefore developed the efficient and stereoselective synthesis of NAA-linked 'dimeric' A-T phosphoramidite building blocks for automated DNA synthesis. Both the (S)- and the (R)-configured NAA-motifs were constructed with high diastereoselectivities to furnish two different phosphoramidite reagents, which were employed for the solid phase-supported automated synthesis of two NAA-modified DNA oligonucleotides. This represents a significant step to further establish the NAA-linkage as a useful addition to the existing 'toolbox' of backbone modifications for the design of bioactive oligonucleotide analogues. PMID:25670992

  13. 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...

  14. 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)

  15. Stimulation of bacterial DNA synthesis by algal exudates in attached algal-bacterial consortia

    International Nuclear Information System (INIS)

    Algal-bacterial consortia attached to polystyrene surfaces were prepared in the laboratory by using the marine diatom Amphora coffeaeformis and the marine bacterium Vibrio proteolytica (the approved name of this bacterium is Vibrio proteolyticus. The organisms were attached to the surfaces at cell densities of approximately 5 x 104 cells cm-2 (diatoms) and 5 x 106 cells cm-2 (bacteria). The algal-bacterial consortia consistently exhibited higher rates of [3H]thymidine incorporation than did biofilms composed solely of bacteria. The rates of [3H]thymidine incorporation by the algal-bacterial consortia were fourfold greater than the rates of incorporation by monobacterial biofilms 16 h after biofilm formation and were 16-fold greater 70 h after biofilm formation. Extracellular material released from the attached Amphora cells supported rates of bacterial activity (0.8 x 10-21 mol to 17.9 x 10-21 mol of [3H]thymidine incorporated cell -1 h-1) and growth (doubling time, 29.5 to 1.4 days) comparable to values reported for a wide variety of marine and freshwater ecosystems. In the presence of sessile diatom populations, DNA synthesis by attached V. proteolytica cells was light dependent and increased with increasing algal abundance. The metabolic activity of diatoms thus appears to be the rate-limiting process in biofilm development on illuminated surfaces under conditions of low bulk-water dissolved organic carbon

  16. 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.

  17. DNA synthesis and pronucleus development in pig zygotes obtained in vivo: an autoradiographic and ultrastructural study

    International Nuclear Information System (INIS)

    Porcine zygotes flushed from oviducts 48, 52, 56, 60, or 64 hr after hCG were incubated 30 min in 3H-thymidine, transferred to nonradioactive medium for 2 hr, and incubated for 30 min with 14C-thymidine. After this procedure, ova were prepared (i.e., at 51, 55, 59, 63, or 67 hr after hCG) for autoradiography and ultrastructural observations, respectively. The first autoradiographic labelling, i.e., DNA synthesis, was observed at 56-56.5 hr after hCG, while the latest labelling was seen at 60-60.5 hr. At 51 hr after hCG, formation of the pronuclear envelope was observed, while no nucleolus precursor bodies or prestages to these structures were found. At 55 hr a few clusters of small electron-dense granules were observed, together with condensed chromatin in the pronuclei. At 59 hr the apposed regions of both pronuclei contained nucleolus precursor bodies and condensed chromatin, in close contact with both clusters of small granules and clusters of an additional category of large granules and the nuclear envelope. Additionally, large accumulations of the small granules were found in the vicinity of similarly sized accumulations of the large granules without chromatin association. At 63 hr the spherical accumulations of large granules on some occasions presented a central vacuole, and condensed chromatin and clusters of small granules were attached to its periphery. Within the vacuole, electron-dense material was found

  18. 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

  19. The mechanism of inhibition of endothelin-1-induced stimulation of DNA synthesis in rat articular chondrocytes.

    Science.gov (United States)

    Khatib, A M; Ribault, D; Quintero, M; Barbara, A; Fiet, J; Mitrovic, D R

    1997-09-19

    Endothelin-1 (ET-1) is a potent mitogen for rat articular chondrocytes (AC) in short term culture (24 h). Prolonged incubation (72 h) of AC with ET-1 resulted in inhibition of [3H]thymidine incorporation. This inhibition seemed to be mediated by prostaglandins (PGs) released in response to ET-1, since indomethacin (INDO) enhanced ET-1-induced [3H]thymidine incorporation. In agreement with this hypothesis, exogenous prostaglandins (PGE2, PGF2alpha and TxB2) blocked all basal, ET-1-induced and ET-1 induced-INDO-enhanced [3H]thymidine incorporation and ET-1 stimulated PGE2 release in a time and concentration-dependent manner. INDO also blocked cGMP production and 6-anilino-5,8-quinolinedione, a relatively specific inhibitor of cGMP formation, enhanced the stimulation and suppressed the inhibition of ET-1-induced DNA synthesis. In addition, 8-bromo-cGMP, an analogue of cGMP, blocked at all time periods studied, both basal and ET-1-induced incorporations of [3H]thymidine. Thus, PGs produced in response to ET-1 counteract the ET-1-induced stimulation of [3H]thymidine incorporation into rat AC by increasing cGMP production. PMID:9324043

  20. 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

  1. HBV cccDNA 检测的研究进展

    Institute of Scientific and Technical Information of China (English)

    陆晖; 江建宁

    2008-01-01

    @@ 自1965年Blumberg发现澳大利亚抗原(HBsAg)以来,病毒性乙型肝炎(viral hepatitis B)有了划时代的研究进展.病毒性乙型肝炎主要流行于亚洲、非洲、南部欧洲和拉丁美洲.全世界约20亿人有既往或持续感染HBV,慢性HBV感染者达3~3.5亿,其中15%~25%最终死于肝衰竭、肝硬化或肝癌,年病死人数约100万;男女性病人的病死率分别约50%和15%.

  2. 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.

  3. 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

  4. 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...

  5. 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.

  6. 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)

  7. Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.

    Science.gov (United States)

    Gürler, H; Malama, E; Heppelmann, M; Calisici, O; Leiding, C; Kastelic, J P; Bollwein, H

    2016-07-15

    The objective was to examine if there are relationships between alterations in sperm viability, reactive oxygen species (ROS) synthesis, and DNA integrity induced by cryopreservation of bovine sperm. Four ejaculates were collected from each of six bulls. Each ejaculate was diluted and divided into two aliquots; one was incubated for 24 hours at 37 °C, and the other frozen, thawed, and incubated for 24 hours at 37 °C. Analyses of quality of sperm were performed after 0, 3, 6, 12, and 24 hours of incubation. Progressive motile sperm was determined with computer assisted sperm analysis. Percentages of plasma membrane- and acrosome-intact sperm, sperm with a high mitochondrial membrane potential, sperm showing a high degree of DNA fragmentation (%DFI), and their reactive oxygen species content were assessed with dichlorofluorescein-diacetate, dihydrorhodamine, diaminofluorescein diacetate, and mitochondrial superoxide indicator using flow cytometry. Although all other sperm parameters showed alterations (P  0.05, 0.91 ± 0.23) in nonfrozen sperm. Cryopreservation induced changes of all sperm parameters (P < 0.05). In contrast to all other sperm parameters, dichlorofluorescein-diacetate-fluoroescence indicating the synthesis of H2O2 showed a similar exponential rise (P < 0.05) like the %DFI values in frozen sperm. In conclusion, changes of DNA integrity in frozen sperm seem to be related to synthesis of H2O2 but not to sperm viability and synthesis of other reactive oxygen species. PMID:27039074

  8. Application of primed in situ DNA synthesis (PRINS) with telomere human commercial kit in molecular cytogenetics of Equus caballus and Sus scrofa scrofa.

    OpenAIRE

    Maciej Wnuk; Monika Bugno; Ewa Slota

    2008-01-01

    Recently, molecular techniques have become an indispensable tools for cytogenetic research. Especially, development of in situ techniques made possible detection at the chromosomal level, genes as well as repetitive sequences like telomeres or the DNA component of telomeres. One of these methods is primed in situ DNA synthesis (PRINS) using an oligonucleotide primer complementary to the specific DNA sequence. In this report we described application of PRINS technique with telomere human comme...

  9. Polymerase synthesis of DNA labelled with benzylidene cyanoacetamide-based fluorescent molecular rotors: fluorescent light-up probes for DNA-binding proteins

    Czech Academy of Sciences Publication Activity Database

    Dziuba, Dmytro; Pohl, Radek; Hocek, Michal

    2015-01-01

    Roč. 51, č. 23 (2015), s. 4880-4882. ISSN 1359-7345 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : enzyme amplification reaction * modified oligonucleotides * nucleoside triphosphates Subject RIV: CC - Organic Chemistry Impact factor: 6.834, year: 2014 http://pubs.rsc.org/en/content/articlepdf/2015/cc/c5cc00530b

  10. X-ray induced inhibition of DNA synthesis and mitosis in internal tissues during the initiation of limb regeneration in the adult newt

    International Nuclear Information System (INIS)

    Left front limbs of adult male newts were given 2000 R of x irradiation at least four weeks prior to amputation of both forelimbs. Internal stump tissues were evaluated for the ability to incorporate 3H-thymidine and accumulate colchicine-blocked mitotic figures. In otherwise uninjured limbs, irradiation stimulated low levels of DNA synthesis which did not increase significantly after amputation. Thus, as soon as DNA synthesis increased significantly in normal limbs as a result of amputation, it was demonstrably higher than in x-rayed limbs. In general, mitotic activity in both groups reflected the DNA synthetic rates. Since others have shown that denervation at the time of amputation blocks subsequent mitosis in internal stump tissues yet allows normal levels of DNA synthesis for eight days, we conclude that x irradiation and denervation prevent cell division in potential blastema cells by different mechanisms

  11. Children's Communication Checklist (CCC) scores in 11-year-old children with communication impairments

    OpenAIRE

    Botting, N.

    2004-01-01

    Background: The pragmatic skills of children with communication disorders and their assessment are currently an issue for speech and language therapy and educational placement. Aims: To explore whether different subgroups of children with communication disorders score differently on the Children’s Communication Checklist (CCC; Bishop, 1998) and how they compare to published normative data. Methods and procedures: A sample of 161 eleven year old children with a history of communication d...

  12. Design of inspection and maintenance models based on the CCC-chart

    OpenAIRE

    Chan, LY

    2003-01-01

    In this research, six maintenance models are constructed based on whether minor inspection, major inspection, minor maintenance and major maintenance are performed on a system. The system to study is a production process in which items produced can be classified as either conforming or nonconforming, and a statistical process control chart called CCC-chart (cumulative count control chart) can be applied to monitor the process. The maintenance models are analyzed quantitatively, and selection ...

  13. 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

  14. DNA synthesis and cell survival after X-irradiation of mammalian cells treated with caffeine or adenine

    International Nuclear Information System (INIS)

    The expression of the transient depression in the rate of DNA synthesis normally observed after exposure of randomly-dividing Chinese hamster V-79 or Chinese hamster CHO cells to ionizing radiation could be postponed by a post-irradiation treatment with 1.0 to 2.0 mM adenine or 1.5 mM caffeine. Caffeine may exert its effect by creating additional sites for replication in irradiated cells. Cells treated with caffeine or adenine for 2 or 4 hours after exposure to 3000 rad of 300 kVp X-rays exhibited depressed synthesis only after the removal of caffeine or adenine. These alterations in the timing of the X-ray-induced depression of the rate of DNA synthesis had no effect on X-ray-induced cell killing. Although a 4 hour post-irradiation treatment of randomly-dividing Chinese hamster V-79 cells with 1.0 or 2.0 mM caffeine potentiated X-ray-induced cell killing, this reduction in survival was due primarily to effects on cells not in S-phase. (author)

  15. 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.

  16. 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 ...

  17. 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...

  18. 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.

  19. Final work plan : investigation of potential contamination at the former CCC/USDA grain storage facility in Hanover, Kansas.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M.; Environmental Science Division

    2008-11-19

    The Commodity Credit Corporation (CCC), an agency of the U.S. Department of Agriculture (USDA), operated a grain storage facility at the northeastern edge of the city of Hanover, Kansas, from 1950 until the early 1970s. During this time, commercial grain fumigants containing carbon tetrachloride were in common use by the grain storage industry to preserve grain in their facilities. In February 1998, trace to low levels of carbon tetrachloride (below the maximum contaminant level [MCL] of 5.0 {micro}g/L) were detected in two private wells near the former grain storage facility at Hanover, as part of a statewide USDA private well sampling program that was implemented by the Kansas Department of Health and Environment (KDHE) near former CCC/USDA facilities. In April 2007, the CCC/USDA collected near-surface soil samples at 1.8-2 ft BGL (below ground level) at 61 locations across the former CCC/USDA facility. All soil samples were analyzed by the rigorous gas chromatograph-mass spectrometer analytical method (purge-and-trap method). No contamination was found in soil samples above the reporting limit of 10 {micro}g/kg. In July 2007, the CCC/USDA sampled indoor air at nine residences on or adjacent to its former facility to address the residents concerns regarding vapor intrusion. Low levels of carbon tetrachloride were detected at four of the nine homes. Because carbon tetrachloride found in private wells and indoor air at the site might be linked to historical use of fumigants containing carbon tetrachloride at its former grain storage facility, the CCC/USDA is proposing to conduct an investigation to determine the source and extent of the carbon tetrachloride contamination associated with the former facility. This investigation will be conducted in accordance with the intergovernmental agreement between the KDHE and the Farm Service Agency (FSA) of the USDA. The investigation at Hanover will be performed, on behalf of the CCC/USDA, by the Environmental Science

  20. 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.

  1. Quantification of covalently closed circular DNA of hepatitis B virus in FFPE liver tissues of chronic hepatitis B patients

    Directory of Open Access Journals (Sweden)

    Jia-qi HAN

    2011-05-01

    Full Text Available Objective To establish a method of detecting HBV covalently closed circular DNA(cccDNA in micro-formalin fixed paraffin imbedding(FFPE liver biopsy samples.Methods FFPE liver biopsies from 37 patients with chronic hepatitis B were studied.The intrahepatic HBV DNA was extracted and pre-treated with plasmid-safe ATP-dependent DNAse(PSAD,and then amplified by rolling circular amplification(RCA.The HBV cccDNA was quantitatively detected by Taqman real-time PCR with primers located on both sides of the gap of HBV DNA.The human β-actin gene served as the internal control.The sensitivity was tested by serially diluting the DNA templates with known concentrations.The repeatability and stability were evaluated with inter-assay and intra-assay.The level of intrahepatic HBV cccDNA,HBV total DNA,serum HBV DNA and ALT were also compared to find the relations between them.Results The quantitative detection method of cccDNA in micro-FFPE liver samples was successfully set up with considerable sensitivity,stability and specificity.The intrahepatic cccDNA level was significantly higher in the HBeAg-positive patients than in the HBeAg-negative patients(P < 0.05.The intrahepatic HBV cccDNA level was positively correlated with the serum and intra-hepatic HBV DNA level(r=0.539,P=0.001.Conclusion The assay established by present study is fit for the detection of HBV cccDNA in micro-FFPE liver biopsies.

  2. 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

  3. Final corrective action study for the former CCC/USDA facility in Ramona, Kansas.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M. (Environmental Science Division)

    2011-04-20

    Past operations at a grain storage facility formerly leased and operated by the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) in Ramona, Kansas, resulted in low concentrations of carbon tetrachloride in groundwater that slightly exceed the regulatory standard in only one location. As requested by the Kansas Department of Health and Environment, the CCC/USDA has prepared a Corrective Action Study (CAS) for the facility. The CAS examines corrective actions to address groundwater impacted by the former CCC/USDA facility but not releases caused by other potential groundwater contamination sources in Ramona. Four remedial alternatives were considered in the CAS. The recommended remedial alternative in the CAS consists of Environmental Use Control to prevent the inadvertent use of groundwater as a water supply source, coupled with groundwater monitoring to verify the continued natural improvement in groundwater quality. The Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) has directed Argonne National Laboratory to prepare a Corrective Action Study (CAS), consistent with guidance from the Kansas Department of Health and Environment (KDHE 2001a), for the CCC/USDA grain storage facility formerly located in Ramona, Kansas. This effort is pursuant to a KDHE (2007a) request. Although carbon tetrachloride levels at the Ramona site are low, they remain above the Kansas Tier 2 risk-based screening level (RBSL) and the U.S. Environmental Protection Agency (EPA) maximum contaminant level (MCL) of 5 {micro}g/L (Kansas 2003, 2004). In its request for the CAS, the KDHE (2007a) stated that, because of these levels, risk is associated with potential future exposure to contaminated groundwater. The KDHE therefore determined that additional measures are warranted to limit future use of the property and/or exposure to contaminated media as part of site closure. The KDHE further requested comparison of at least two corrective

  4. 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.)

  5. Technical justification for a request to reclassify the former CCC/USDA facility at Canada, Kansas.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M.; Environmental Science Division

    2007-12-21

    Contamination in groundwater at Canada, Kansas, was discovered in 1997, during limited private well sampling near former grain storage facilities of the Commodity Credit Corporation, U.S. Department of Agriculture (CCC/USDA). Subsequent investigations by the Kansas Department of Health and Environment (KDHE) confirmed carbon tetrachloride and nitrate concentrations in groundwater above the respective maximum contaminant levels (MCLs) of 5.0 {micro}g/L and 10.0 mg/L. The KDHE investigations identified both the former CCC/USDA grain storage facility and a private grain storage facility as likely sources for the carbon tetrachloride contamination. The CCC/USDA funded extension of a rural water district line to provide a permanent alternate water supply, and the KDHE has conducted long-term monitoring under the State Water Plan. This document presents an analysis of the available information for the Canada site, acquired in previous investigations and the long-term KDHE monitoring. This analysis forms the technical justification for a request to reclassify the former CCC/USDA grain storage facility at Canada as a site requiring no further action under the Intergovernmental Agreement (IGA) between the KDHE and the USDA's Farm Service Agency. The KDHE's long-term water level monitoring results indicate a consistent groundwater flow direction to the east-southeast. Consequently, the wells with the highest overall concentrations of carbon tetrachloride are downgradient from the private grain storage facility but not downgradient from the former CCC/USDA facility. The KDHE criterion for reclassification of a site is that contamination there should not pose an unacceptable risk, on the basis of analytical results for four consecutive, equally timed, sequenced sampling episodes over a period of no less than two years. In seven KDHE sampling events over a period of six years (2001-2007), the concentrations of carbon tetrachloride in the monitoring well on the former

  6. Autoradiographic studies of the rate of DNA synthesis in the rat epididymis duct epithelium and brain subependimal zone cells after the whole body X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Gracheva, N.D.; Shatalin, G.I. (Tsentral' nyj Nauchno-Issledovatel' skij Rentgeno-Radiologicheskij Inst., Leningrad (USSR))

    1982-11-01

    DNA synthesis rate was analyzed on the basis of labelled cell distribution in epithelium of epididymis duct and subependyma zone of rat brain from the number of reduced silver grains under a nucleus calculated on recorders of histologic sections (5 ..mu..m) during different time after /sup 3/H hymidine intake and total X-ray irradiation in 300 Gy dose. Results of observations served as the additional substation of an earlier conclusion that in a series of truncal-semitruncal-differentiated cell per stage decrease of DNA synthesis rate occurs. During the period of maximum postradiation repair the proliferation increase took place at the expense of cell self-reproducibility, which in norm have medium and high rates of DNA synthesis against the background of cell preproduction deceleration which are characterized in norm with low rates of DNA synthesis and after mitosis should initiate differentiation. These facts conditioned the increase in the mean number of the reduced silver grains per a nucleus at a height of the postradiation proliferation, while DNA synthesis rates themselves peculiar to successive generations of truncal cells didn't change.

  7. Down-regulation of SMT3A gene expression in association with DNA synthesis induction after X-ray irradiation in nevoid basal cell carcinoma syndrome (NBCCS) cells

    International Nuclear Information System (INIS)

    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

  8. 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.

  9. 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.

  10. TWINKLE is an essential mitochondrial helicase required for synthesis of nascent D-loop strands and complete mtDNA replication.

    Science.gov (United States)

    Milenkovic, Dusanka; Matic, Stanka; Kühl, Inge; Ruzzenente, Benedetta; Freyer, Christoph; Jemt, Elisabeth; Park, Chan Bae; Falkenberg, Maria; Larsson, Nils-Göran

    2013-05-15

    Replication of the mammalian mitochondrial DNA (mtDNA) is dependent on the minimal replisome, consisting of the heterotrimeric mtDNA polymerase (POLG), the hexameric DNA helicase TWINKLE and the tetrameric single-stranded DNA-binding protein (mtSSB). TWINKLE has been shown to unwind DNA during the replication process and many disease-causing mutations have been mapped to its gene. Patients carrying Twinkle mutations develop multiple deletions of mtDNA, deficient respiratory chain function and neuromuscular symptoms. Despite its importance in human disease, it has been unclear whether TWINKLE is the only replicative DNA helicase in mammalian mitochondria. Furthermore, a substantial portion of mtDNA replication events is prematurely terminated at the end of mitochondrial control region (D-loop) and it is unknown whether TWINKLE also has a role in this abortive replication. Here, we present a conditional mouse knockout for Twinkle and demonstrate that TWINKLE is essential for mouse embryonic development and thus is the only replicative DNA helicase in mammalian mitochondria. Conditional knockout of Twinkle results in severe and rapid mtDNA depletion in heart and skeletal muscle. No replication intermediates or deleted mtDNA molecules are observed after Twinkle knockout, suggesting that TWINKLE once loaded is very processive. We also demonstrate that TWINKLE is essential for nascent H-strand synthesis in the D-loop, thus showing that there is no separate DNA helicase responsible for replication of this region. Our data thus suggest that the relative levels of abortive D-loop synthesis versus complete mtDNA replication are regulated and may provide a mechanism to control progression to complete mtDNA replication. PMID:23393161

  11. 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; Wiuf, Carsten; Willerslev, Eske; Poinar, Hendrik; Carlson, John E; Leebens-Mack, James H; Schuster, Stephan C

    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. Furt...

  12. X-ray inhibition of DNA synthesis at discrete times during S phase in synchronous human diploid fibroblasts

    International Nuclear Information System (INIS)

    Synchronous human diploid fibroblasts were exposed to 1000 rad of X rays at various times in S phase, and thymidine incorporation, an indicator of DNA synthesis, was measured throughout the cell cycle. When cells were irradiated just before the time when thymidine incorporation peaked in control cells, the peak was strongly suppressed. Irradiation after appearance of an incorporation peak in control cells resulted in some depression of thymidine incorporation but had much less effect than irradiation before the peak. These results are in agreement with the interpretation that, in human diploid fibroblasts, most replicons initiate during relatively restricted times in S phase, leading to the multiple thymidine incorporation peaks observed in these cells

  13. 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

  14. Modifying action of DNA synthesis precursors on Aspergillus nidulans conidium irradiated by ultraviolet and X-rays

    International Nuclear Information System (INIS)

    Modification of inactivation action of radiation on conidia Aspergillus nidulans, UVS+ and UVS strains, by desoxynucleosides, purine and pyrimidine bases is shown. The modification manifested in increased conidia survival is revealed when the precursor of DNA synthesis is added to the suspension before exposure to ultraviolet or X-rays. In the case of postradiation application of the substance no modification is observed. The modifying effect of different precursors becomes equally apparent with equimolar solutions and increases at higher concentration of the latter. An increase in thymidine endogenic pool in the exposed conidia does not affect their survival. When conidia are exposed to ultraviolet rays through a thymidine filter the survival rate increases to the same extent as in the case when they are exposed to irradiation in thymidine solution. The authors suggest that modification of the inactivating radiation action by DNA precursors at exposure of conidia Aspergillus nidulans is caused by the radioprotective effect of precursors not related to reparation

  15. 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

  16. Anthraquinone as a Redox Label for DNA: Synthesis, Enzymatic Incorporation, and Electrochemistry of Anthraquinone-Modified Nucleosides, Nucleotides, and DNA

    Czech Academy of Sciences Publication Activity Database

    Balintová, Jana; Pohl, Radek; Horáková Brázdilová, Petra; Vidláková, Pavlína; Havran, Luděk; Fojta, Miroslav; Hocek, Michal

    2011-01-01

    Roč. 17, č. 50 (2011), s. 14063-14073. ISSN 0947-6539 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk LC512; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : anthraquinone * DNA * electrochemistry * nucleosides * oligonucleotides Subject RIV: CC - Organic Chemistry Impact factor: 5.925, year: 2011

  17. Analysis of Early Human Immunodeficiency Virus Type 1 DNA Synthesis by Use of a New Sensitive Assay for Quantifying Integrated Provirus

    OpenAIRE

    Brussel, Audrey; Sonigo, Pierre

    2003-01-01

    A novel Alu-long terminal repeat (LTR)-based real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type 1 (HIV-1) DNA in infected cells with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents). Parallel assays for total HIV-1 DNA and two-LTR HIV-1 DNA circles allowed the synthesis and fate of the different HIV-1 DNA species to be monitored upon a single round of viral replication.

  18. Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes.

    Science.gov (United States)

    Ewert, Kai K; Kotamraju, Venkata Ramana; Majzoub, Ramsey N; Steffes, Victoria M; Wonder, Emily A; Teesalu, Tambet; Ruoslahti, Erkki; Safinya, Cyrus R

    2016-03-15

    Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles. PMID:26874401

  19. φ29 DNA polymerase

    OpenAIRE

    Blanco, Luis; Bernad, Antonio; Salas, Margarita

    1996-01-01

    An improved method for determining the nucleotide base sequence of a DNA molecule employs a φ-29 type DNA polymerase modified to have reduced or no exonuclease activity. The method includes annealing the DNA molecule with a primer molecule able to hybridize to the DNA molecule; incubating the annealed mixture in a vessel containing four different deoxynucleoside triphosphates, a DNA polymerase, and one or more DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleo...

  20. 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.

  1. 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

  2. Flow cytometric measurement of RNA synthesis based on bromouridine labelling and combined with measurement of DNA content or cell surface antigen

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J; Larsen, J K

    1993-01-01

    RNA synthesis can be analysed in nuclei or cells labelled with 5-bromouridine (BrUrd) and stained using cross-reacting anti-bromodeoxyuridine (BrdUrd) antibody. Flow cytometric dual parameter analysis of BrUrd incorporation and DNA content in nuclear suspensions of human blood lymphocytes showed ...... synthesis in HL-60 and K-562 cells was measured simultaneous with CD13 expression....

  3. 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...

  4. 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...

  5. In vitro RNA synthesis with UV-irradiated phage lambda DNA

    International Nuclear Information System (INIS)

    Irradiation of phage lambda DNA with UV light at a dose of 10 Jm-2 leads to a 40% decrease in DNA template activity and at a dose of 100 Jm-2 - to its complete suppression. This is apparently due to the transcription-terminating effect mainly of pyrimidine dimers. Electrophoretic analysis of RNA shows that RNA chains, homogeneous on their molecular weight but shorter, are produced in vitro with 10 Jm-2 UV-irradiated DNA. (author)

  6. PARP-1 mechanism for coupling DNA damage detection to poly(ADP-ribose) synthesis

    OpenAIRE

    Langelier, Marie-France; Pascal, John M.

    2013-01-01

    Poly(ADP-ribose) polymerase 1 (PARP-1) regulates gene transcription, cell death signaling, and DNA repair through production of the posttranslational modification poly(ADP-ribose). During the cellular response to genotoxic stress PARP-1 rapidly associates with DNA damage, which robustly stimulates poly(ADP-ribose) production over a low basal level of PARP-1 activity. DNA damage-dependent PARP-1 activity is central to understanding PARP-1 biological function, but structural insights into the m...

  7. 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.

  8. 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

  9. Extraction and purification of C-phycocyanin from Spirulina platensis (CCC540)

    OpenAIRE

    Kumar, Devendra; Dhar, Dolly Wattal; Pabbi, Sunil; Kumar, Neeraj; Walia, Suresh

    2014-01-01

    In this study a simple protocol was developed for purifying phycocyanin (PC) from Spirulina platensis (CCC540) by using ammonium sulphate precipitation, followed by a single step chromatography by using DEAE-Cellulose-11 and acetate buffer. Precipitation with 65 % ammonium sulphate resulted in 80 % recovery of phycocyanin with purity of 1.5 (A620/A280). Thro1ugh chromatography an 80 % recovery of phycocyanin with a purity of 4.5 (A620/A280) was achieved. In SDS_PAGE analysis, the purified PC ...

  10. Service-dominant logic as a future strategy : Case CCC Corporation Oy

    OpenAIRE

    Mattila, Heidi

    2011-01-01

    The focus of this thesis is service-dominant logic, a framework created by Stephen Vargo and Robert Lusch (2004), in the software business. The case company, CCC Corporation is used as an analysis point. The subject is approached through research questions which are as follows: 1) How does the case company implement service-dominant logic in Finland? 2) What is service-dominant logic in the software business? 3) Why is service-dominant logic important for the software business and 4) What com...

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. RADIO SEARCH FOR H{sub 2}CCC TOWARD HD 183143 AS A CANDIDATE FOR A DIFFUSE INTERSTELLAR BAND CARRIER

    Energy Technology Data Exchange (ETDEWEB)

    Araki, Mitsunori; Yamabe, Hiromichi; Tsukiyama, Koichi [Department of Chemistry, Faculty of Science Division I, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku 162-8601, Tokyo (Japan); Takano, Shuro [Nobeyama Radio Observatory and Department of Astronomical Science, The Graduate University for Advanced Studies (Sokendai), 462-2 Nobeyama, Minamimaki, Minamisaku, Nagano 384-1305 (Japan); Kuze, Nobuhiko, E-mail: araki@rs.kagu.tus.ac.jp [Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku 102-8554, Tokyo (Japan)

    2012-07-01

    To clarify the authenticity of a recently proposed identification of H{sub 2}CCC (linear-C{sub 3}H{sub 2}) as a diffuse interstellar band (DIB) carrier, we searched for the rotational transition of H{sub 2}CCC at a frequency of 103 GHz toward HD 183143 using the 45 m telescope at the Nobeyama Radio Observatory. Although rms noise levels of 32 mK in the antenna temperature were achieved, detection of H{sub 2}CCC was unsuccessful, producing a 3{sigma} upper limit corresponding to a column density of 2.0 Multiplication-Sign 10{sup 13} cm{sup -2}. The upper limit indicates that the contribution of H{sub 2}CCC to the DIB at 5450 Angstrom-Sign is less than 1/25; thus, it is unlikely that the laboratory bands of the B{sup 1} B{sub 1}-X{sup 1} A{sub 1} transition of H{sub 2}CCC and the DIBs at 5450 Angstrom-Sign (and also 4881 Angstrom-Sign ) toward HD 183143 are related.

  16. 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.

  17. Synthesis and detection of 3'-OH terminal biotin-labeled DNA probes

    International Nuclear Information System (INIS)

    Nick translation has been used to prepare biotin-dUTP-containing DNA probes. These stable DNA probes have been identified, following hybridization to target DNA, by fluorescence using antibiotin antibodies or by enzyme reactions in which the enzyme has been linked to avidin or streptavidin. It is probable that this technology will be applicable to certain diagnostic determinations and that, with sufficient sensitivity, this technology might provide a system for obtaining rapid and specific diagnoses in situations presently requiring time-consuming growth assays. The sensitivity of this assay can be increased in two ways: (1) by increasing the amount of biotin contained in the DNA probes, and (2) by increasing the response to individual biotin molecules in the DNA probes. This report demonstrates that terminal deoxynucleotide transferase can be employed to increase the biotin content of DNA probes. We also introduce a new streptavidin-linked enzyme system that produces a greater response to biotinylated DNA probes than does streptavidin-linked horseradish peroxidase

  18. 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

  19. 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 ...

  20. 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.

  1. Association between early inhibition of DNA synthesis and the MICs and MBCs of carboxyquinolone antimicrobial agents for wild-type and mutant [gyrA nfxB(ompF) acrA] Escherichia coli K-12.

    OpenAIRE

    Chow, R T; Dougherty, T J; Fraimow, H S; Bellin, E Y; Miller, M H

    1988-01-01

    Quinolone antimicrobial agents are known to interact with DNA gyrase, but the mechanism by which bacterial cell death occurs is not fully understood. In order to determine whether there is a correlation between quinolone-induced inhibition of early (i.e., 10 to 15 min) DNA synthesis and potency (MICs and MBCs), we measured the rate of DNA synthesis in log-phase Escherichia coli K-12 by using [3H]thymidine incorporation. Three quinolones (ciprofloxacin, norfloxacin, and difloxacin) were select...

  2. In vitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling.

    OpenAIRE

    Hirose, S; Suzuki, Y

    1988-01-01

    In a posterior silk gland extract, covalently closed circular (ccc) DNA is in a superhelical state that supports more transcription of fibroin gene than does linear DNA. A HeLa cell extract showed neither the supercoiling activity nor the preference for the transcription of ccc DNA over linear DNA. These activities could be added to the HeLa cell extract. Phosphocellulose fractionation of the posterior silk gland extract yielded a flow-through fraction and a 0.6 M KCl eluate fraction that wer...

  3. Synthesis and structure formation in dilute aqueous solution of a chitosan-DNA hybrid

    Czech Academy of Sciences Publication Activity Database

    Safir, I.; Ngo, K. X.; Abraham, J. N.; Afshar, M. G.; Pavlova, Ewa; Nardin, C.

    2015-01-01

    Roč. 79, 19 November (2015), s. 29-36. ISSN 0032-3861 Institutional support: RVO:61389013 Keywords : chitosan * DNA * self-assembly Subject RIV: CD - Macromolecular Chemistry Impact factor: 3.562, year: 2014

  4. 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.

  5. 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。

  6. 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.

  7. Nuclear Reorganization of Mammalian DNA Synthesis Prior to Cell Cycle Exit

    OpenAIRE

    Barbie, David A; Kudlow, Brian A.; Frock, Richard; Zhao, Jiyong; Johnson, Brett R.; Dyson, Nicholas; Harlow, Ed; Kennedy, Brian K.

    2004-01-01

    In primary mammalian cells, DNA replication initiates in a small number of perinucleolar, lamin A/C-associated foci. During S-phase progression in proliferating cells, replication foci distribute to hundreds of sites throughout the nucleus. In contrast, we find that the limited perinucleolar replication sites persist throughout S phase as cells prepare to exit the cell cycle in response to contact inhibition, serum starvation, or replicative senescence. Proteins known to be involved in DNA sy...

  8. A SELEX Protocol to Identify ssDNA Biotemplates for Gold Nanoparticle Synthesis

    OpenAIRE

    Calabrese, Philip

    2015-01-01

    A modified ssDNA SELEX protocol was developed in order to evolve a randomized library of imidazole modified ssDNA sequences towards sequences that mediate the formation of gold nanoparticles from Au3+ precursor ions in aqueous solution. Active sequences bound to nanoparticles were partitioned from inactive sequences based on density via ultracentrifugation through a discontinuous sucrose gradient. Colloidal gold solutions produced by the evolved pool had a distinct absorbance spectra and prod...

  9. 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

  10. 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.

  11. 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

  12. Physical Characterization of Gemini Surfactant-Based Synthetic Vectors for the Delivery of Linear Covalently Closed (LCC DNA Ministrings.

    Directory of Open Access Journals (Sweden)

    Chi Hong Sum

    Full Text Available In combination with novel linear covalently closed (LCC DNA minivectors, referred to as DNA ministrings, a gemini surfactant-based synthetic vector for gene delivery has been shown to exhibit enhanced delivery and bioavailability while offering a heightened safety profile. Due to topological differences from conventional circular covalently closed (CCC plasmid DNA vectors, the linear topology of LCC DNA ministrings may present differences with regards to DNA interaction and the physicochemical properties influencing DNA-surfactant interactions in the formulation of lipoplexed particles. In this study, N,N-bis(dimethylhexadecyl-α,ω-propanediammonium(16-3-16gemini-based synthetic vectors, incorporating either CCC plasmid or LCC DNA ministrings, were characterized and compared with respect to particle size, zeta potential, DNA encapsulation, DNase sensitivity, and in vitro transgene delivery efficacy. Through comparative analysis, differences between CCC plasmid DNA and LCC DNA ministrings led to variations in the physical properties of the resulting lipoplexes after complexation with 16-3-16 gemini surfactants. Despite the size disparities between the plasmid DNA vectors (CCC and DNA ministrings (LCC, differences in DNA topology resulted in the generation of lipoplexes of comparable particle sizes. The capacity for ministring (LCC derived lipoplexes to undergo complete counterion release during lipoplex formation contributed to improved DNA encapsulation, protection from DNase degradation, and in vitro transgene delivery.

  13. Physical Characterization of Gemini Surfactant-Based Synthetic Vectors for the Delivery of Linear Covalently Closed (LCC) DNA Ministrings.

    Science.gov (United States)

    Sum, Chi Hong; Nafissi, Nafiseh; Slavcev, Roderick A; Wettig, Shawn

    2015-01-01

    In combination with novel linear covalently closed (LCC) DNA minivectors, referred to as DNA ministrings, a gemini surfactant-based synthetic vector for gene delivery has been shown to exhibit enhanced delivery and bioavailability while offering a heightened safety profile. Due to topological differences from conventional circular covalently closed (CCC) plasmid DNA vectors, the linear topology of LCC DNA ministrings may present differences with regards to DNA interaction and the physicochemical properties influencing DNA-surfactant interactions in the formulation of lipoplexed particles. In this study, N,N-bis(dimethylhexadecyl)-α,ω-propanediammonium(16-3-16)gemini-based synthetic vectors, incorporating either CCC plasmid or LCC DNA ministrings, were characterized and compared with respect to particle size, zeta potential, DNA encapsulation, DNase sensitivity, and in vitro transgene delivery efficacy. Through comparative analysis, differences between CCC plasmid DNA and LCC DNA ministrings led to variations in the physical properties of the resulting lipoplexes after complexation with 16-3-16 gemini surfactants. Despite the size disparities between the plasmid DNA vectors (CCC) and DNA ministrings (LCC), differences in DNA topology resulted in the generation of lipoplexes of comparable particle sizes. The capacity for ministring (LCC) derived lipoplexes to undergo complete counterion release during lipoplex formation contributed to improved DNA encapsulation, protection from DNase degradation, and in vitro transgene delivery. PMID:26561857

  14. Repair of DNA double-strand breaks in Escherichia coli cells requires synthesis of proteins that can be induced by UV light

    International Nuclear Information System (INIS)

    The repair of DNA double-strand breaks in Escherichia coli cells irradiated with γ rays occurs only after new proteins are synthesized in response to damage introduced in the genome DNA. One protein whose synthesis is thus induced is the recA protein, and previous work has shown that recA- cells do not repair double-strand breaks. However, inducing recA protein by treating cells with nalidixic acid does not induce repair of double-strand breaks, so this repair requires more than the presence of the recA protein. When repair of double-strand breaks is blocked, the genome DNA is degraded by an endonuclease-like action. Evidence is presented to show that the inducible inhibition of DNA degradation after x-irradiation [Pollard, E.C. and Randall, E.P. (1973) Radiat. Res. 55, 265] is probably caused by the inducible repair of DNA double-strand breaks

  15. Final report : results of the 2007 investigation of potential contamination at the former CCC/USDA facility in Powhattan, Kansas.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M.; Environmental Science Division

    2008-08-15

    The 2007 investigation of carbon tetrachloride and chloroform contamination at Powhattan, Kansas, was conducted at the request of the Kansas Department of Health and Environment (KDHE 2006a). The Environmental Science Division of Argonne National Laboratory implemented the investigation on behalf of the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA). The primary purposes of the investigation were to evaluate potential contaminant source areas on the former CCC/USDA property, determine the horizontal and vertical extent of potential contamination, conduct groundwater monitoring, and provide recommendations for future action.

  16. Negative effect of the M184V mutation in HIV-1 reverse transcriptase on initiation of viral DNA synthesis

    International Nuclear Information System (INIS)

    The M184V mutation in HIV reverse transcriptase (RT) is associated with high-level resistance against the nucleoside inhibitor lamivudine as well as diminished viral replication capacity. We have previously demonstrated that HIV variants containing the M184V mutation were relatively unable to successfully undergo compensatory mutagenesis following deletion of an A-rich loop located upstream of the primer binding site (PBS). To understand the mechanisms involved, we synthesized viral RNA templates containing different compensatory mutations that were emergent during the long-term culture of the A-rich loop-deleted viruses. These templates were then used in cell-free reverse transcription initiation assays and in tRNA primer placement assays performed with either recombinant wild-type RT or recombinant RT containing the M184V substitution. The results showed that the RNA template that contained the A-rich loop deletion was impaired in ability to initiate reverse transcription and that the presence of the M184V substitution in RT amplified this effect. Clearance from pausing at position +3 during synthesis of viral DNA was identified as a sensitive step in this reaction that could not be efficiently bypassed with the M184V mutant enzyme. Increased efficiency of initiation was seen with the deleted RNA templates that also contained mutations identified in the revertant viruses, provided that these mutations facilitated formation of a competent binary tRNA/RNA complex. These findings provide biochemical evidence that initiation of tRNALys3-primed DNA synthesis is an important rate-limiting step in reverse transcription that correlates with viral replication fitness

  17. Synthesis and Characterization of Chitosan-Saponin Nanoparticle for Application in Plasmid DNA Delivery

    Directory of Open Access Journals (Sweden)

    Faruku Bande

    2015-01-01

    Full Text Available Nonviral delivery system receives attention over the last decade. Chitosan (CS is a cationic polymer whereas saponin (SP is classified as glycoside. In this study, a spherically-shaped CS-SP nanoparticle was synthesized and characterized. The ability of the nanoparticle to protect DNA from enzymatic degradation, its thermostability and cytotoxicity were evaluated. The particle size was found below 100 nm as determined by Zetasizer, transmission electron microscopy (TEM, and field scanning electron microscopy (FSEM results. The surface charge ranges from 43.7 mV to 38.5 mV before and after encapsulation with DNA plasmid, respectively. In terms of thermostability, Thermal Gravimetric Analysis (TGA and Differential Scanning Calorimetry (DSC revealed that CS-SP nanoparticle had a melting temperature of 110°C, with rapid decomposition occurring at 120°C. Encapsulation of DNA with the synthesized nanoparticle was evidenced by changes in the FTIR spectra including characteristic peaks at 3267.39 and 1635.58 cm−1, wavenumbers. Additional peak was also observed at 1169.7 cm−1 following encapsulation. Electrophoretic mobility showed that CS-SP nanoparticle protected plasmid DNA from enzymatic degradation, while cell viability assays confirmed that the synthesized nanoparticle exhibited low cytotoxicity at different concentrations in avian cells. Taken together these, CS-SP nanoparticle showed potentials for applications as a DNA delivery system.

  18. 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

  19. Synthesis, Characterization, Molecular Modeling, and DNA Interaction Studies of Copper Complex Containing Food Additive Carmoisine Dye.

    Science.gov (United States)

    Shahabadi, Nahid; Akbari, Alireza; Jamshidbeigi, Mina; Khodarahmi, Reza

    2016-06-01

    A copper complex of carmoisine dye; [Cu(carmoisine)2(H2O)2]; was synthesized and characterized by using physico-chemical and spectroscopic methods. The binding of this complex with calf thymus (ct) DNA was investigated by circular dichroism, absorption studies, emission spectroscopy, and viscosity measurements. UV-vis results confirmed that the Cu complex interacted with DNA to form a ground-state complex and the observed binding constant (2× 10(4) M(-1)) is more in keeping with the groove bindings with DNA. Furthermore, the viscosity measurement result showed that the addition of complex causes no significant change on DNA viscosity and it indicated that the intercalation mode is ruled out. The thermodynamic parameters are calculated by van't Hoff equation, which demonstrated that hydrogen bonds and van der Waals interactions played major roles in the reaction. The results of circular dichroism (CD) suggested that the complex can change the conformation of DNA from B-like form toward A-like conformation. The cytotoxicity studies of the carmoisine dye and its copper complex indicated that both of them had anticancer effects on HT-29 (colon cancer) cell line and they may be new candidates for treatment of the colon cancer. PMID:27152751

  20. Synthesis of new heterometallic macromolecules: Their DNA binding, cleavage activity and in vitro model electrochemotherapy study

    Science.gov (United States)

    Tabassum, Sartaj; Bhat, Irshad-ul-Haq; Arjmand, Farukh

    2009-12-01

    The homodinuclear C 16H 30N 8O 5Sn 2Cl 4 ( 1), heterotetranuclear C 16H 38N 8O 9Sn 2Cu 2Cl 8 ( 2) and C 16H 38N 8O 9Sn 2Mn 2Cl 8 ( 3) macrocyclic complexes were synthesized and characterized by elemental analysis, spectroscopic techniques and molar conductance measurements. The interaction studies of 1-3 with calf thymus DNA (CT-DNA) were carried out by UV-vis titration, fluorescence, cyclic voltammetry and viscosity measurements. These results were further authenticated by carrying out interaction studies of 1-3 with plasmid pBR322 DNA employing gel electrophoresis. To overcome the dose resistance, auto toxicity of the drugs, a model study based on electrochemotherapy (ECT) was carried out and the results were compared in the presence and in the absence of the applied electrical potential.

  1. Synthesis and characterisation of platinum (II) salphen complex and its interaction with calf thymus DNA

    International Nuclear Information System (INIS)

    A platinum (II) salphen complex was synthesised by condensation reaction of 2,4-dihydroxylbenzaldehyde and o-phenylenediamine with potassium tetrachloroplatinate to obtain N,N′-Bis-4-(hydroxysalicylidene)-phenylenediamine-platinum (II). The structure of the complex was confirmed by 1H and 13C NMR spectroscopy, FTIR spectroscopy, CHN elemental analyses and ESI-MS spectrometry. The platinum (II) salphen complex with four donor atoms N2O2 from its salphen ligand coordinated to platinum (II) metal centre were determined. The binding mode and interaction of this complex with calf thymus DNA was determined by UV/Vis DNA titration and emission titration. The intercalation between the DNA bases by π-π stacking due to its square planar geometry and aromatic rings structures was proposed

  2. Synthesis, characterization, DNA binding and cleavage studies of chiral Ru(II) salen complexes

    Science.gov (United States)

    Khan, Noor-ul H.; Pandya, Nirali; Kureshy, Rukhsana I.; Abdi, Sayed H. R.; Agrawal, Santosh; Bajaj, Hari C.; Pandya, Jagruti; Gupte, Akashya

    2009-09-01

    Interaction of chiral Ru(II) salen complexes (S)-1 and (R)-1 with Calf Thymus DNA (CT-DNA) was studied by absorption spectroscopy, competitive binding study, viscosity measurements, CD measurements, thermal denaturation study and cleavage studies by agarose gel electrophoresis. The DNA binding affinity of (S)-1 (6.25 × 10 3 M -1) was found to be greater than (R)-1 (3.0 × 10 3 M -1). The antimicrobial studies of these complexes on five different gram (+)/(-) bacteria and three different fungal organisms showed selective inhibition of the growth of gram (+) bacteria and were not affective against gram (-) and fungal organisms. Further, the (S)-1 enantiomer inhibited the growth of organisms to a greater extent as compared to (R)-1 enantiomer.

  3. Synthesis and characterisation of platinum (II) salphen complex and its interaction with calf thymus DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sukri, Shahratul Ain Mohd; Heng, Lee Yook; Karim, Nurul Huda Abd [School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43650 Bangi, Selangor (Malaysia)

    2014-09-03

    A platinum (II) salphen complex was synthesised by condensation reaction of 2,4-dihydroxylbenzaldehyde and o-phenylenediamine with potassium tetrachloroplatinate to obtain N,N′-Bis-4-(hydroxysalicylidene)-phenylenediamine-platinum (II). The structure of the complex was confirmed by {sup 1}H and {sup 13}C NMR spectroscopy, FTIR spectroscopy, CHN elemental analyses and ESI-MS spectrometry. The platinum (II) salphen complex with four donor atoms N{sub 2}O{sub 2} from its salphen ligand coordinated to platinum (II) metal centre were determined. The binding mode and interaction of this complex with calf thymus DNA was determined by UV/Vis DNA titration and emission titration. The intercalation between the DNA bases by π-π stacking due to its square planar geometry and aromatic rings structures was proposed.

  4. Structural Insight into Processive Human Mitochondrial DNA Synthesis and Disease-Related Polymerase Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young-Sam; Kennedy, W. Dexter; Yin, Y. Whitney; (Texas)

    2010-09-07

    Human mitochondrial DNA polymerase (Pol {gamma}) is the sole replicase in mitochondria. Pol {gamma} is vulnerable to nonselective antiretroviral drugs and is increasingly associated with mutations found in patients with mitochondriopathies. We determined crystal structures of the human heterotrimeric Pol {gamma} holoenzyme and, separately, a variant of its processivity factor, Pol {gamma}B. The holoenzyme structure reveals an unexpected assembly of the mitochondrial DNA replicase where the catalytic subunit Pol {gamma}A interacts with its processivity factor primarily via a domain that is absent in all other DNA polymerases. This domain provides a structural module for supporting both the intrinsic processivity of the catalytic subunit alone and the enhanced processivity of holoenzyme. The Pol {gamma} structure also provides a context for interpreting the phenotypes of disease-related mutations in the polymerase and establishes a foundation for understanding the molecular basis of toxicity of anti-retroviral drugs targeting HIV reverse transcriptase.

  5. Synthesis, antioxidant, enzyme inhibition and DNA binding studies of novel N-benzylated derivatives of sulfonamide

    Science.gov (United States)

    Abbas, Aadil; Murtaza, Shahzad; Tahir, Muhammad Nawaz; Shamim, Saima; Sirajuddin, Muhammad; Rana, Usman Ali; Naseem, Khadija; Rafique, Hummera

    2016-08-01

    A series of novel N-benzylated derivatives of sulfonamide were synthesized and characterized by FT-IR, NMR and XRD analysis. The synthesized compounds were assayed for their biological potential. The biological studies involved antioxidant, enzyme inhibition, and DNA interaction studies. Antioxidant potential was investigated by Ferric Reducing Antioxidant Power assay (FRAP) and DPPH free radical scavenging method, the capacity of synthesized compounds to inhibit the enzyme's activity was assayed by using the well-known Elman method whereas DNA interaction studies were carried out with the help UV-Vis absorption titration method. Moreover, a direct correlation between enzyme inhibition activity and concentration of the compounds was observed both in experimental and molecular docking studies. DNA interaction studies of the synthesized compounds showed weak interaction.

  6. Synthesis of a new conjugated polymer for DNA alkylation and gene regulation.

    Science.gov (United States)

    Nie, Chenyao; Zhu, Chunlei; Feng, Liheng; Lv, Fengting; Liu, Libing; Wang, Shu

    2013-06-12

    A new polyfluorene derivative containing pendent alkylating chlorambucil (PFP-Cbl) was synthesized and characterized. Under direct incubation with DNA in vitro, PFP-Cbl could undergo an efficient DNA alkylating reaction and induce DNA cross-linking. In vitro transcription and translation experiment exhibited that the PFP-Cbl significantly down-regulated the gene expression of luciferase reporter plasmid. The down-regulation of gene expression was also verified through the transfection experiment of p-EGFP plasmid, which showed decreased green fluorescent protein (GFP) in cells. Meanwhile, the self-luminous property of PFP-Cbl could make it able to trace the internalized PFP-Cbl and plasmid complexes resulted from cross-linking in cells by fluorescent microscopy. Combining the features of alkylating function, multivalent binding sites, and fluorescent characteristics, PFP-Cbl provides a new insight in the area of gene regulation and extends the new applications of conjugated polymers (CPs). PMID:23548104

  7. Palladium polypyridyl complexes: synthesis, characterization, DNA interaction and biological activity on Leishmania (L.) mexicana

    Energy Technology Data Exchange (ETDEWEB)

    Navarro, Maribel [Instituto Venezolano de Investigaciones Cientificas, Caracas (Venezuela). Centro de Quimica; Betancourt, Adelmo [Universidad de Carabobo, Valencia (Venezuela). Facultad Experimental de Ciencia y Tecnologia. Dept. de Quimica; Hernandez, Clara [Universidad de Carabobo Sede Aragua, Maracay (Venezuela). Facultad de Ciencias de la Salud. Dept. de Ciencias Basicas; Marchan, Edgar [Universidad de Oriente, Cumana (Venezuela). Inst. de Investigaciones en Biomedicina y Ciencias Aplicadas. Nucleo de Sucre

    2008-07-01

    This paper describes the search for new potential chemotherapeutic agents based on transition metal complexes with planar ligands. In this study, palladium polypyridyl complexes were synthesized and characterized by elemental analysis, NMR, UV-VIS and IR spectroscopies. The interaction of the complexes with DNA was also investigated by spectroscopic methods. All metal-to-ligand charge transfer (MLCT) bands of the palladium polypyridyl complexes exhibited hypochromism and red shift in the presence of DNA. The binding constant and viscosity data suggested that the complexes [PdCl{sub 2}(phen)] and [PdCl{sub 2}(phendiamine)] interact with DNA by electrostatic forces. Additionally, these complexes induced an important leishmanistatic effect on L. (L.) mexicana promastigotes at the final concentration of 10 {mu}mol L{sup -1} in 48 h. (author)

  8. Palladium polypyridyl complexes: synthesis, characterization, DNA interaction and biological activity on Leishmania (L.) mexicana

    International Nuclear Information System (INIS)

    This paper describes the search for new potential chemotherapeutic agents based on transition metal complexes with planar ligands. In this study, palladium polypyridyl complexes were synthesized and characterized by elemental analysis, NMR, UV-VIS and IR spectroscopies. The interaction of the complexes with DNA was also investigated by spectroscopic methods. All metal-to-ligand charge transfer (MLCT) bands of the palladium polypyridyl complexes exhibited hypochromism and red shift in the presence of DNA. The binding constant and viscosity data suggested that the complexes [PdCl2(phen)] and [PdCl2(phendiamine)] interact with DNA by electrostatic forces. Additionally, these complexes induced an important leishmanistatic effect on L. (L.) mexicana promastigotes at the final concentration of 10 μmol L-1 in 48 h. (author)

  9. Synthesis, characterization and DNA binding of the complexes of rare earth with phenanthroline and demethylcantharate

    Institute of Scientific and Technical Information of China (English)

    ZHANG Fan; ZHU Wenzhong; LIN Qiuyue; GUO Weidong; ZHANG Lingling; LI Shikun

    2011-01-01

    Three novel rare earth complexes, [Ln2(DCA)2(phen)2](NO3)2·6H2O (Ln(Ⅲ)=Sm(Ⅲ)(1), Er(Ⅲ)(2), Yb(Ⅲ)(3); DCA2-=demethylcantharate, 7-oxabicyclo[2.2.1] heptane-2,3-dicarboxylate, C8H8O52-; phen=1,10-phenanthroline, C12H8N2) were synthesized. The structures were characterized by elemental analysis, molar conductance, IR and TGA. The results suggested that the structural features of the complexes were: in each DCA2-, one carboxylate group, as bidentate bridging group, connected two rare earth ions; the other carboxylate group, as bidentate chelate group took part in the coordination with rare earth ion. And cyclic ether oxygen of DCA2- and nitrogen atoms of phen took part in the coordination. The probable coordination number was seven. The interaction of the complexes with DNA was studied by UV-spectra, fluorescence spectra and viscosity measurements. Following increasing the concentration of DNA, the UV absorption bands nearby 265 mn of the three complexes appeared hypochromism and red-shift phenomena. And the values of binding constants Kb were 1.89× 105 L/mol (1), 3.54× 104 L/mol (2) and 3.83× 104 L/mol (3). The complexes could quench the fluorescence of EB-DNA system, and the values of equilibrium constants Ksq were 1.72(1), 0.56(2) and 1.09(3). The relative viscosity of DNA steadily decreased with increasing theconcentration of complexes. So, we could infer that the complexes may partially insert into DNA. The study of agarose gel electrophoresis showed that the complexes could cleave plasmid DNA, and the process of the reaction was through unclassical redox mechanism.

  10. Final master work plan : environmental investigations at former CCC/USDA facilities in Kansas, 2002 revision.

    Energy Technology Data Exchange (ETDEWEB)

    Burton, J. C.; Environmental Research

    2003-01-23

    The Commodity Credit Corporation (CCC) of the U.S. Department of Agriculture (USDA) has entered into an interagency agreement with the U.S. Department of Energy (DOE) under which Argonne National Laboratory provides technical assistance for hazardous waste site characterization and remediation for the CCC/USDA. Carbon tetrachloride is the contaminant of primary concern at sites in Kansas where former CCC/USDA grain storage facilities were located. Argonne applies its QuickSite(reg sign) Expedited Site Characterization (ESC) approach to these former facilities. The QuickSite environmental site characterization methodology is Argonne's proprietary implementation of the ESC process (ASTM 1998). Argonne has used this approach at several former CCC/USDA facilities in Kansas, including Agenda, Agra, Everest, and Frankfort. The Argonne ESC approach revolves around a multidisciplinary, team-oriented approach to problem solving. The basic features and steps of the QuickSite methodology are as follows: (1) A team of scientists with diverse expertise and strong field experience is required to make the process work. The Argonne team is composed of geologists, geochemists, geophysicists, hydrogeologists, chemists, biologists, engineers, computer scientists, health and safety personnel, and regulatory staff, as well as technical support staff. Most of the staff scientists are at the Ph.D. level; each has on average, more than 15 years of experience. The technical team works together throughout the process. In other words, the team that plans the program also implements the program in the field and writes the reports. More experienced scientists do not remain in the office while individuals with lesser degrees or experience carry out the field work. (2) The technical team reviews, evaluates, and interprets existing data for the site and the contaminants there to determine which data sets are technically valid and can be used in initially designing the field program. A basic

  11. Unscheduled DNA synthesis: a quantitative indicator of residual immunodetectable pyrimidine dimers in human fibroblasts after ultraviolet-B irradiation

    International Nuclear Information System (INIS)

    The authors studied the use of the rate of unscheduled DNA synthesis (UDS) as an indicator of UV-B induced DNA damage. Human fibroblasts were irradiated with UV radiation at 290, 313 or 365 nm. The LD50 was 85 J/m2 at 290 nm, 4500 J/m2 as 313 nm, and 70 kJ/m2 at 365 nm. Analysis of UDS measurements indicated complete arrest of repair processes within 24 h after irradiation, irrespective of dose (in the range 10-60 J/m2 at 290 nm, and 250-1000 J/m2 at 313 nm). Irradiation at 365 nm failed to yield detectable evidence of UDS. Incubation of irradiated cells with an antiserum directed against both 6-4 type and cyclobutane-type pyrimidine dimers shows a clear parallelism between disappearance of antibody-binding determinants and variation of the rate of UDS vs time after the end of irradiation. It is concluded that in UV-B irradiated normal cultured human fibroblasts, lack of UDS reflects absence of immunodetectable pyrimidine dimers. (author)

  12. Effect of carbenoxolone on the synthesis of glycoproteins and DNA in rat gastric epithelial cells.

    OpenAIRE

    van Huis, G A; Kramer, M.F.

    1981-01-01

    The influence of carbenoxolone on the synthesis of glycoproteins in the surface mucous cells and the production of new cells in the rat gastric mucosa was studied by means of a vascular perfusion system. The rate of incorporation of tritiated galactose, glucosamine, serine, and sulphate in surface mucous cells, studied by autoradiography, was not affected by the addition of carbenoxolone to the drinking water. The sugar composition (determined by gas-liquid chromatography) of the gastric glyc...

  13. 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.)

  14. Steroidal pyrimidines: Synthesis, characterization, molecular docking studies with DNA and in vitro cytotoxicity

    Science.gov (United States)

    Shamsuzzaman; Dar, Ayaz Mahmood; Yaseen, Zahid; Alam, Khursheed; Hussain, Altaf; Gatoo, Manzoor Ahmad

    2013-08-01

    A series of new steroid pyrimidines (7-9) were synthesized by reacting steroidal thiosemicarbazones (4-6) with diethyl malonate. The new compounds were characterized by IR, 1H NMR, 13C NMR, MS and analytical data. The interaction studies of compounds (7-9) with DNA were carried out by employing gel electrophoresis, UV-vis and fluorescence spectroscopy. The acting force between the compounds (7-9) and DNA was mainly hydrophobic while the other interactions like van der Waals, hydrogen bonding cannot be ruled out. The gel electrophoresis pattern also demonstrated that the compound 7 alone or in presence of Cu (II) causes the nicking of supercoiled pBR322 and it seems to follow the mechanistic pathway involving generation of hydroxyl radicals that are responsible for initiating DNA strand scission. The docking study of compounds (7-9) suggested that the intercalation of compounds in between the nucleotide base pairs might be due to the presence of pyrimidine moiety in steroid molecule. MTT assay was carried out to check the toxicity of new compounds (7-9) against the different human cancer as well as non-cancer cell lines A545, MCF-7, HeLa, HL-60, SW480, HepG2, HT-29, A549, 184B5, MCF10A, NL-20, HPC and HPLF. Apoptotic degradation of DNA in presence of steroidal pyrimidines (7-9) was analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining (comet assay).

  15. Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7-Substituted 7-Deazaguanine Nucleobases

    Czech Academy of Sciences Publication Activity Database

    Mačková, Michaela; Boháčová, Soňa; Perlíková, Pavla; Poštová Slavětínská, Lenka; Hocek, Michal

    2015-01-01

    Roč. 16, č. 15 (2015), s. 2225-2236. ISSN 1439-4227 R&D Projects: GA ČR GA14-04289S Institutional support: RVO:61388963 Keywords : DNA * nucleotides * polymerases * pyrrolopyrimidines Subject RIV: CC - Organic Chemistry Impact factor: 3.088, year: 2014

  16. Synthesis, Characterization and DNA Binding Affinities of Water Soluble Benzoheterocycle Triosmium Clusters

    Czech Academy of Sciences Publication Activity Database

    Rosenberg, E.; Spada, F.; Sugden, K.; Martin, B.; Milone, L.; Gobetto, R.; Viale, A.; Fiedler, Jan

    2003-01-01

    Roč. 668, 1/2 (2003), s. 51-58. ISSN 0022-328X R&D Projects: GA MŠk OC D15.10 Institutional research plan: CEZ:AV0Z4040901 Keywords : DNA * metal clusters * water soluble Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.042, year: 2003

  17. Synthesis, crystal structure, DNA interaction and anticancer activity of tridentate copper(II) complexes.

    Science.gov (United States)

    Li, Guan-Ying; Du, Ke-Jie; Wang, Jin-Quan; Liang, Jie-Wen; Kou, Jun-Feng; Hou, Xiao-Juan; Ji, Liang-Nian; Chao, Hui

    2013-02-01

    Three new tridentate copper(II) complexes [Cu(dthp)Cl(2)] (1) (dthp=2,6-di(thiazol-2-yl)pyridine), [Cu(dmtp)Cl(2)] (2) (dmtp=2,6-di(5-methyl-4H-1,2,4-triazol-3-yl)pyridine) and [Cu(dtp)Cl(2)] (3) (dtp=2,6-di(4H-1,2,4-triazol-3-yl)pyridine) have been synthesized and characterized. Crystal structure of complex 1 shows that the complex existed as distorted square pyramid with five co-ordination sites occupied by the tridentate ligand and the two chlorine anions. Ethidium bromide displacement assay, viscosity measurements, circular dichroism studies and cyclic voltammetric experiments suggested that these complexes bound to DNA via an intercalative mode. Three Cu(II) complexes were found to efficiently cleave DNA in the presence of sodium ascorbate, and singlet oxygen ((1)O(2)) and hydrogen peroxide were proved to contribute to the DNA cleavage process. They exhibited anticancer activity against HeLa, Hep-G2 and BEL-7402 cell lines. Nuclear chromatin cleavage has also been observed with AO/EB staining assay and the alkaline single-cell gel electrophoresis (comet assay). The results demonstrated that three Cu(II) complexes cause DNA damage that can induce the apoptosis of BEL-7402 cells. PMID:23186647

  18. Synthesis of new heteroaryldi(diindolyl)methanes: Colorimetric detection of DNA by di(diindolylmethyl)carbazoles

    Indian Academy of Sciences (India)

    Ramu Meesala; Rajagopal Nagarajan

    2009-03-01

    We have synthesized di(diindolylmethyl)carbazoles and di(diindolylmethyl)pyrroles by the reaction of substituted indoles with the corresponding carbazole and pyrroledicarboxaldehydes by employing a new catalyst PPh3.CF3SO3H. We have also demonstrated the utility of di(diindolylmethyl) carbazole derivatives for the colourimetric and fluorometric detection of DNA.

  19. Synthesis, DNA Binding and Topoisomerase I Inhibition Activity of Thiazacridine and Imidazacridine Derivatives

    Directory of Open Access Journals (Sweden)

    Elizabeth Almeida Lafayette

    2013-12-01

    Full Text Available Thiazacridine and imidazacridine derivatives have shown promising results as tumors suppressors in some cancer cell lines. For a better understanding of the mechanism of action of these compounds, binding studies of 5-acridin-9-ylmethylidene-3-amino-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-imidazolidin-4-one and 3-acridin-9-ylmethyl-thiazolidin-2,4-dione with calf thymus DNA (ctDNA by electronic absorption and fluorescence spectroscopy and circular dichroism spectroscopy were performed. The binding constants ranged from 1.46 × 104 to 6.01 × 104 M−1. UV-Vis, fluorescence and circular dichroism measurements indicated that the compounds interact effectively with ctDNA, both by intercalation or external binding. They demonstrated inhibitory activities to human topoisomerase I, except for 5-acridin-9-ylmethylidene-2-thioxo-1,3-thiazolidin-4-one. These results provide insight into the DNA binding mechanism of imidazacridines and thiazacridines.

  20. Plasmodium species: Flow cytometry and microfluorometry assessments of DNA content and synthesis

    NARCIS (Netherlands)

    Janse, C.J.; Vianen, P.H. van; Tanke, H.J.; Mons, B.; Ponnudurai, T.; Overdulve, J.P.

    1987-01-01

    Fluorescence intensities were established by flow cytometry of different erythrocytic stages of Plasmodium berghei after staining of their DNA with Hoechst-33258 or Hoechst-33342. Parasites were obtained from highly synchronized infections or in vitro cultures. Most fluorescence measurements were pe

  1. Synthesis of a drug delivery vehicle for cancer treatment utilizing DNA-functionalized gold nanoparticles

    Science.gov (United States)

    Brann, Tyler

    The treatment of cancer with chemotherapeutic agents has made great strides in the last few decades but still introduces major systemic side effects. The potent drugs needed to kill cancer cells often cause irreparable damage to otherwise healthy organs leading to further morbidity and mortality. A therapy with intrinsic selective properties and/or an inducible activation has the potential to change the way cancer can be treated. Gold nanoparticles (GNPs) are biocompatible and chemically versatile tools that can be readily functionalized to serve as molecular vehicles. The ability of these particles to strongly absorb light with wavelengths in the therapeutic window combined with the heating effect of surface plasmon resonance makes them uniquely suited for noninvasive heating in biologic applications. Specially designed DNA aptamers have shown their ability to serve as drug carriers through intercalation as well as directly acting as therapeutic agents. By combining these separate molecules a multifaceted drug delivery vehicle can be created with great potential as a selective and controllable treatment for cancer. Oligonucleotide-coated GNPs have been created using spherical GNPs but little work has been reported using gold nanoplates in this way. Using the Diasynth method gold nanoplates were produced to absorb strongly in the therapeutic near infrared (nIR) window. These particles were functionalized with two DNA oligonucleotides: one serving as an intercalation site for doxorubicin, and another, AS1411, serving directly as an anticancer targeting/therapeutic agent. These functional particles were fully synthesized and processed along with confirmation of DNA functionalization and doxorubicin intercalation. Doxorubicin is released via denaturation of the DNA structure into which doxorubicin is intercalated upon the heating of the gold nanoplate well above the DNA melting temperature. This temperature increase, due to light stimulation of surface plasmon

  2. Templated synthesis of monodisperse mesoporous maghemite/silica microspheres for magnetic separation of genomic DNA

    International Nuclear Information System (INIS)

    A novel method is described for the preparation of superparamagnetic mesoporous maghemite (γ-Fe2O3)/silica (SiO2) composite microspheres to allow rapid magnetic separation of DNA from biological samples. With magnetite (Fe3O4) and silica nanoparticles as starting materials, such microspheres were synthesized by the following two consecutive steps: (1) formation of monodispersed organic/inorganic hybrid microspheres through urea-formaldedyde (UF) polymerization and (2) removal of the organic template and phase transformation of Fe3O4 to γ-Fe2O3 by calcination at elevated temperatures. The as-synthesized particles obtained by heating at temperature 300 deg. C feature spherical shape and uniform particle size (dparticle=1.72 μm), high saturation magnetization (Ms=17.22 emu/g), superparamagnetism (Mr/Ms=0.023), high surface area (SBET=240 m2/g), and mesoporosity (dpore=6.62 nm). The composite microsphere consists of interlocked amorphous SiO2 nanoparticles, in which cubic γ-Fe2O3 nanocrystals are homogeneously dispersed and thermally stable against γ- to α-phase transformation at temperatures up to 600 deg. C. With the exposed iron oxide nanoparticles coated with a thin layer of silica shell, the magnetic microspheres were used as a solid-phase adsorbent for rapid extraction of genomic DNA from plant samples. The results show that the DNA templates isolated from pea and green pepper displayed single bands with molecular weights greater than 8 kb and A260/A280 values of 1.60-1.72. The PCR amplification of a fragment encoding the endogenous chloroplast ndhB gene confirmed that the DNA templates obtained were inhibitor-free and amenable to sensitive amplification-based DNA technologies.

  3. 7 CFR 1486.301 - How is the working relationship established between CCC and the Recipient of program funding?

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false How is the working relationship established between... relationship established between CCC and the Recipient of program funding? (a) FAS will send an approval letter... agreement and submit it to the Director, Marketing Operations Staff, FAS, USDA. The applicant may not...

  4. Highly Active and Durable Co-Doped Pt/CCC Cathode Catalyst for Polymer Electrolyte Membrane Fuel Cells

    International Nuclear Information System (INIS)

    Highlights: •Co-doped Pt core–shell type catalyst having 0.75 nm thick Pt shell is synthesized. •Co-doped Pt exhibited mass activity of 0.44 A mgPt−1 at 0.9 ViR-free. •Co-doped Pt cathode catalyst showed high stability under cycling conditions. •Co-doped Pt catalyst showed only 16% power density loss after 30,000 cycles. •The enhanced stability is due to the increase in onset potential for PtO2 formation. -- Abstract: Cathode catalyst based on Co-doped Pt deposited on carbon composite catalyst (CCC) support with high measured activity and stability under potential cycling conditions for polymer electrolyte membrane (PEM) fuel cells was developed in this study. The catalyst was synthesized through platinum deposition on Co-doped CCC support containing pyridinic-nitrogen active sites followed by controlled heat-treatment. High resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) studies confirmed uniform Pt deposition (Pt/CCC catalyst, dPt = 2 nm) and formation of Co-doped Pt/CCC catalyst (dPt = 5.4 nm) respectively. X-ray energy dispersive spectrometry (XEDS) line-scan studies showed the formation of Co-core Pt-shell type catalyst with a Pt-shell thickness of ∼0.75 nm. At 0.9 ViR-free, the Co-doped Pt/CCC catalyst showed initial mass activity of 0.44 A mgPt−1 and 0.25 A mgPt−1 after 30,000 potential cycles between 0.6 and 1.0 V corresponding to an overall measured activity loss of 42.8%. The commercial Pt-Co/C showed initial mass activity of 0.38 A mgPt−1 and ∼70% loss of activity after 30,000 cycles. The enhanced catalytic activity at high potentials and stability of mass activity for the Co-doped Pt/CCC catalyst are attributed to the formation of compressive Pt lattice catalyst due to Co doping. The Co-doped Pt/CCC showed stable open circuit potential close to 1.0 V under H2-air with an initial power density of 857 mW cm−2 and only 16% loss after 30,000 cycles. Catalyst durability studies performed between 0

  5. Synthesis, characterization and DNA cleavage activity of nickel(II adducts with aromatic heterocyclic bases

    Directory of Open Access Journals (Sweden)

    G. H. PHILIP

    2010-01-01

    Full Text Available Mixed ligand complexes of nickel(II with 2,4-dihydroxyaceto-phenone oxime (DAPO and 2,4-dihydroxybenzophenone oxime (DBPO as primary ligands, and pyridine (Py and imidazole (Im as secondary ligands were synthesized and characterized by molar conductivity, magnetic moments measurements, as well as by electronic, IR, and 1H-NMR spectroscopy. Electrochemical studies were performed by cyclic voltammetry. The active signals are assignable to the NiIII/II and NiII/I redox couples. The binding interactions between the metal complexes and calf thymus DNA were investigated by absorption and thermal denaturation. The cleavage activity of the complexes was determined using double-stranded pBR322 circular plasmid DNA by gel electrophoresis. All complexes showed increased nuclease activity in the presence of the oxidant H2O2. The nuclease activities of mixed ligand complexes were compared with those of the parent copper(II complexes.

  6. Synthesis, Cytotoxicity, DNA Binding and Apoptosis of Rhein-Phosphonate Derivatives as Antitumor Agents

    Directory of Open Access Journals (Sweden)

    Man-Yi Ye

    2013-04-01

    Full Text Available Several rhein-phosphonate derivatives (5a–c were synthesized and evaluated for in vitro cytotoxicity against HepG-2, CNE, Spca-2, Hela and Hct-116 cell lines. Some compounds showed relatively high cytotoxicity. Especially compounds 5b exhibited the strongest cytotoxicity against HepG-2 and Spca-2 cells (IC50 was 8.82 and 9.01 µM, respectively. All the synthesized compounds exhibited low cytotoxicity against HUVEC cells. Further experiments proved that 5b could disturb the cell cycle in HepG-2 cells and induce apoptosis. In addition, the binding properties of a model conjugate 5b to DNA were investigated by methods (UV-Vis, fluorescence, CD spectroscopy. Results indicated that 5b showed moderate ability to interact ct-DNA.

  7. Synthesis of isatin thiosemicarbazones derivatives: In vitro anti-cancer, DNA binding and cleavage activities

    Science.gov (United States)

    Ali, Amna Qasem; Teoh, Siang Guan; Salhin, Abdussalam; Eltayeb, Naser Eltaher; Khadeer Ahamed, Mohamed B.; Majid, A. M. S. Abdul

    New derivatives of thiosemicarbazone Schiff base with isatin moiety were synthesized L1-L6. The structures of these compounds were characterized based on the spectroscopic techniques. Compound L6 was further characterized by XRD single crystal. The interaction of these compounds with calf thymus (CT-DNA) exhibited high intrinsic binding constant (kb = 5.03-33.00 × 105 M-1) for L1-L3 and L5 and (6.14-9.47 × 104 M-1) for L4 and L6 which reflect intercalative activity of these compounds toward CT-DNA. This result was also confirmed by the viscosity data. The electrophoresis studies reveal the higher cleavage activity of L1-L3 than L4-L6. The in vitro anti-proliferative activity of these compounds against human colon cancer cell line (HCT 116) revealed that the synthesized compounds (L3, L6 and L2) exhibited good anticancer potency.

  8. Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases

    OpenAIRE

    Wang, Xuxiang; Zhang, Jianye; Li, Yingjia; Chen, Gang; Wang, Xiaolong

    2015-01-01

    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 on...

  9. Thermodynamics of translesion synthesis across a major DNA adduct of antitumor oxaliplatin: Differential scanning calorimetric study

    Czech Academy of Sciences Publication Activity Database

    Florian, Jakub; Brabec, Viktor

    2012-01-01

    Roč. 18, č. 6 (2012), s. 1634-1639. ISSN 0947-6539 R&D Projects: GA ČR(CZ) GD301/09/H004; GA ČR(CZ) GAP301/10/0598 Institutional research plan: CEZ:AV0Z50040702 Keywords : antitumor platinum * DNA * calorimetry Subject RIV: BO - Biophysics Impact factor: 5.831, year: 2012

  10. Synthesis, DNA Binding and Antitumor Evaluation of Styelsamine and Cystodytin Analogues

    Directory of Open Access Journals (Sweden)

    Hugo K. H. Fong

    2013-01-01

    Full Text Available A series of N-14 sidechain substituted analogues of styelsamine (pyrido[4,3,2-mn]acridine and cystodytin (pyrido[4,3,2-mn]acridin-4-one alkaloids have been prepared and evaluated for their DNA binding affinity and antiproliferative activity towards a panel of human tumor cell lines. Overall it was found that styelsamine analogues were stronger DNA binders, with the natural products styelsamines B and D having particularly high affinity (Kapp 5.33 × 106 and 3.64 × 106 M−1, respectively. In comparison, the cystodytin iminoquinone alkaloids showed lower affinity for DNA, but were typically just as active as styelsamine analogues at inhibiting proliferation of tumor cells in vitro. Sub-panel selectivity towards non-small cell lung, melanoma and renal cancer cell lines were observed for a number of the analogues. Correlation was observed between whole cell activity and clogP, with the most potent antiproliferative activity being observed for 3-phenylpropanamide analogues 37 and 41 (NCI panel average GI50 0.4 μM and 0.32 μM, respectively with clogP ~4.0–4.5.

  11. Synthesis, spectral characterization, DNA interaction, radical scavenging and cytotoxicity studies of ruthenium(II) hydrazone complexes.

    Science.gov (United States)

    Mohanraj, Maruthachalam; Ayyannan, Ganesan; Raja, Gunasekaran; Jayabalakrishnan, Chinnasamy

    2016-05-01

    Three new ruthenium(II) complexes with hydrazone ligands, furan-2-carboxylic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(1)), furan-2-carboxylic acid [4-(ethyl-propyl-amino)-2-hydroxy-benzylidene]-hydrazide (HL(2)) and furan-2-carboxylic acid (3-ethoxy-2-hydroxy-benzylidene)-hydrazide (HL(3)) were synthesized and characterized by various spectro-analytical techniques. The hydrazone ligands act as a tridendate ligand with ONO as the donor sites and are preferably found in the enol form in all the complexes. The molecular structure of the ligands was determined by single crystal X-ray diffraction technique. The interaction of the ligands and the complexes with CT-DNA were evaluated by an absorption titration method which revealed that the compounds interact with CT-DNA through intercalation. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the calf thymus DNA hydrolytically. Antioxidant studies showed that the ruthenium(II) complexes have a strong radical-scavenging properties. Further, the cytotoxic effect of the compounds examined on cancerous cell lines showed that the complexes exhibited substantial anticancer activity. PMID:26974577

  12. Quantification of covalently closed circular DNA of hepatitis B virus in FFPE liver tissues of chronic hepatitis B patients

    OpenAIRE

    Jia-qi HAN; Yan-wei ZHONG; Xiao-qiang REN; Zheng-sheng ZOU; Liu, Shu-Hong; Xue-en LIU; Jing-min ZHAO; Dong-ping XU

    2011-01-01

    Objective To establish a method of detecting HBV covalently closed circular DNA(cccDNA) in micro-formalin fixed paraffin imbedding(FFPE) liver biopsy samples.Methods FFPE liver biopsies from 37 patients with chronic hepatitis B were studied.The intrahepatic HBV DNA was extracted and pre-treated with plasmid-safe ATP-dependent DNAse(PSAD),and then amplified by rolling circular amplification(RCA).The HBV cccDNA was quantitatively detected by Taqman real-time PCR with primers located on both sid...

  13. Detection of the covalently closed circular DNA in peripheral blood mononuclear cells of hepatitis B patients and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    朱圣涛

    2014-01-01

    Objective To analyze the correlation between covalently closed circular DNA(ccc DNA)in the peripheral blood mononuclear cells(PBMC)of hepatitis B virus(HBV)-infected patients and serum HBV DNA,hepatitis B surface antigen(HBsA g),hepatitis B e antigen(HBe Ag)and liver histology of hepatitis B patients,and to explore the clinical significance of HBV ccc DNA detection in PBMC.Methods One hundred and eight patients with chronic HBV infection were involved in this

  14. Peripheral SLC6A4 DNA methylation is associated with in vivo measures of human brain serotonin synthesis and childhood physical aggression.

    Directory of Open Access Journals (Sweden)

    Dongsha Wang

    Full Text Available The main challenge in addressing the role of DNA methylation in human behaviour is the fact that the brain is inaccessible to epigenetic analysis in living humans. Using positron emission tomography (PET measures of brain serotonin (5-HT synthesis, we found in a longitudinal sample that adult males with high childhood-limited aggression (C-LHPA had lower in vivo 5-HT synthesis in the orbitofrontal cortex (OBFC. Here we hypothesized that 5-HT alterations associated with childhood aggression were linked to differential DNA methylation of critical genes in the 5-HT pathway and these changes were also detectable in peripheral white blood cells. Using pyrosequencing, we determined the state of DNA methylation of SLC6A4 promoter in T cells and monocytes isolated from blood of cohort members (N = 25 who underwent a PET scan, and we examined whether methylation status in the blood is associated with in vivo brain 5-HT synthesis. Higher levels of methylation were observed in both T cells and monocytes at specific CpG sites in the C-LHPA group. DNA methylation of SLC6A4 in monocytes appears to be associated more reliably with group membership than T cells. In both cell types the methylation state of these CpGs was associated with lower in vivo measures of brain 5-HT synthesis in the left and right lateral OBFC (N = 20 where lower 5-HT synthesis in C-LHPA group was observed. Furthermore, in vitro methylation of the SLC6A4 promoter in a luciferase reporter construct suppresses its transcriptional activity supporting a functional role of DNA methylation in SLC6A4 promoter regulation. These findings indicate that state of SLC6A4 promoter methylation is altered in peripheral white blood cells of individuals with physical aggression during childhood. This supports the relevance of peripheral DNA methylation for brain function and suggests that peripheral SLC6A4 DNA methylation could be a marker of central 5-HT function.

  15. Final report : phase I investigation at the former CCC/USDA grain storage facility in Savannah, Missouri.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M.; Environmental Science Division

    2010-08-05

    From approximately 1949 until 1970, the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) operated a grain storage facility on federally owned property approximately 0.25 mi northwest of Savannah, Missouri (Figure 1.1). During this time, commercial grain fumigants containing carbon tetrachloride were commonly used by the CCC/USDA and the private grain storage industry to preserve grain in their facilities. In November 1998, carbon tetrachloride was detected in a private well (Morgan) roughly 50 ft south of the former CCC/USDA facility, as a result of state-wide screening of private wells near former CCC/USDA facilities, conducted in Missouri by the U.S. Environmental Protection Agency (EPA 1999). The 1998 and subsequent investigations by the EPA and the Missouri Department of Natural Resources (MoDNR) confirmed the presence of carbon tetrachloride in the Morgan well, as well as in a second well (on property currently owned and occupied by the Missouri Department of Transportation [MoDOT]), described as being approximately 400 ft east of the former CCC/USDA facility. The identified concentrations in these two wells were above the EPA maximum contaminant level (MCL) and the default target level (DTL) values of 5.0 {micro}g/L for carbon tetrachloride in water used for domestic purposes (EPA 1999; MoDNR 2000a,b, 2006). (The DTL is defined in Section 4.) Because the observed contamination in the Morgan and MoDOT wells might be linked to the past use of carbon tetrachloride-based fumigants at its former grain storage facility, the CCC/USDA is conducting an investigation to (1) characterize the source(s), extent, and factors controlling the subsurface distribution and movement of carbon tetrachloride at Savannah and (2) evaluate the potential risks to human health, public welfare, and the environment posed by the contamination. This work is being performed in accord with the Intergovernmental Agreement established between the Farm Service

  16. Peptidyl anthraquinones as potential antineoplastic drugs: synthesis, DNA binding, redox cycling, and biological activity.

    Science.gov (United States)

    Gatto, B; Zagotto, G; Sissi, C; Cera, C; Uriarte, E; Palù, G; Capranico, G; Palumbo, M

    1996-08-01

    A series of new compounds containing a 9,10-anthracenedione moiety and one or two peptide chains at position 1 and/or 4 have been synthesized. The amino acid residues introduced are glycine (Gly), lysine (Lys), and tryptophan (Trp), the latter two in both the L- and D-configurations. The peptidyl anthraquinones maintain the ability of intercalating efficiently into DNA, even though the orientation within the base-pair pocket may change somewhat with reference to the parent drugs mitoxantrone (MX) and ametantrone (AM). The interaction constants of the mono-, di-, and triglycyl derivatives are well comparable to those found for AM but 5-10 times lower than the value reported for MX. On the other hand, the glycyl-lysyl compounds bind DNA to the same extent as (L-isomer) or even better than (D-isomer) MX. As for the parent drugs without peptidyl chains, the new compounds prefer alternating CG binding sites, although to different extents. The bis-Gly-Lys derivatives are the least sensitive to base composition, which may be due to extensive aspecific charged interactions with the polynucleotide backbone. As far as redox properties are concerned, all peptidyl anthraquinones show a reduction potential very close to that of AM and 60-80 mV less negative than that of MX; hence, they can produce free-radical-damaging species to an extent similar to the parent drugs. The biological activity has been tested in human tumor and murine leukemia cell lines. Most of the test anthraquinones exhibit cytotoxic properties close to those of AM and considerably lower than those of MX. Stimulation of topoisomerase-mediated DNA cleavage is moderately present in representatives of the glycylanthraquinone family, whereas inhibition of the background cleavage occurs when Lys is present in the peptide chain. For most of the test anthraquinones, the toxicity data are in line with the DNA affinity scale and the topoisomerase II stimulation activity. However, in the lysyl derivatives, for which

  17. Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus)

    International Nuclear Information System (INIS)

    Ultraviolet (uv)-induced unscheduled DNA synthesis of xeroderma pigmentosum cells, belonging to complementation groups, A, B, C, D, and E, was restored to the normal level by concomitant treatment of the cells with T4 endonuclease V and uv-inactivated HVJ (Sendai virus). The present results suggest that T4 endonuclease molecules were inserted effectively into the cells by the interaction of HVJ with the cell membranes, the enzyme was functional on human chromosomal DNA which had been damaged by uv irradiation in the viable cells, all the studied groups of xeroderma pigmentosum (variant was not tested) were defective in the first step (incision) of excision repair

  18. Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups.

    OpenAIRE

    Jonge, A.J.R.; Vermeulen, Wim; Keijzer, W.; Hoeijmakers, Jan; 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 the UV-induced UDS, in some cells to the repair-proficient human level, was observed. Another prokaryotic DNA-repair enzyme, T4 endonuclease V, restored the UV-induced UDS in a similar way after mi...

  19. Effects of microinjected photoreactivating enzyme on thymine dimer removal and DNA repair synthesis in normal human and xeroderma pigmentosum fibroblasts

    International Nuclear Information System (INIS)

    UV-induced thymine dimers (10 J/m2 of UV-C) were assayed in normal human and xeroderma pigmentosum (XP) fibroblasts with a monoclonal antibody against these dimers and quantitative fluorescence microscopy. In repair-proficient cells dimer-specific immunofluorescence gradually decreased with time, reaching about 25% of the initial fluorescence after 27 h. Rapid disappearance of dimers was observed in cells which had been microinjected with yeast photoreactivating enzyme prior to UV irradiation. This photoreactivation (PHR) was light dependent and (virtually) complete within 15 min of PHR illumination. In general, PHR of dimers strongly reduces UV-induced unscheduled DNA synthesis (UDS). However, when PHR was applied immediately after UV irradiation, UDS remained unchanged initially; the decrease set in only after 30 min. When PHR was performed 2 h after UV exposure, UDS dropped without delay. An explanation for this difference is preferential removal of some type(s) of nondimer lesions, which is responsible for the PHR-resistant UDS immediately following UV irradiation. After the rapid removal of these photoproducts, the bulk of UDS is due to dimer repair. From the rapid effect of dimer removal by PHR on UDS it can be deduced that the excision of dimers up to the repair synthesis step takes considerably less than 30 min. Also in XP fibroblasts of various complementation groups the effect of PHR was investigated. The immunochemical dimer assay showed rapid PHR-dependent removal comparable to that in normal cells. However, the decrease of (residual) UDS due to PHR was absent (in XP-D) or much delayed (in XP-A and -E) compared to normal cells. This supports the idea that in these XP cells preferential repair of nondimer lesions does occur, but at a much lower rate

  20. Macrocyclic nickel(II) complexes: Synthesis, characterization, superoxide scavenging activity and DNA-binding

    Science.gov (United States)

    Ramadan, Abd El-Motaleb M.

    2012-05-01

    A new series of nickel(II) complexes with the tetraaza macrocyclic ligand have been synthesized as possible functional models for nickel-superoxide dismutase enzyme. The reaction of 5-amino-3-methyl-1-phenylpyrazole-4-carbaldehyde (AMPC) with itself in the presence of nickel(II) ion yields, the new macrocyclic cationic complex, [NiL(NO3)2], containing a ligand composed of the self-condensed AMPC (4 mol) bound to a single nickel(II) ion. A series of metathetical reactions have led to the isolation of a number of newly complexes of the types [NiL]X2; X = ClO4 and BF4, [NiLX2], X = Cl and Br (Scheme 1). Structures and characterizations of these complexes were achieved by several physicochemical methods namely, elemental analysis, magnetic moment, conductivity, and spectral (IR and UV-Vis) measurements. The electrochemical properties and thermal behaviors of these chelates were investigated by using cyclic voltammetry and thermogravimetric analysis (TGA and DTG) techniques. A distorted octahedral stereochemistry has been proposed for the six-coordinate nitrato, and halogeno complexes. For the four-coordinate, perchlorate and fluoroborate, complex species a square-planar geometry is proposed. The measured superoxide dismutase mimetic activities of the complexes indicated that they are potent NiSOD mimics and their activities are compared with those obtained previously for nickel(II) complexes. The probable mechanistic implications of the catalytic dismutation of O2rad - by the synthesized nickel(II) complexes are discussed. The DNA-binding properties of representative complexes [NiLCl2] and [NiL](PF4)2 have been investigated by the electronic absorption and fluorescence measurements. The results obtained suggest that these complexes bind to DNA via an intercalation binding mode and the binding affinity for DNA follows the order: [NiLCl2] □ [NiL](PF4)2.

  1. 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 of both sexes, using 3H-thymidine and radioautography were studied. The labeled cells frequency and proportion were determined and the data were statiscally analysed. The labeled cells frenquency is higher in female than in male animals, but difference is statiscally significant for adult animals only; this result suggests a hormonal influence, possibly of estrogen on the epithelial tissue. (Author)

  2. DNA Three Way Junction Core Decorated with Amino Acids-Like Residues-Synthesis and Characterization.

    Science.gov (United States)

    Addamiano, Claudia; Gerland, Béatrice; Payrastre, Corinne; Escudier, Jean-Marc

    2016-01-01

    Construction and physico-chemical behavior of DNA three way junction (3WJ) functionalized by protein-like residues (imidazole, alcohol and carboxylic acid) at unpaired positions at the core is described. One 5'-C(S)-propargyl-thymidine nucleotide was specifically incorporated on each strand to react through a post synthetic CuACC reaction with either protected imidazolyl-, hydroxyl- or carboxyl-azide. Structural impacts of 5'-C(S)-functionalization were investigated to evaluate how 3WJ flexibility/stability is affected. PMID:27563857

  3. Synthesis of biaryl-substituted fluorescent nucleosides and nucleoside triphosphates and their incorporation to DNA

    Czech Academy of Sciences Publication Activity Database

    Riedl, Jan; Hocek, Michal

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i., 2011 - (Hocek, M.), s. 233-235 ISBN 978-80-86241-37-1. - (Collection Symposium Series. 12). [ Chemistry of Nucleic Acid Components /15./. Český Krumlov (CZ), 05.06.2011-10.06.2011] R&D Projects: GA MŠk LC512; GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleotides * fluorescence * DNA Subject RIV: CC - Organic Chemistry

  4. Polymerase synthesis of base-modified DNA: New methods and new applications

    Czech Academy of Sciences Publication Activity Database

    Balintová, Jana; Daďová, Jitka; Kielkowski, Pavel; Ménová, Petra; Vaníková, Zuzana; Riedl, Jan; Raindlová, Veronika; Fojta, Miroslav; Hocek, Michal

    Praha: Institute of Organic Chemistry and Biochemistry AS CR, v. v. i, 2014 - (Hocek, M.), s. 66-67. (Collection Symposium Series. 14). ISBN 978-80-86241-50-0. [Symposium on Chemistry of Nucleic Acid Components /16./. Český Krumlov (CZ), 08.06.2014-13.06.2014] R&D Projects: GA ČR GA203/09/0317; GA ČR GBP206/12/G151; GA ČR GA14-04289S Institutional support: RVO:61388963 ; RVO:68081707 Keywords : nucleic acids * base-modified DNA * dNTPs Subject RIV: CC - Organic Chemistry; BO - Biophysics (BFU-R)

  5. Synthesis, DNA-binding, photocleavage, cytotoxicity and antioxidant activity of ruthenium (II) polypyridyl complexes.

    Science.gov (United States)

    Liu, Yun-Jun; Zeng, Cheng-Hui; Huang, Hong-Liang; He, Li-Xin; Wu, Fu-Hai

    2010-02-01

    Two new ligands maip (1a), paip (1b) with their ruthenium (II) complexes [Ru(bpy)(2)(maip)](ClO(4))(2) (2a) and [Ru(bpy)(2)(paip)](ClO(4))(2) (2b) have been synthesized and characterized. The results show that complexes 2a and 2b interact with DNA through intercalative mode. The cytotoxicity of these compounds has been evaluated by MTT assay. The experiments on antioxidant activity show that these compounds exhibit good antioxidant activity against hydroxyl radical (OH). PMID:19932529

  6. Normal inhibition of DNA synthesis following γ-irradiation of radiosensitive cell lines from patients with Down's syndrome and Alzheimer's disease

    International Nuclear Information System (INIS)

    Inhibition of DNA synthesis was studied in γ-iradiated lymphoblastoid cells from patients with Alzheimer's disease and Down's syndrome. A normal biphasic pattern of inhibition was observed over a dose range of 0-4 krad of γ-rays in all of the cell lines 3 out of 4 Down's and all the Alzheimer's cell lines were shown to be hypersensitive to ionizing radiation based on induced chromosomal aberrations. Increased G2 phase delay, comparable to that occurring in ataxia-telangiectasia cells, was observed for some of the cell lines, after exposure to γ-rays. Contrary to other data in the literature these results demonstrate that radioresistand DNA synthesis is not an intrinsic feature of all disorders characterized by radiosensitivitey. (author).; 25 refs.; 2 figs.; 1 tab

  7. Utilization of 3H from deoxyuridine and thymidine for synthesis of DNA and other macromolecules in various organs of the rat

    International Nuclear Information System (INIS)

    The reported investigation showed that liver and intestinal mucosa, but not spleen or thymus of ACI strain rats incorporated significant amounts of 3H from deoxyuridine 6-3H and thymidine- methyl-3H into a RNA fraction obtained by alkaline hydrolysis of tissue homogenates. In bone marrow, 3H for thymidine but not from deoxyuridine was incorporated into this fraction. Inhibition of DNA synthesis by 5-fluorouracils (5-FU) increased 3H in this RNA fraction for only intestinal mucosa and bone marrow, while enhancement of DNA synthesis during recovery from 5-FU toxicity was associated with an increase in 3H for the alkali-solubilized fractions of liver, intestinal mucosa, spleen and thymus but not Morris hepatoma 3924A. (U.K.)

  8. Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI

    International Nuclear Information System (INIS)

    The authors demonstrated the existence of human cytokine synthesis inhibitory factor (DSIF) [interleukin 10 (IL-10)]. cDNA clones encoding human IL-10 (hIL-10) were isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus, BDRFL. hIL-10 and the BCRFI product inhibit cytokine synthesis by activated human peripheral blood mononuclear cells and by a mouse Th1 clone. Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but BCRFI lacks comparable activity in this way, suggesting that BCRFI may have conserved only a subset of hIL-10 activities

  9. Final work plan : supplemental upward vapor intrusion investigation at the former CCC/USDA grain storage facility in Hanover, Kansas.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M.; Environmental Science Division

    2008-12-15

    The Commodity Credit Corporation (CCC), an agency of the U.S. Department of Agriculture (USDA), operated a grain storage facility at the northeastern edge of the city of Hanover, Kansas, from 1950 until the early 1970s. During this time, commercial grain fumigants containing carbon tetrachloride were in common use by the grain storage industry to preserve grain in their facilities. In February 1998, trace to low levels of carbon tetrachloride (below the maximum contaminant level [MCL] of 5.0 {micro}g/L) were detected in two private wells near the former grain storage facility at Hanover, as part of a statewide USDA private well sampling program that was implemented by the Kansas Department of Health and Environment (KDHE) near former CCC/USDA facilities. In 2007, the CCC/USDA conducted near-surface soil sampling at 61 locations and also sampled indoor air at nine residences on or adjacent to its former Hanover facility to address the residents concerns regarding vapor intrusion. Low levels of carbon tetrachloride were detected at four of the nine homes. The results were submitted to the KDHE in October 2007 (Argonne 2007). On the basis of the results, the KDHE requested sub-slab sampling and/or indoor air sampling (KDHE 2007). This Work Plan describes, in detail, the proposed additional scope of work requested by the KDHE and has been developed as a supplement to the comprehensive site investigation work plan that is pending (Argonne 2008). Indoor air samples collected previously from four homes at Hanover were shown to contain the carbon tetrachloride at low concentrations (Table 2.1). It cannot be concluded from these previous data that the source of the detected carbon tetrachloride is vapor intrusion attributable to former grain storage operations of the CCC/USDA at Hanover. The technical objective of the vapor intrusion investigation described here is to assess the risk to human health due to the potential for upward migration of carbon tetrachloride and

  10. Synthesis, Crystal Structures, and DNA Binding Properties of Zinc(II Complexes with 3-Pyridine Aldoxime

    Directory of Open Access Journals (Sweden)

    Konstantis F. Konidaris

    2010-01-01

    Full Text Available The employment of 3-pyridine aldoxime, (3-pyCHNOH, in ZnII chemistry has afforded two novel compounds: [Zn(acac2{(3-pyCHNOH}]⋅H2O (1⋅H2O [where acac- is the pentane-2,4-dionato(-1 ion] and [Zn2(O2CMe4{(3-pyCHNOH}2] (2. Complex 1⋅H2O crystallizes in the monoclinic space group P21/n. The ZnII ion is five-coordinated, surrounded by four oxygen atoms of two acac- moieties and by the pyridyl nitrogen atom of the (3-pyCHNOH ligand. Molecules of 1 interact with the water lattice molecules forming a 2D hydrogen-bonding network. Complex 2 crystallizes in the triclinic P-1 space group and displays a dinuclear paddle-wheel structure. Each ZnII exhibits a perfect square pyramidal geometry, with four carboxylate oxygen atoms at the basal plane and the pyridyl nitrogen of one monodentate (3-pyCHNOH ligand at the apex. DNA mobility shift assays were performed for the determination of the in vitro effect of both complexes on the integrity and the electrophoretic mobility of pDNA.

  11. Synthesis, structural characterization, cytotoxic properties and DNA binding of a dinuclear copper(II) complex.

    Science.gov (United States)

    Ferreira, B J M Leite; Brandão, P; Meireles, M; Martel, Fátima; Correia-Branco, Ana; Fernandes, Diana M; Santos, T M; Félix, V

    2016-08-01

    In this study a novel dinuclear copper(II) complex with adenine and phenanthroline has been synthesized and its structure determined by single crystal X-ray diffraction. In the dinuclear complex [Cu₂(μ-adenine)₂(phen)₂(H2O)2](NO3)4·0.5H2O (phen=1,10-phenanthroline) (1) the two Cu(II) centres exhibit a distorted square pyramidal coordination geometry linked by two nitrogen donors from adenine bridges leading to a Cu-Cu distance of 3.242(3)Å. Intramolecular and intermolecular π⋯π interactions as well as an H-bonding network were observed. The antitumor capacity of the complex has been tested in vitro against human cancer cell lines, cervical carcinoma (HeLa) and colorectal adenocarcinoma (Caco-2), by metabolic tests, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide as reagent. The complex 1 has remarkable low IC50 values of 0.87±0.06μM (HeLa) and 0.44±0.06μM (Caco-2), when compared with values for cisplatin against the same cell lines. The interaction of complex 1 with calf thymus DNA (CT DNA) was further investigated by absorption and fluorescence spectroscopic methods. A binding constant of 5.09×10(5)M(-1) was obtained from UV-vis absorption studies. PMID:27157979

  12. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

    Energy Technology Data Exchange (ETDEWEB)

    Sinnett-Smith, James; Kisfalvi, Krisztina; Kui, Robert [Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, David Geffen School of Medicine and Molecular Biology Institute, University of California at Los Angeles, CA (United States); Rozengurt, Enrique, E-mail: erozengurt@mednet.ucla.edu [Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, David Geffen School of Medicine and Molecular Biology Institute, University of California at Los Angeles, CA (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Metformin inhibits cancer cell growth but the mechanism(s) are not understood. Black-Right-Pointing-Pointer We show that the potency of metformin is sharply dependent on glucose in the medium. Black-Right-Pointing-Pointer AMPK activation was enhanced in cancer cells incubated in physiological glucose. Black-Right-Pointing-Pointer Reciprocally, metformin potently inhibited mTORC1, DNA synthesis and proliferation. Black-Right-Pointing-Pointer Metformin, at low concentrations, inhibited DNA synthesis through AMPK. -- Abstract: Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser{sup 79} and Raptor at Ser{sup 792}, was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05-0.1 mM) that were 10-100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the {alpha}{sub 1} and {alpha}{sub 2} catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.

  13. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

    International Nuclear Information System (INIS)

    Highlights: ► Metformin inhibits cancer cell growth but the mechanism(s) are not understood. ► We show that the potency of metformin is sharply dependent on glucose in the medium. ► AMPK activation was enhanced in cancer cells incubated in physiological glucose. ► Reciprocally, metformin potently inhibited mTORC1, DNA synthesis and proliferation. ► Metformin, at low concentrations, inhibited DNA synthesis through AMPK. -- Abstract: Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser79 and Raptor at Ser792, was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05–0.1 mM) that were 10–100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the α1 and α2 catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.

  14. Synthesis, spectral characterization and eukaryotic DNA degradation of thiosemicarbazones and their platinum(IV) complexes

    Science.gov (United States)

    Al-Hazmi, G. A.; El-Metwally, N. M.; El-Gammal, O. A.; El-Asmy, A. A.

    2008-01-01

    The condensation products of acetophenone (or its derivatives), salicylaldehyde and o-hydroxy- p-methoxybenzophenone with thiosemicarbazide and ethyl- or phenyl-thiosemicarbazide are the investigated thiosemicarbazones. Their reactions with H 2PtCl 6 produced Pt(IV) complexes characterized by elemental, thermal, mass, IR and electronic spectral studies. The coordination modes were found mononegative bidentate in the acetophenone derivatives and binegative tridentate in the salicylaldehyde derivatives. The complexes were analyzed thermogravimetrically and found highly stable. Some ligands and their complexes were screened against Sarcina sp. and E. coli using the cup-diffusion technique. [Pt( oHAT)(OH)Cl] shows higher activity against E. coli than the other compounds. The degradation power of the tested compounds on the calf thymus DNA supports their selectivity against bacteria and not against the human or related eukaryotic organisms.

  15. Final master work plan : environmental investigations at former CCC/USDA facilities in Kansas, 2002 revision.

    Energy Technology Data Exchange (ETDEWEB)

    Burton, J. C.; Environmental Research

    2003-01-23

    The Commodity Credit Corporation (CCC) of the U.S. Department of Agriculture (USDA) has entered into an interagency agreement with the U.S. Department of Energy (DOE) under which Argonne National Laboratory provides technical assistance for hazardous waste site characterization and remediation for the CCC/USDA. Carbon tetrachloride is the contaminant of primary concern at sites in Kansas where former CCC/USDA grain storage facilities were located. Argonne applies its QuickSite(reg sign) Expedited Site Characterization (ESC) approach to these former facilities. The QuickSite environmental site characterization methodology is Argonne's proprietary implementation of the ESC process (ASTM 1998). Argonne has used this approach at several former CCC/USDA facilities in Kansas, including Agenda, Agra, Everest, and Frankfort. The Argonne ESC approach revolves around a multidisciplinary, team-oriented approach to problem solving. The basic features and steps of the QuickSite methodology are as follows: (1) A team of scientists with diverse expertise and strong field experience is required to make the process work. The Argonne team is composed of geologists, geochemists, geophysicists, hydrogeologists, chemists, biologists, engineers, computer scientists, health and safety personnel, and regulatory staff, as well as technical support staff. Most of the staff scientists are at the Ph.D. level; each has on average, more than 15 years of experience. The technical team works together throughout the process. In other words, the team that plans the program also implements the program in the field and writes the reports. More experienced scientists do not remain in the office while individuals with lesser degrees or experience carry out the field work. (2) The technical team reviews, evaluates, and interprets existing data for the site and the contaminants there to determine which data sets are technically valid and can be used in initially designing the field program. A basic

  16. Effects of anticonvulsant drugs on the synthesis of DNA and protein by human bone marrow cells in vitro

    International Nuclear Information System (INIS)

    Suspensions of human bone marrow cells were incubated with various concentrations of phenobarbitone or phenytoin sodium for 2 h, and the effects of this incubation on the subsequent incorporation of 3H-thymidine and 3H-leucine into DNA and protein, respectively, were studied. Both drugs caused a depression of 3H-thymidine incorporation and this phenomenon was not prevented by the addition of 100 μg of pteroylglutamic acid, folinic acid or 5-methyltetrahydrofolate per ml of marrow culture. The lowest concentration of drug which caused a statistically significant depression of 3H-thymidine incorporation was 200μg per ml for phenobarbitone and 50 μg per ml for phenytoin sodium. Both phenobarbitone and phenytoin sodium also caused an increase in the incorporation of 3H-leucine at concentrations of 50 and 20 μg per ml., respectively, suggesting the possibility that a stimulation of protein synthesis within erythropoietic cells may play an important role in the development of anticonvulsant-induced macrocytosis. (authod)

  17. Microwave assisted synthesis, spectroscopic, electrochemical and DNA cleavage studies of lanthanide(III) complexes with coumarin based imines

    Science.gov (United States)

    Kapoor, Puja; Fahmi, Nighat; Singh, R. V.

    2011-12-01

    The present work stems from our interest in the synthesis, characterization and biological evaluation of lanthanide(III) complexes of a class of coumarin based imines which have been prepared by the interaction of hydrated lanthanide(III) chloride with the sodium salts of 3-acetylcoumarin thiosemicarbazone (ACTSZH) and 3-acetylcoumarin semicarbazone (ACSZH) in 1:3 molar ratio using thermal as well as microwave method. Characterization of the ligands as well as the metal complexes have been carried out by elemental analysis, melting point determinations, molecular weight determinations, magnetic moment, molar conductance, IR, 1H NMR, 13C NMR, electronic, EPR, X-ray powder diffraction and mass spectral studies. Spectral studies confirm ligands to be monofunctional bidentate and octahedral environment around metal ions. The redox behavior of one of the synthesized metal complex was investigated by cyclic voltammetry. Further, free ligands and their metal complexes have been screened for their antimicrobial as well as DNA cleavage activity. The results of these findings have been presented and discussed.

  18. Prenatal diagnosis of ataxia-telangiectasia and Nijmegen Breakage Syndrome by the assay of radioresistant DNA synthesis

    International Nuclear Information System (INIS)

    Prenatal diagnosis was performed in 16 pregnancies at risk of ataxia-telangiectasia (A-T) or Nijmegen Breakage Syndrome (NBS). Radioresistant DNA synthesis (RDS) was investigated in cultured chorionic villus (CV) cells and/or amniotic fluid (AF) cells. In four pregnancies, an affected foetus was diagnosed with increased RDS in cultured CV cells. In three of the four cases confirmation of the diagnosis was obtained by analysis of AF cells and/or skin fibroblasts from the foetus cultured after termination of the pregnancy; in the fourth case a fibroblast culture from the aborted foetus failed. In one case, only AF cells could be analysed in a late stage of pregnancy; pregnancy was terminated due to intermediate/equivocal results but the foetus fibroblasts showed normal RDS. Normal RDS was demonstrated in the other 11 pregnancies at 25% risk either by analysis of CB cells (nine cases) or of AF cells (two cases). In some cases the (normal) results on the CV cells were corroborated by subsequent analysis of Af cells. The results suggest that RDS analysis of CV cells allows reliable prenatal diagnosis of A-T/NBS. However, amniocentesis may be necessary to confirm normal results on CV cells if the foetus is female (because of the risk of maternal cell contamination) or in the rare case of equivocal results. (author)

  19. Synthesis, crystal structure and electrochemical and DNA binding studies of oxygen bridged-copper(II) carboxylate

    Science.gov (United States)

    Iqbal, Muhammad; Ali, Saqib; Tahir, Muhammad Nawaz; Muhammad, Niaz; Shah, Naseer Ali; Sohail, Manzar; Pandarinathan, Vedapriya

    2015-08-01

    A new binuclear O-bridged Cu(II) complex with 4-chlorophenyl acetate and 2,2‧-bipyridine has been synthesized and characterized using FT-IR, powder and single crystal XRD and electrochemical solution studies. The results revealed that the two penta-coordinated Cu(II) centers are linked by two carboxylate ligands in end-on bonding fashion. The coordination geometry is slightly distorted square pyramidal (SP) with bridging oxygen atoms occupying the apical position and other ligands lying in the equatorial plane. The striking difference in Cu-O bond distance of the bridging oxygen atom in the complex may be responsible for the SP geometry of Cu(II) ion. The complex gave rise to metal centered irreversible electro-activity where one electron Cu(II)/Cu(III) oxidation process and a single step two electron Cu(II)/Cu(0) reduction process was observed. The redox processes were found predominantly adsorption controlled. The values of diffusion coefficient and heterogeneous rate constant for oxidation process were 6.98 × 10-7 cm2 s-1 and 4.60 × 10-5 cm s-1 while the corresponding values for reduction were 5.30 × 10-8 cm2 s-1 and 5.41 × 10-6 cm s-1, respectively. The formal potential and charge transfer coefficient were also calculated. The DNA-binding ability was explored through cyclic voltammetry and UV-Visible spectroscopy. Diminution in the value of Do for oxidation indicated the binding of the complex with DNA corresponding to Kb = 8.58 × 104 M-1. UV-Visible spectroscopy yielded ε = 49 L mol-1 cm-1 and Kb = 2.96 × 104 M-1. The data of both techniques support each other. The self-induced redox activation of the complex, as indicated by cyclic voltammetry heralds its potential applications in redox catalysis and anticancer activity.

  20. Separate Roles of Escherichia coli Replication Proteins in Synthesis and Partitioning of pSC101 Plasmid DNA

    OpenAIRE

    Miller, Christine; Cohen, Stanley N.

    1999-01-01

    We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditi...

  1. Comparative studies on the effect of ionizing and nonionizing radiations on the kinetics of DNA synthesis and postirradiation degradation in Micrococcus radiodurans R115

    International Nuclear Information System (INIS)

    The kinetics of degradation and synthesis of DNA and the nature of radioactive substances released from M. radiodurans R115 labeled with thymidine-methyl-3H after UV and gamma irradiations were investigated. The release of labeled material from the DNA began immediately upon incubation and terminated in due time 90 min and 180 min for UV and gamma irradiations, respectively. When acriflavine was added to the medium, post-irradiation degradation process did not terminate even after 9 h in the case of UV exposure. However, it terminated after 6 h in the case of gamma irradiation. In the presence of acriflavine, DNA synthesis resumed after termination of DNA degradation in the case of gamma irradiation and this was not observed in the case of UV irradiation. Degradation products were chromatographed and it was found that they were located in one major radioactive peak. However their locations were different for UV and gamma radiations. For UV irradiation, the peak fell in the thymine region, while for gamma irradiation it fell in the thymidine region. (author)

  2. Insulin-like growth factor binding protein-2 mediates the inhibition of DNA synthesis by transforming growth factor-beta in mink lung epithelial cells.

    Science.gov (United States)

    Dong, Feng; Wu, Hai-Bin; Hong, Jiang; Rechler, Matthew M

    2002-01-01

    Insulin-like growth factor binding protein-3 (IGFBP-3) has been proposed to mediate the growth inhibitory effects of transforming growth factor (TGF)-beta in breast and prostate cancer cells. Both TGF-beta and exogenous IGFBP-3 inhibit DNA synthesis in Mv1 mink lung epithelial cells (CCL64). The present study asks whether IGFBPs synthesized by CCL64 cells mediate growth inhibition by TGF-beta. CCL64 cells synthesize and secrete a single 34-kDa IGFBP that was identified as IGFBP-2 by immunoprecipitation and immunodepletion. Recombinant bovine IGFBP-2 inhibited CCL64 DNA synthesis in serum-free media in an IGF-independent manner. Coincubation with Leu(60)-IGF-I, an IGF-I analog that binds to IGFBPs with higher affinity than to IGF-I receptors, decreased the inhibition by bIGFBP-2. Leu(60)-IGF-I also decreased the inhibition of CCL64 DNA synthesis by TGF-beta by up to 70%, whereas Long-R3-IGF-I, an IGF-I analog with higher affinity for IGF-I receptors than for IGFBPs, did not decrease inhibition, suggesting that the effect of Leu(60)-IGF-I resulted from its forming complexes with endogenous IGFBPs. Leu(60)-IGF-I did not decrease TGF-beta stimulation of a Smad3-dependent reporter gene. Following incubation of intact CCL64 cells with bIGFBP-2 at 0 degrees C, bIGFBP-2 was recovered in membrane fractions; membrane association was abolished by coincubation with Leu(60)-IGF-I. If exogenous and secreted IGFBP-2 must bind to CCL64 cells to inhibit DNA synthesis, Leu(60)-IGF-I might reduce the inhibition of DNA synthesis by bIGFBP-2 or TGF-beta by inhibiting the association of IGFBP-2 in the media with CCL64 cells. Since TGF-beta does not increase IGFBP-2 abundance, we propose that TGF-beta sensitizes CCL64 cells to the latent growth inhibitory activity of endogenous IGFBP-2 by potentiating an intracellular IGFBP-2 signaling pathway or by promoting the association of secreted IGFBP-2 with the plasma membrane. PMID:11807812

  3. Detecting DNA synthesis of neointimal formation after catheter balloon injury in GK and in Wistar rats: using 5-ethynyl-2'-deoxyuridine

    Directory of Open Access Journals (Sweden)

    Guo Jingsheng

    2012-12-01

    Full Text Available Abstract Background Neointimal formation plays an important role in the pathogenesis of coronary restenosis after percutaneous coronary intervention (PCI, especially in patients with diabetes mellitus. Recently, some studies have shown that 5-ethynyl-2'-deoxyuridine (EdU incorporation can serve as a novel alternative to the 5-bromo-2'-deoxyuridine (BrdU antibody detection method for detection of DNA synthesis in regenerating avian cochlea, chick embryo and the adult nervous system. However, few studies have been performed to assess the suitability of EdU for detecting DNA synthesis in vascular neointima. Methods The carotid artery balloon injury model was established in Goto-Kakizaki (GK and Wistar rats. A Cell-LightTM EdU Kit was used to detect EdU-labeled cell nuclei of common carotid arteries at day 7 after catheter balloon injury. Different methods of injecting EdU were tested. The protein levels of proliferating cell nuclear antigen (PCNA and p-Akt (Ser473, as well as the mRNA levels of PCNA were evaluated by Western blotting and quantitative real-time PCR (qRT-PCR, respectively. Immunohistochemical staining was also employed to visualize PCNA-positive cells. Results At day 7 after catheter balloon injury, far more EdU-positive and PCNA-positive cells were observed in GK rats. When comparing groups that received different EdU doses, it was found that the percentage of EdU-positive cells at a dose of 100 mg/kg body weight was than at doses of 25 mg/kg and 50 mg/kg. The number of positive cells was significantly higher in the repeated injection group compared to the single injection group. Further, after balloon injury DNA synthesis in GK rats was more notable than in Wistar rats. Neointimal formation in GK rats was more obvious than in Wistar rats. The protein levels of PCNA and p-Akt (Ser473 and the mRNA levels of PCNA were increased in injured rats as compared to uninjured rats, and were significantly higher in GK rats than in Wistar rats

  4. Lack of effect of furfural on unscheduled DNA synthesis in the in vivo rat and mouse hepatocyte DNA repair assays and in precision-cut human liver slices.

    Science.gov (United States)

    Lake, B G; Edwards, A J; Price, R J; Phillips, B J; Renwick, A B; Beamand, J A; Adams, T B

    2001-10-01

    The ability of furfural to induce unscheduled DNA synthesis (UDS) in hepatocytes of male and female B6C3F(1) mice and male F344 rats after in vivo administration and in vitro in precision-cut human liver slices has been studied. Preliminary toxicity studies established the maximum tolerated dose (MTD) of furfural to be 320 and 50 mg/kg in the mouse and rat, respectively. Furfural was dosed by gavage at levels of 0 (control), 50, 175 and 320 mg/kg to male and female mice and 0, 5, 16.7 and 50 mg/kg to male rats. Hepatocytes were isolated by liver perfusion either 2-4 h or 12-16 h after treatment, cultured in medium containing [3H]thymidine for 4 h and assessed for UDS by grain counting of autoradiographs. Furfural treatment did not produce any statistically significant increase or any dose-related effects on UDS in mouse and rat hepatocytes either 2-4 h or 12-16 h after dosing. In contrast, UDS was markedly induced in mice and rats 2-4 h after treatment with 20 mg/kg dimethylnitrosamine and 12-16 h after treatment of mice and rats with 200 mg/kg o-aminoazotoluene and 50 mg/kg 2-acetylaminofluorene (2-AAF), respectively. Precision-cut human liver slices from four donors were cultured for 24 h in medium containing [3H]thymidine and 0-10 mM furfural. Small increases in the net grain count (i.e. nuclear grain count less mean cytoplasmic grain count) observed with 2-10 mM furfural were not due to any increase in the nuclear grain count. Rather, it was the result of concentration-dependent decreases in the mean cytoplasmic grain counts and to a lesser extent in nuclear grain counts, due to furfural-induced cytotoxicity. In contrast, marked increases in UDS (both net grain and nuclear grain counts) were observed in human liver slices treated with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 mM aflatoxin B(1) and 0.005 and 0.05 mM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. This study demonstrates that furfural does not induce UDS in the hepatocytes of male and female B6C3F

  5. Synthesis, spectral characterization of Schiff base transition metal complexes: DNA cleavage and antimicrobial activity studies

    Indian Academy of Sciences (India)

    N Raman; J Dhaveethu Raja; A Sakthivel

    2007-07-01

    A new series of transition metal complexes of Cu(II), Ni(II), Co(II), Mn(II), Zn(II), VO(IV), Hg(II) and Cd(II) have been synthesized from the Schiff base (L) derived from 4-aminoantipyrine, 3-hydroxy-4-nitrobenzaldehyde and -phenylenediamine. Structural features were obtained from their elemental analyses, magnetic susceptibility, molar conductance, mass, IR, UV-Vis, 1H NMR and ESR spectral studies. The data show that these complexes have composition of ML type. The UV-Vis, magnetic susceptibility and ESR spectral data of the complexes suggest a square-planar geometry around the central metal ion except VO(IV) complex which has square-pyramidal geometry. The redox behaviour of copper and vanadyl complexes was studied by cyclic voltammetry. Antimicrobial screening tests gave good results in the presence of metal ion in the ligand system. The nuclease activity of the above metal complexes shows that Cu, Ni and Co complexes cleave DNA through redox chemistry whereas other complexes are not effective.

  6. Bio-inspired benzo[k,l]xanthene lignans: synthesis, DNA-interaction and antiproliferative properties.

    Science.gov (United States)

    Spatafora, Carmela; Barresi, Vincenza; Bhusainahalli, Vedamurthy M; Di Micco, Simone; Musso, Nicolò; Riccio, Raffaele; Bifulco, Giuseppe; Condorelli, Daniele; Tringali, Corrado

    2014-05-01

    In this work twelve benzo[k,l]xanthene lignans were synthesized by biomimetic, Mn-mediated oxidative coupling of caffeic esters and amides. These compounds, bearing different flexible pendants at position C1/C2 of the aromatic core, interact with DNA in a dual mode, as confirmed by DF-STD NMR analysis and molecular docking: the planar core acts as a base pair intercalant, whereas the flexible pendants act as minor groove binders. Their antiproliferative activity was evaluated on a panel of six tumor cell lines: HT-29, Caco-2, HCT-116 (human colon carcinoma), H226, A549 (human lung carcinoma), and SH-SY5Y (human neuroblastoma). All compounds under study, except 29, resulted in activity against one or more cell lines, and the markedly lipophilic esters 13 and 28 showed the highest activity. Compound 13 was more active than the anticancer drug 5-fluorouracil (5-FU) towards HCT-116 (colon, GI50 = 3.16 μM) and H226 (lung, GI50 = 4.33 μM) cell lines. PMID:24647864

  7. Synthesis of an oligonucleotide with a nicotinamide mononucleotide residue and its molecular recognition in DNA helices.

    Science.gov (United States)

    Göckel, A; Richert, C

    2015-11-01

    Nicotinamide adenine dinucleotide (NAD) is a pivotal redox cofactor of primary metabolism. Its redox reactivity is based on the nicotinamide mononucleotide (NMN) moiety. We investigated whether NMN(+) can engage in pairing interactions, when incorporated into an oligonucleotide. Here we describe the incorporation of NMN(+) at the 3'-terminus of an oligodeoxynucleotide via a phosphoramidate coupling in solution. The stability of duplexes and triplexes with the NMN(+)-containing strand was measured in UV-melting curves. While the melting points of duplexes with different bases facing the nicotinamide were similar, triplex stabilities varied greatly between different base combinations, suggesting specific pairing. The most stable triplexes were found when a guanine and an adenine were facing the NMN(+) residue. Their triplex melting points were higher than those of the corresponding triplexes with a thymidine residue at the same position. These results show that NMN(+) residues can be recognized selectively in DNA helices and are thus compatible with the molecular recognition in nucleic acids. PMID:26371420

  8. Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III.

    OpenAIRE

    Wright, G E; Baril, E F; Brown, V M; Brown, N C

    1982-01-01

    The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, w...

  9. Intramolecular telomeric G-quadruplexes dramatically inhibit DNA synthesis by replicative and translesion polymerases, revealing their potential to lead to genetic change.

    Directory of Open Access Journals (Sweden)

    Deanna N Edwards

    Full Text Available Recent research indicates that hundreds of thousands of G-rich sequences within the human genome have the potential to form secondary structures known as G-quadruplexes. Telomeric regions, consisting of long arrays of TTAGGG/AATCCC repeats, are among the most likely areas in which these structures might form. Since G-quadruplexes assemble from certain G-rich single-stranded sequences, they might arise when duplex DNA is unwound such as during replication. Coincidentally, these bulky structures when present in the DNA template might also hinder the action of DNA polymerases. In this study, single-stranded telomeric templates with the potential to form G-quadruplexes were examined for their effects on a variety of replicative and translesion DNA polymerases from humans and lower organisms. Our results demonstrate that single-stranded templates containing four telomeric GGG runs fold into intramolecular G-quadruplex structures. These intramolecular G quadruplexes are somewhat dynamic in nature and stabilized by increasing KCl concentrations and decreasing temperatures. Furthermore, the presence of these intramolecular G-quadruplexes in the template dramatically inhibits DNA synthesis by various DNA polymerases, including the human polymerase δ employed during lagging strand replication of G-rich telomeric strands and several human translesion DNA polymerases potentially recruited to sites of replication blockage. Notably, misincorporation of nucleotides is observed when certain translesion polymerases are employed on substrates containing intramolecular G-quadruplexes, as is extension of the resulting mismatched base pairs upon dynamic unfolding of this secondary structure. These findings reveal the potential for blockage of DNA replication and genetic changes related to sequences capable of forming intramolecular G-quadruplexes.

  10. CERN control centre (CCC) ou de la conception à l'exécution

    CERN Document Server

    Poehler, M

    2005-01-01

    Dès fin 2001, la section Design Office TS-CE est mandatée pour l’étude et la conception d’une nouvelle salle de contrôle des accélérateurs. Après de multiples variantes d’études et d’implantation, la direction CERN retient en début 2004, sur la base de facteurs économiques, le projet CCC, consistant en l’extension et le réaménagement de la PCR existante. Un Working Group TS Infrastructure est mis en place sous le pilotage TS-CE, avec pour objectifs de passer de la phase d’avant projet à la phase de projet définitif et d’étayer la faisabilité technique et économique de cette solution, dans le respect du cahier des charges des utilisateurs. Appuyé sur le rapport d’étude présenté à la direction CERN, cette dernière confirme son feu vert de lancement du projet dans les limites des coûts et des délais présentés. L’ouvrage et son infrastructure technique devant être livrés aux utilisat...

  11. B cells in the appendix and other lymphoid organs of the rabbit: stimulation of DNA synthesis by anti-immunoglobulin

    International Nuclear Information System (INIS)

    Lymphocytes from rabbit lymphoid organs were cultured in the presence of class specific anti-immunoglobulin sera and of anti-allotype sera. Stimulation of tritiated thymidine uptake into DNA was taken to indicate the presence of the corresponding immunoglobulins on the cell surfaces. Thymus and bone marrow lymphocytes were unresponsive to all reagents tested. Popliteal lymph node contained cells responsive to anti-μ, anti-γ, and anti-α and therefore presumably bearing IgM, IgG, and IgA. Spleen had only IgM- and IgG-bearing cells, and the appendix contained cells with IgM and IgA receptors only. The lymph node, spleen, and appendix cells of rabbits depleted of B lymphocytes by irradiation (900 R) and injection of thymocytes were unresponsive to anti-immunoglobulin but were stimulated at almost normal levels by PHA and Con A. T cell-depleted animals (thymectomy, irradiation with three divided doses of 450 R and bone marrow shielding) had immunoglobulin-bearing lymphocytes but were unresponsive to the mitogens. However a moderate level of mitogen-responsiveness reappeared by 3 to 4 wk after irradiation. Cells of morphologically distinct regions of the appendix, separated manually, showed different responses corresponding to the inferred origins of these anatomic areas. The ''dome'' and ''corona'' contained functional IgM- and IgA-bearing cells. The ''TDA'' reacted well to PHA, Con A, and PWM, but was depleted of immunoglobulin-bearing cells. The ''follicle'' cells, which are almost all in active DNA synthesis or mitosis, were relatively unresponsive to either T or B cell stimuli. Anti-allotype serum stimulated the same populations which responded to class-specific heteroantisera but at a slightly lower level. It was inferred that gut-associated lymphoid tissues like the appendix may play a special role as an amplification site for B-cells destined to produce IgM and IgA elsewhere in the organism

  12. Premitotic DNA synthesis in the brain of the adult frog (Rana esculenta L.): An autoradiographic 3H-thymidine study

    International Nuclear Information System (INIS)

    Replicative synthesis of DNA in the brain of the adult frog was studied by light microscope autoradiography. Animals collected during the active period (May-June) and in hibernation (January) were used. In active frogs, 3H-thymidine labelling occurred mainly in the ependymal cells which line the ventricles. The mean labelling index (LI%) was higher in the ependyma of the lateral and fourth ventricles than in the ependyma of the lateral diencephalon and tectal parts of the mesencephalon. In the recessus infundibularis and preopticus the number of labelled cells (LCs) was several times greater than in the lateral parts of the third ventricle. LCs were seen subependymally only occasionally. The incidence of LCs in the parenchyma of the brain was much lower in most regions than in the ventricular ependyma; LCs were mainly small and, from their nuclear morphology, they were glial cells. The LI% reached the highest value in the septum hippocampi and in the nucleus entopeduncularis. In these locations, LCs were larger and closer in size to the nerve cells of these regions. From comparison with data obtained earlier in the brain of mammals, it is evident that the distribution of proliferating cells in the olfactory and limbic system is phylogenetically conservative. The occurrence of pyknotic cells in the same areas which contain LCs, suggests that cell division reflects in part the process of cell renewal observed in mammals. However, proliferating cells could also be linked to the continuous growth observed in non-mammalian vertebrates. In hibernating frogs, LCs and pyknoses were not seen or were found occasionally, which further indicates the functional significance of both processes

  13. Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin

    Directory of Open Access Journals (Sweden)

    Hansen Peter J

    2008-01-01

    Full Text Available Abstract Background Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS, inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3 cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes. Results Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 μg/ml as determined by measurements of [3H]thymidine incorporation or ATP content per well. Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of lymphocytes into S phase following activation of proliferation with phytohemagglutinin. Conclusion Results indicate that OvUS acts to block cell proliferation through disruption of the cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth.

  14. Evidence that ultraviolet light-induced DNA replication death of recA bacteria is prevented by protein synthesis in repair-proficient bacteria

    International Nuclear Information System (INIS)

    The ultraviolet light (UV) survival curve of Escherichia coli WP10 recA trp is almost biphasic, with a greatly reduced shoulder but demonstrating a transition to decreased slope with increasing fluences, indicating the presence in the culture of a low frequency of resistant cells. Treatment of the culture with chloramphenicol before UV exposure brought almost all of the cells to a high degree of UV resistance, by bringing them to the end of their DNA replication cycle. The survival curves of the repair-proficient E. coli Wp2 trp showed a similar pattern with chloramphenicol treatment or tryptophan starvation before UV exposure, but only if protein synthesis after UV exposure, death occurs unless the cells are in the resistant state characteristic of bacteria at the end of their DNA replication cycle. With repair-proficient bacteria treated before UV exposure with chloramphenicol, when protein synthesis is not blocked after UV exposure, a marked expansion of the shoulder occurs because of the function of another resistance-conferring mechanism. This mechanism also depends on the recA+ gene since expansion of the shoulder does not occur in recA bacteria when protein synthesis is inhibited before UV exposure. (author). 11 refs.; 5 figs

  15. Protective effects of adrenochrome monoaminoguanidine methanesulfonate (AMM) and cytochrome C (CCC) on natural killer cells in the peripheral blood of cancer patients during radiotherapy

    International Nuclear Information System (INIS)

    The purpose of this study was to examine radioprotective effects of adrenochrome monoaminoguanidine methanesulfonate (AMM) and cytochrome C (CCC) on lymphocyte subset during radiotherapy (RT). Sixty five patients received irradiation of 50 Gy or more to the chest field of 100 cm2 or larger. The patients were classified into four groups: those treated with RT alone (Group I, n=15), combined RT and OK-432 (Group II, n=15), combined RT and AMMM + CCC (Group III, n=17), and combined RT and AMM + CCC + OK-432 (Group IV, n=18). Lymphocytes decreased by 64% after RT in Group I and by 50% in Group III. AMM and CCC prevented a decrease in the absolute number of potent natural killer cells and activated T cells, with statistically significant differences. In Group III, the absolute number of activated T cells tended to increase even after RT. Cytotoxic T cells decreased by only 11% after RT in Group III, as compared with 58% in Group I. As found in Groups II and IV, the combination of OK-432 did not have so protective effects on lymphocytes associated with RT as AMM + CCC combined with RT in Groups III. In view of the selective protection for potent natural killer cells and activated T cells, AMM + CCC seemed to serve as biological response modifiers, as well as radioprotective agents. (N.K.)

  16. Synthesis, characterization and crystal structure of cobalt(III) complexes containing 2-acetylpyridine thiosemicarbazones: DNA/protein interaction, radical scavenging and cytotoxic activities.

    Science.gov (United States)

    Manikandan, Rajendran; Viswanathamurthi, Periasamy; Velmurugan, Krishnaswamy; Nandhakumar, Raju; Hashimoto, Takeshi; Endo, Akira

    2014-01-01

    The synthesis, structure and biological studies of cobalt(III) complexes supported by NNS-tridentate ligands are reported. Reactions of 2-acetylpyridine N-substituted thiosemicarbazone (HL(1-3)) with [CoCl2(PPh3)2] resulted [Co(L(1-3))2]Cl (1-3) which were characterized by elemental analysis and various spectral studies. The molecular structure of the complex 1 has been determined by single crystal X-ray diffraction studies. In vitro DNA binding studies of complexes 1-3 carried out by fluorescence studies and the results revealed the binding of complexes to DNA via intercalation. The binding constant (Kb) values of complexes 1-3 from fluorescence experiments showed that the complex 3 has greater binding propensity for DNA. The DNA cleavage activity of the complexes 1 and 3 were ascertained by gel electrophoresis assay which revealed that the complexes are good DNA cleavage agents. Further, the interactions of the complexes with bovine serum albumin (BSA) were also investigated using fluorescence spectroscopic method, which showed that the complexes 1-3 could bind strongly with BSA. The antioxidant property of the complexes was evaluated to test their free-radical scavenging ability. Furthermore, in vitro cytotoxicity of the complexes against MCF-7 and A431 cell lines was assayed which showed higher activity and efficiently vanished the cancer cells even at low concentrations. PMID:24342132

  17. A 28-fold increase in secretory protein synthesis is associated with DNA puff activity in the salivary gland of Bradysia hygida (Diptera, Sciaridae

    Directory of Open Access Journals (Sweden)

    de-Almeida J.C.

    1997-01-01

    Full Text Available When the first group of DNA puffs is active in the salivary gland regions S1 and S3 of Bradysia hygida larvae, there is a large increase in the production and secretion of new salivary proteins demonstrable by [3H]-Leu incorporation. The present study shows that protein separation by SDS-PAGE and detection by fluorography demonstrated that these polypeptides range in molecular mass from about 23 to 100 kDa. Furthermore, these proteins were synthesized mainly in the S1 and S3 salivary gland regions where the DNA puffs C7, C5, C4 and B10 are conspicuous, while in the S2 region protein synthesis was very low. Others have shown that the extent of amplification for DNA sequences that code for mRNA in the DNA puffs C4 and B10 was about 22 and 10 times, respectively. The present data for this group of DNA puffs are consistent with the proposition that gene amplification is necessary to provide some cells with additional gene copies for the production of massive amounts of proteins within a short period of time (Spradling AC and Mahowald AP (1980 Proceedings of the National Academy of Sciences, USA, 77: 1096-1100.

  18. Solid-phase synthesis of amidine-substituted phenylbenzimidazoles and incorporation of this DNA binding and recognition motif into amino acid and peptide conjugates.

    Science.gov (United States)

    Garner, Matthew L; Georgiadis, Taxiarchis M; Li, Jessica Bo; Wang, Tianxiu; Long, Eric C

    2014-05-01

    Amidine-substituted phenylbenzimidazoles are well-established DNA-binding structural motifs that have contributed to the development of diverse classes of DNA-targeted agents; this ring system not only assists in increasing the overall DNA affinity of an agent, but can also influence its site selectivity. Seeking a means to conveniently exploit these attributes, a protocol for the on-resin synthesis of amino acid- and peptide-phenylbenzimidazole-amidine conjugates was developed to facilitate installation of phenylbenzimidazole-amidines into peptide chains during the course of standard solid-phase syntheses. Building from a resin-bound amino acid or peptide on Rink amide resin, 4-formyl benzoic acid was coupled to the resin-bound free amine followed by introduction of 3,4-diamino-N'-hydroxybenzimidamide (in the presence of 1,4-benzoquinone) to construct the benzimidazole heterocycle. Finally, the resin-bound N'-hydroxybenzimidamide functionality was reduced to an amidine via 1 M SnCl2·2H2O in DMF prior to resin cleavage to release final product. This procedure permits the straightforward synthesis of amino acids or peptides that are N-terminally capped by a phenylbenzimidazole-amidine ring system. Employing this protocol, a series of amino acid-phenylbenzimidazole-amidine (Xaa-R) conjugates was synthesized as well as dipeptide conjugates of the general form Xaa-Gly-R (where R is the phenylbenzimidazole-amidine and Xaa is any amino acid). PMID:24562478

  19. Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium.

    Science.gov (United States)

    Stammen, Simon; Schuller, Franziska; Dietrich, Sylvia; Gamer, Martin; Biedendieck, Rebekka; Jahn, Dieter

    2010-09-01

    Gene "7" of Escherichia coli phage K1E was proposed to encode a novel DNA-dependent RNA polymerase (RNAP). The corresponding protein was produced recombinantly, purified to apparent homogeneity via affinity chromatography, and successfully employed for in vitro RNA synthesis. Optimal assay conditions (pH 8, 37 degrees C, 10 mM magnesium chloride and 1.3 mM spermidine) were established. The corresponding promoter regions were identified on the phage genome and summarized in a sequence logo. Surprisingly, next to K1E promoters, the SP6 promoter was also recognized efficiently in vitro by K1E RNAP, while the T7 RNAP promoter was not recognized at all. Based on these results, a system for high-yield in vitro RNA synthesis using K1E RNAP was established. The template plasmid is a pUC18 derivative, which enables blue/white screening for positive cloning of the target DNA. Production of more than 5 microg of purified RNA per microgram plasmid DNA was achieved. Finally, in vivo protein production systems for Bacillus megaterium were established based on K1E and SP6 phage RNAP transcription. Up to 61.4 mg g (CDW) (-1) (K1E RNAP) of the reporter protein Gfp was produced in shaking flask cultures of B. megaterium. PMID:20596705

  20. In situ DNA-templated synthesis of silver nanoclusters for ultrasensitive and label-free electrochemical detection of microRNA.

    Science.gov (United States)

    Yang, Cuiyun; Shi, Kai; Dou, Baoting; Xiang, Yun; Chai, Yaqin; Yuan, Ruo

    2015-01-21

    On the basis of the use of silver nanoclusters (AgNCs) in situ synthesized by cytosine (C)-rich loop DNA templates as signal amplification labels, the development of a label-free and highly sensitive method for electrochemical detection of microRNA (miRNA-199a) is described. The target miRNA-199a hybridizes with the partial dsDNA probes to initiate the target-assisted polymerization nicking reaction (TAPNR) amplification to produce massive intermediate sequences, which can be captured on the sensing electrode by the self-assembled DNA secondary probes. These surface-captured intermediate sequences further trigger the hybridization chain reaction (HCR) amplification to form dsDNA polymers with numerous C-rich loop DNA templates on the electrode surface. DNA-templated synthesis of AgNCs can be realized by subsequent incubation of the dsDNA polymer-modified electrode with AgNO3 and sodium borohydride. With this integrated TAPNR and HCR dual amplification strategy, the amount of in situ synthesized AgNCs is dramatically enhanced, leading to substantially amplified current response for highly sensitive detection of miRNA-199a down to 0.64 fM. In addition, the developed method also shows high selectivity toward the target miRNA-199a. Featured with high sensitivity and label-free capability, the proposed sensing scheme can thus offer new opportunities for achieving sensitive, selective, and simple detection of different types of microRNA targets. PMID:25537119

  1. Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK, DNA Synthesis and Proliferation in Pancreatic Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Ming Ming

    Full Text Available Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC cells (PANC-1, MiaPaCa-2 with the isoquinoline alkaloid berberine (0.3-6 µM inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70% the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC at Ser79. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr389 and S6 at Ser240/244 and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α1 and α2 catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.

  2. Induction of DNA and RNA synthesis in murine B lymphocytes does not correlate with early changes in cytosolic free calcium concentration

    International Nuclear Information System (INIS)

    In order to ascertain if early changes in cytosolic free calcium concentration [Ca2+] are correlated with either activation, as defined by 3H-uridine incorporation or increase in cell size, or induction of DNA synthesis, 3H-thymidine incorporation, murine B lymphocytes were stimulated with preparations of lipopolysaccharide (LPS), rabbit anti-mouse Fab (RAMFab), and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). LPS, although a potent inducer of 3H-thymidine incorporation does not cause an increase in [Ca2+]. F(ab')2RAMFab at 50 μs ml causes a 6X increase in 3H-thymidine incorporation as opposed to a 2-3X increase at 10 μg/ml. IgG-RAMFab at concentrations up to 50 μg/ml neither induces DNA synthesis nor activates cells by any criteria, including 3H-uridine incorporation, increase in cell size, and increase in I-A expression. Both RAMFab causes increases in [Ca2+] that saturate at 10 μg/ml. Minimally proliferative doses of TPA inhibit the increase in [Ca2+] caused by both preparations of RAMFab. However, B cells pretreated with TPA and then stimulated with 2 or 10 μg/ml of either preparation of RAMFab showed increases of 10 X in 3H-uridine and 100X in 3H-thymidine incorporation. These data demonstrate that there appears to be no correlation between early changes in [Ca2+] and either activation or induction of DNA synthesis in murine B cells

  3. Modelling the Structure of a Protein Domain (N-terminal of XPB) Linked with Protein Synthesis, DNA Damage Repair, Rare Diseases, Cancer Therapeutics, and Tuberculosis

    OpenAIRE

    Saha, Mitul

    2016-01-01

    In this work, we develop first near-complete 3D models for NTD-hXPB - the N-terminal protein domain of the human transcription factor XPB. The results are very significant as NTD-hXPB plays a critical role in the synthesis of proteins (specifically transcription) and DNA damage repair (specifically nucleotide excision repair). NTD-hXPB is directly implicated in rare diseases XP-B, XP-CS, and TTD2, whose symptoms include neurodegenerative disorders, premature aging, and decreased fertility. NT...

  4. Synthesis, restriction analysis, and molecular cloning of near full length DNA complementary to bovine parathyroid hormone mRNA.

    OpenAIRE

    Gordon, D. F.; Kemper, B

    1980-01-01

    DNA complementary (cDNA) to a partially purified preparation of bovine parathyroid hormone mRNA was synthesized using avian myeloblastosis viral reverse transcriptase. The PTH cDNA contained about 750 bases and was greater than 95% sensitive to digestion by S1 nuclease. Analysis of the mRNA preparation by excess RNA hybridization to the PTH cDNA revealed one rapidly hybridizing component consisting of 50% of the PTH cDNA. Sequential incubation of the PTH mRNA with reverse transcriptase and E....

  5. Synthesis and Evaluation of a Rationally Designed Click-Based Library for G-Quadruplex Selective DNA Photocleavage

    Directory of Open Access Journals (Sweden)

    Dominic McBrayer

    2015-09-01

    Full Text Available DNA containing repeating G-rich sequences can adopt higher-order structures known as G-quadruplexes (G4. These structures are believed to form within telomeres and the promoter regions of some genes, particularly in a number of proto-oncogenes, where they may play a role in regulating transcription. Alternatively, G4 DNA may act as a barrier to replication. To investigate these potential biological roles, probes that combine highly selective G4 DNA targeting with photocleavage activity can allow temporal detection of G4 DNA, providing opportunities to obtain novel insights about the biological roles of G4 DNA. We have designed, synthesized, and screened a small library of potential selective G-quadruplex DNA photocleavage agents incorporating the G-quadruplex targeting moiety of 360A with known photocleavage groups linked via “click” chemistry.

  6. Human Cytomegalovirus UL44 Concentrates at the Periphery of Replication Compartments, the Site of Viral DNA Synthesis

    OpenAIRE

    Strang, Blair L.; Boulant, Steeve; Chang, Lynne; Knipe, David M.; Kirchhausen, Tomas; Coen, Donald M

    2012-01-01

    The formation of replication compartments, the subnuclear structures in which the viral DNA genome is replicated, is a hallmark of herpesvirus infections. The localization of proteins and viral DNA within human cytomegalovirus replication compartments is not well characterized. Immunofluorescence analysis demonstrated the accumulation of the viral DNA polymerase subunit UL44 at the periphery of replication compartments and the presence of different populations of UL44 in infected cells. In co...

  7. UAP56 is an important mediator of Angiotensin II/platelet derived growth factor induced vascular smooth muscle cell DNA synthesis and proliferation

    International Nuclear Information System (INIS)

    Highlights: ► Knockdown of UAP56 inhibits Angiotensin II/PDGF induced vascular smooth muscle cell proliferation. ► UAP56 is a positive regulator of E2F transcriptional activation. ► UAP56 is present in the vessel wall of low flow carotid arteries. -- Abstract: Angiotensin (Ang) II and platelet-derived growth factor (PDGF) are important mediators of pathologic vascular smooth muscle cell (VSMC) proliferation. Identifying downstream mediators of Ang II and PDGF signaling may provide insights for therapies to improve vascular proliferative diseases. We have previously demonstrated that breakpoint cluster region (Bcr) is an important mediator of Ang II/PDGF signaling in VSMC. We have recently reported that the DExD/H box protein UAP56 is an interacting partner of Bcr in regulating VSMC DNA synthesis. We hypothesized that UAP56 itself is an important regulator of VSMC proliferation. In this report we demonstrate that knockdown of UAP56 inhibits Ang II/PDGF induced VSMC DNA synthesis and proliferation, and inhibits E2F transcriptional activity. In addition, we demonstrate that UAP56 is present in the vessel wall of low-flow carotid arteries. These findings suggest that UAP56 is a regulator of VSMC proliferation and identify UAP56 as a target for preventing vascular proliferative disease

  8. Activation of anthocyanin synthesis genes by white light in eggplant hypocotyl tissues, and identification of an inducible P-450 cDNA

    International Nuclear Information System (INIS)

    Eggplant seedlings (Solanum melongena) grown under red light irradiation showed a normal morphology with green, fully expanded cotyledons. When the seedlings grown under red light were irradiated with ultraviolet-containing white light, anthocyanin synthesis was induced in the hypocotyl tissues, especially when a UV light supplement was added. The accumulation of pigments was closely associated with the expression of genes involved in flavonoid synthesis. These genes include chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR). Using subtracted probes, which had been enriched for the accumulated mRNA, one white light-responsive cDNA was identified as being a P450 gene by comparison with database sequences. The maximal amino acid homology this cDNA had with other P450s was 36%. This was with CYP71 from avocado (Persea americana). Thus it represents a new P-450 family, which has been named CYP75. The mRNA of this gene was localized in the hypocotyl tissues of eggplant seedlings, which had been white light-irradiated. The transcript was accumulated by changing the light source, as in the case of other flavonoid biosynthesis genes. In delphinidin producing petunia plants, the mRNAs corresponding to the eggplant P-450 and flavonoid biosynthesis genes such as CHS and DFR were most abundant during the mid stage of flower bud development, but could not be detected in leaf tissues. These results suggest that this P-450 gene encodes a hydroxylating enzyme involved in flavonoid biosynthesis. (author)

  9. DNA-templated synthesis of PtAu bimetallic nanoparticle/graphene nanocomposites and their application in glucose biosensor

    OpenAIRE

    Leng, Jing; Wang, Wen-Min; Lu, Li-min; Bai, Ling; Qiu, Xin-Lan

    2014-01-01

    In this paper, single-stranded DNA (ss-DNA) is demonstrated to functionalize graphene (GR) and to further guide the growth of PtAu bimetallic nanoparticles (PtAuNPs) on GR with high densities and dispersion. The obtained nanocomposites (PtAuNPs/ss-DNA/GR) were characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectrometer (EDS), and electrochemical techniques. Then, an enzyme nanoassembly was prepared by self-assembling glucose oxidase (GOD) on PtAuNP/ss-DNA/GR ...

  10. CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL

    Science.gov (United States)

    Bartuzi, Paulina; Billadeau, Daniel D.; Favier, Robert; Rong, Shunxing; Dekker, Daphne; Fedoseienko, Alina; Fieten, Hille; Wijers, Melinde; Levels, Johannes H.; Huijkman, Nicolette; Kloosterhuis, Niels; van der Molen, Henk; Brufau, Gemma; Groen, Albert K.; Elliott, Alison M.; Kuivenhoven, Jan Albert; Plecko, Barbara; Grangl, Gernot; McGaughran, Julie; Horton, Jay D.; Burstein, Ezra; Hofker, Marten H.; van de Sluis, Bart

    2016-01-01

    The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal sorting of LDLR and for its function. We find that patients with X-linked intellectual disability caused by mutations in CCDC22 are hypercholesterolaemic, and that COMMD1-deficient dogs and liver-specific Commd1 knockout mice have elevated plasma LDL cholesterol levels. Furthermore, Commd1 depletion results in mislocalization of LDLR, accompanied by decreased LDL uptake. Increased total plasma cholesterol levels are also seen in hepatic COMMD9-deficient mice. Inactivation of the CCC-associated WASH complex causes LDLR mislocalization, increased lysosomal degradation of LDLR and impaired LDL uptake. Furthermore, a mutation in the WASH component KIAA0196 (strumpellin) is associated with hypercholesterolaemia in humans. Altogether, this study provides valuable insights into the mechanisms regulating cholesterol homeostasis and LDLR trafficking. PMID:26965651

  11. DNA ligase I, the replicative DNA ligase

    OpenAIRE

    Howes, Timothy R.L.; Tomkinson, Alan E.

    2012-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each...

  12. Thermodynamic and mechanistic insights into translesion DNA synthesis catalyzed by Y-family DNA polymerase across a bulky double-base lesion of an antitumor platinum drug

    Czech Academy of Sciences Publication Activity Database

    Brabec, Viktor; Malina, Jaroslav; Margiotta, N.; Natile, G.; Kašpárková, Jana

    2012-01-01

    Roč. 18, č. 48 (2012), s. 15439-15448. ISSN 0947-6539 R&D Projects: GA ČR(CZ) GAP205/11/0856; GA ČR(CZ) GAP301/10/0598 Institutional research plan: CEZ:AV0Z50040702 Keywords : DNA polymerase * platinum * calorimetry Subject RIV: BO - Biophysics Impact factor: 5.831, year: 2012

  13. CCC.UGA: A new site of ribosomal frameshifting in escherichia coli

    NARCIS (Netherlands)

    M.H. de Smit (Maarten); J. van Duin (Jan); P.H. van Knippenberg (Peter); H.G. van Eijk (Henk)

    1994-01-01

    textabstractTo activate expression of a human transferrin(Tf)-encoding cDNA in Escherichia coli by translational coupling, it was placed in an expression plasmid downstream from a 5'-terminal fragment from the replicase(R)-encoding gene of bacteriophage MS2. The resulting construct was found to prod

  14. Co(III and Ni(II Complexes Containing Bioactive Ligands: Synthesis, DNA Binding, and Photocleavage Studies

    Directory of Open Access Journals (Sweden)

    M. C. Prabhakara

    2007-02-01

    Full Text Available DNA binding and photocleavage characteristics of a series of mixed ligand complexes of the type [M(bpy2qbdp](PF6n⋅xH2O (where M=Co(III or Ni(II, bpy=2.2′-bipryidine, qbdp = Quinolino[3,2-b]benzodiazepine, n=3 or 2 and x=5 or 2 have been investigated. The DNA binding property of the complexes with calf thymus DNA has been investigated by using absorption spectra, viscosity measurements, as well as thermal denaturation studies. Intrinsic binding constant (Kb has been estimated under similar set of experimental conditions. Absorption spectral studies indicate that the Co(III and Ni(II complexes intercalate between the base pairs of the CT-DNA tightly with intrinsic DNA binding constant of 1.3×106 and 3.1×105 M-1 in Tris-HCl buffer containing 50 mM NaCl, respectively. The proposed DNA binding mode supports the large enhancement in the relative viscosity of DNA on binding to quinolo[3,2-b]benzodiazepine. The oxidative as well as photo-induced cleavage reactions were monitered by gel electrophoresis for both complexes. The photocleavage experiments showed that the cobalt(III complex can cleave pUC19 DNA effectively in the absence of external additives as an effective inorganic nuclease.

  15. Design, synthesis and DNA interactions of a chimera between a platinum complex and an IHF mimicking peptide.

    Science.gov (United States)

    Rao, Harita; Damian, Mariana S; Alshiekh, Alak; Elmroth, Sofi K C; Diederichsen, Ulf

    2015-12-28

    Conjugation of metal complexes with peptide scaffolds possessing high DNA binding affinity has shown to modulate their biological activities and to enhance their interaction with DNA. In this work, a platinum complex/peptide chimera was synthesized based on a model of the Integration Host Factor (IHF), an architectural protein possessing sequence specific DNA binding and bending abilities through its interaction with a minor groove. The model peptide consists of a cyclic unit resembling the minor grove binding subdomain of IHF, a positively charged lysine dendrimer for electrostatic interactions with the DNA phosphate backbone and a flexible glycine linker tethering the two units. A norvaline derived artificial amino acid was designed to contain a dimethylethylenediamine as a bidentate platinum chelating unit, and introduced into the IHF mimicking peptides. The interaction of the chimeric peptides with various DNA sequences was studied by utilizing the following experiments: thermal melting studies, agarose gel electrophoresis for plasmid DNA unwinding experiments, and native and denaturing gel electrophoresis to visualize non-covalent and covalent peptide-DNA adducts, respectively. By incorporation of the platinum metal center within the model peptide mimicking IHF we have attempted to improve its specificity and DNA targeting ability, particularly towards those sequences containing adjacent guanine residues. PMID:26477860

  16. Synthesis of N-methyl-N-nitrosourea linked to a methidium chloride analogue and its reactions with 32P-end-labeled DNA

    International Nuclear Information System (INIS)

    The synthesis and characterization of an N-methyl-N-nitrosourea (MNU) analogue that is covalently linked to methidium nucleus (9) is described. At 37/degrees/C in pH 8.0 buffer 9 hydrolyzes via pseudo-first-order kinetics, with a calculated t/sub 1/2/ = 77 min. By use of polyacrylamide sequencing gels the formation of piperidine-labile N7-methylguanine adducts from the reaction of 9 and MNU with 5'-32P-end-labeled DNA restriction fragments is reported. DNA methylation by 9 in 10 mM Tris buffer is enhanced with increasing ionic strength (50-200 mM NaCl), which contrasts to the inhibition of MNU-induced cleavage with increasing salt. In addition, 9 methylates all G sites equally, while MNU shows a clear preference for d(G)/sub n/ (n ≥ 3) runs and an asymmetrical methylation pattern within these G-rich regions. The results are discussed in terms of the delivery of the MNU moiety to the DNA target by a non-sequence-specific intercalation process and the subsequent hydrolytic generation of a nondiffusible alkylating intermediate

  17. Mixed ligand ruthenium(III) complexes of benzaldehyde 4-methyl-3-thiosemicarbazones with triphenylphosphine/triphenylarsine co-ligands: Synthesis, DNA binding, DNA cleavage, antioxidative and cytotoxic activity

    Science.gov (United States)

    Sampath, K.; Sathiyaraj, S.; Raja, G.; Jayabalakrishnan, C.

    2013-08-01

    The new ruthenium(III) complexes with 4-methyl-3-thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL1) and (E)-2-(2-nitrobenzylidene)-N-methylhydrazinecarbothioamide (HL2), were prepared and characterized by various physico-chemical and spectroscopic methods. The title compounds act as bidentate, monobasic chelating ligands with S and N as the donor sites and are preferably found in the thiol form in all the complexes studied. The molecular structure of HL1 and HL2 were determined by single crystal X-ray diffraction method. DNA binding of the ligands and complexes were investigated by absorption spectroscopy and IR spectroscopy. It reveals that the compounds bind to nitrogenous bases of DNA via intercalation. The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage are dose dependent. Antioxidant study of the ligands and complexes showed the significant antioxidant activity against DPPH radical. In addition, the in vitro cytotoxicity of the ligands and complexes against MCF-7 cell line was assayed which showed higher cytotoxic activity with the lower IC50 values indicating their efficiency in killing the cancer cells even at low concentrations.

  18. Comparative studies on performance of CCC and preparative RP-HPLC in separation and purification of steroid saponins from Dioscorea zingiberensis C.H.Wright

    OpenAIRE

    Zhang, Xinxin; Liang, Jinru; Zhang, Yongmin; Liu, Jianli; Sun, Wenji; Ito, Yoichiro

    2015-01-01

    Steroid saponins from Dioscorea zingiberensis C.H.Wright were separated for the first time using two chromatographic methods for comparison: counter-current chromatography (CCC) coupled with evaporative light scattering detector (ELSD) and preparative reversed phase high-performance liquid chromatography (RP-HPLC) with an ultraviolet detector. Ethyl acetate-n-butanol-methanol-water (4:1:2:4, v/v) was chosen as the two-phase solvent system for CCC, while the acetonitrile-water (25:75 for the f...

  19. Final report : results of the 2006-2007 investigation of potential contamination at the former CCC/USDA facility in Barnes, Kansas.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M.; Environmental Science Division

    2008-08-28

    The 2006-2007 investigation of carbon tetrachloride and chloroform contamination at Barnes, Kansas, was conducted at the request of the Kansas Department of Health and Environment (KDHE). The Environmental Science Division of Argonne National Laboratory implemented the investigation on behalf of the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA). The overall goal of the investigation was to establish criteria for monitoring leading to potential site reclassification. The investigation objectives were to (1) determine the hydraulic gradient near the former CCC/USDA facility, (2) delineate the downgradient carbon tetrachloride plume, and (3) design and implement an expanded monitoring network at Barnes (Argonne 2006a).

  20. Roles of DNA polymerase epsilon and TopBP1 in DNA replication and damage response

    OpenAIRE

    Hillukkala, T.

    2006-01-01

    Abstract During DNA replication cells accurately copy their DNA to transfer the genetic information to daughter cells. DNA polymerases synthesise the new DNA strand using the old strand as a template. Other functions of DNA polymerases are recombination linked and DNA iamage repair linked DNA synthesis, regulation of replication complex formation and regulation of transcription – a process in which the genetic information is transformed into an RNA sequence needed to guide protein synthesi...

  1. Predictors of hepatitis B cure using gene therapy to deliver DNA cleavage enzymes: a mathematical modeling approach.

    Directory of Open Access Journals (Sweden)

    Joshua T Schiffer

    Full Text Available Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA, the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs, TAL effector nucleases (TALENs, and CRISPR-associated system 9 (Cas9 proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which

  2. The use of (3H) deoxyadenosine to measure the rate of DNA synthesis in germinating wheat embryos

    International Nuclear Information System (INIS)

    The pattern of DNA labelling in germinating wheat embryos was studied with the use of deoxy(G-3H) adenosine as the radioactive precursor and a newly-developed method for the measurement of the DNA radioactivity. The method included alkaline extraction, precipitation on a filter paper disc and mild acid hydrolysis of the precipitated material to release DNA purines selectively. Radioactive DNA could be detected in embryos already 30 min after imbibition started and its amounts (total counts) increased further at a gradually decreasing rate throughout the investigated germination period (24 h). The pattern observed differs markedly from those reported for embryos germinating in the presence of 3H- and 14C-labelled thymidine. (author)

  3. Mixed-ligand complexes of ruthenium(II) incorporating a diazo ligand: Synthesis, characterization and DNA binding

    Indian Academy of Sciences (India)

    Megha S Deshpande; Avinash S Kumbhar

    2005-03-01

    Mixed-ligand complexes of the type [Ru(N-N)2(dzdf)]Cl2, where N-N is 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen) and 9-diazo-4,5-diazafluorene (dzdf), have been synthesized and characterized by elemental analysis, UV-Vis, IR and NMR spectroscopy. Binding of these complexes with calf thymus DNA (CT-DNA) has been investigated by absorption spectroscopy, steady-state emission spectroscopy and viscosity measurements. The experimental results indicate that the size and shape of the intercalating ligands have marked effect on the binding affinity of the complexes to CT-DNA. The complex [Ru(phen)2(dzdf)]Cl2 binds with CT-DNA through an intercalative binding mode, while the complex [Ru(bpy)2(dzdf)]Cl2 binds electrostatically.

  4. In vitro synthesis and purification of PhIP-deoxyguanosine and PhIP-DNA oligomer covalent complexes

    Energy Technology Data Exchange (ETDEWEB)

    Freeman, J.

    1994-12-01

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine compound formed when meats are cooked at high temperatures. PhIP damages DNA by forming covalent complexes with DNA carcinogen. In an effort to understand how the binding of PhIP to DNA may cause cancer, it is important to characterize the structures of PhIP-damaged DNA molecules. Our HPLC data support fluorescence and {sup 32}P Post-labeling studies which indicate the formation of several species of 2{prime}deoxyguanosine-(dG) or oligodeoxynucleotide-PhIP adducts. The reaction of PhIP with dG resulted in a reddish precipitate that was likely the major adduct, N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP) adduct, with a more polar adduct fraction remaining in the supernatant. Reversed-phase HPLC analysis of the adducts in the supernatant revealed the existence of species of much shorter retention times than the dG-C8-PhIP adduct, confirming that these species are more polar than dG-C8-PhIP. At least four adducts were formed in the reaction of PhIP with DNA oligomer. HPLC analysis of the PhIP-DNA oligomer supernatant after butanol extractions revealed four unresolved peaks which spectra had maximum wavelengths between 340 and 360 nm. Though adduct peaks were not completely resolved, there was {approximately}3 minutes interval between the DNA oligomer peak and the adduct peaks. Furthermore, fluorescence emission data of the DNA oligomer-PhIP adduct solution show heterogeneous binding. The more polar PhIP adducts were fraction-collected and their structures will be solved by nuclear magnetic resonance or x-ray crystallography.

  5. 5-azacytidine induces micronuclei in and morphological transformation of Syrian hamster embryo fibroblasts in the absence of unscheduled DNA synthesis

    OpenAIRE

    Stopper, Helga; Pechan, R; Schiffmann, D

    2012-01-01

    lt is known that 5-azacytidine (5-AC) induces tumors in several organs of rats and mice. The mechanisms of these effects are still poorly understood although it is known that 5-AC can be incorporated into DNA. Furthermore, it can inhibit DNA methylation. The known data on its clastogenic andjor gene mutation-inducing potential are still controversial. Therefore, we have investigated the kinds of genotoxic effects caused by 5-AC in Syrian hamster embryo (SHE) fibroblasts. Three different endp6...

  6. 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

  7. DNA-templated synthesis of PtAu bimetallic nanoparticle/graphene nanocomposites and their application in glucose biosensor

    Science.gov (United States)

    Leng, Jing; Wang, Wen-Min; Lu, Li-Min; Bai, Ling; Qiu, Xin-Lan

    2014-02-01

    In this paper, single-stranded DNA (ss-DNA) is demonstrated to functionalize graphene (GR) and to further guide the growth of PtAu bimetallic nanoparticles (PtAuNPs) on GR with high densities and dispersion. The obtained nanocomposites (PtAuNPs/ss-DNA/GR) were characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectrometer (EDS), and electrochemical techniques. Then, an enzyme nanoassembly was prepared by self-assembling glucose oxidase (GOD) on PtAuNP/ss-DNA/GR nanocomposites (GOD/PtAuNPs/ss-DNA/GR). The nanocomposites provided a suitable microenvironment for GOD to retain its biological activity. The direct and reversible electron transfer process between the active site of GOD and the modified electrode was realized without any extra electron mediator. Thus, the prepared GOD/PtAuNP/ss-DNA/GR electrode was proposed as a biosensor for the quantification of glucose. The effects of pH, applied potential, and temperature on the performance of the biosensor were discussed in detail and were optimized. Under optimal conditions, the biosensor showed a linearity with glucose concentration in the range of 1.0 to 1,800 μM with a detection limit of 0.3 μM (S/N = 3). The results demonstrate that the developed approach provides a promising strategy to improve the sensitivity and enzyme activity of electrochemical biosensors.

  8. Biological Impact of Pd (II) Complexes: Synthesis, Spectral Characterization, In Vitro Anticancer, CT-DNA Binding, and Antioxidant Activities

    Science.gov (United States)

    Sharma, Nitin Kumar; Ameta, Rakesh Kumar; Singh, Man

    2016-01-01

    A new series of Pd (II) complexes of methyl substituted benzylamine ligands (BLs) has been synthesized and characterized via spectroscopic techniques such as UV/Vis. FTIR, LCMS, 1H, and 13C NMR. The UV/Vis study in DMSO, DMSO + water, and DMSO + PBS buffer (pH = 7.2) confirmed their molecular sustainability in liquids. Their in vitro anticancer activity against breast cancer cell lines such as MCF-7 and MDA-MB-231 makes them interesting for in vivo analysis. Their stronger DNA binding activity (DBA) compared with free ligand suggested them as a good DNA binder. DBA was further confirmed by physicochemical studies such as surface tension and viscosity of complex + DNA which inferred the disruption of DNA and intercalation of complexes, respectively. Their % binding activity, % disruption of DNA base pairs (DNABP), and % intercalating strength are reported in this paper for the first time for better understanding of DNA binding mechanism. Along with this, their scavenging activity (SA) determined through DPPH free radical and the results indicate good antioxidant behaviour of complexes. PMID:26989511

  9. Peculiarities of the incorporation of [3H]thymidine into AT-rich regions of DNA during replicative synthesis

    International Nuclear Information System (INIS)

    The authors studied the role of the AT-rich regions in DNA replication in vivo. The authors selected cells of humans and Drosophila - organisms belonging to different types of alternation of unique and repetitive sequences - as the objects of investigation. The authors then studied the behavior of the AT-rich sequences in replication by the method of thermoelution of [3H]thymidine-labeled DNA, fragmented by ultrasound to 350 nucleotide pairs. By measuring the amount of DNA and the amount of the label in the fractions, the authors were able to construct curves of the change in the specific activity of DNA as a function of the temperature of elution from HAP and, consequently, as a function of the AT composition. The authors call them differential temperature chromatograms (DTC). Human peripheral blood lymphocytes were cultured according to the standard procedure with PHA (Difco P). A culture of D. melanogaster cells was labeled with [3H]thymidine in the logarithmic phase of growth for 1.2 and 42 h. At the end of the labeling, cell DNA was isolated from the lymphocytes and cell and nuclear DNA from a Drosophila tissue culture by the standard methods

  10. Copper(II) complexes with 4-hydroxyacetophenone-derived acylhydrazones: Synthesis, characterization, DNA binding and cleavage properties

    Science.gov (United States)

    Gup, Ramazan; Gökçe, Cansu; Aktürk, Selçuk

    2015-01-01

    Two new Cu(II) complexes of Schiff base-hydrazone ligands, hydroxy-N‧-[(1Z)-1-(4-hydroxyphenyl)ethylidene]benzohydrazide [H3L1] and ethyl 2-(4-(1-(2-(4-(2-ethoxy-2-oxoethoxy)benzoyl)hydrazono)ethyl)phenoxy)acetate (HL2) have been synthesized and then characterized by microcopy and spectral studies. X-ray powder diffraction illustrates that [Cu(L2)2] complex is crystalline in nature whereas [Cu(H2L1)2]·2H2O has an amorphous structure. Binding of the copper complexes with Calf thymus DNA (CT-DNA) has been investigated by UV-visible spectra, exhibiting non-covalent binding to CT-DNA. DNA cleavage experiments have been also investigated by agarose gel electrophoresis in the presence and absence of an oxidative agent (H2O2). The effect of complex concentration on the DNA cleavage reaction has been also studied. Both copper complexes show nuclease activity, which significantly depends on concentrations of the complexes, in the presence of H2O2 through oxidative mechanism whereas they slightly cleavage DNA in the absence an oxidative agent.

  11. One-pot solvent free synthesis and DNA binding studies of thieno[2,3-b]-1,8-naphthyridines.

    Science.gov (United States)

    Naik, Tangali R Ravikumar; Naik, Halehatty S Bhojya; Prabhakara, Mustur C

    2008-01-01

    With the aim of evaluating interaction between double-stranded calf thymus (ds)DNA and sulphur containing fused planar rings, the derivatives of 1,8-naphthyridine containing thiono groups were synthesized by the condensation of 2-mercapto-3-formyl[1,8]naphthyridines using 1-chloroacetone, 2-chloroacetamide, chloroaceticacid, and 2-chloro-1-phenylethanone in the presence of anhydrous potassium carbonate as s catalyst under solvent free microwave irradiation. The structures of the compounds were elucidated on the basis of elemental analysis, IR, (1)H NMR, and mass spectra. The interaction of thieno[2,3-b]-1,8-naphthyridine-2-carboxylic acid (TNC) (3a) with ct-DNA was studied by UV-Vis spectrophotometry, viscosity, thermal denaturation, as well as cyclic voltammetry experiments. On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts. Binding parameters, determined from spectrophotometric measurements indicated a binding constant of Kb=2.1 x 10(6) M(-1). The thieno[2,3-b]-1,8-naphthyridine-2-carboxylic acid (3a) increases the viscosity of sonicated rod-like DNA fragments. The binding of TNC to DNA increased the melting temperature by about 4 degrees C. The decrease in peak current heights and shifts of peak potential values are observed by the addition of calf thymus DNA in cyclic voltammetry studies. PMID:18080916

  12. Synthesis and bioactive evaluations of novel benzotriazole compounds as potential antimicrobial agents and the interaction with calf thymus DNA

    Indian Academy of Sciences (India)

    Yu Ren; Hui Zhen Zhang; Shao Lin Zhang; Yun Lei Luo; Ling Zhang; Cheng He Zhou; Rong Xia Geng

    2015-12-01

    A novel series of benzotriazole derivatives were synthesized and characterized by NMR, IR and MS spectra. The bioactive assay manifested that most of the new compounds exhibited moderate to good antibacterial and antifungal activities against the tested strains in comparison to reference drugs chloromycin, norfloxacin and fluconazole. Especially, 2,4-dichlorophenyl substituted benzotriazole derivative 6f displayed good antibacterial activity against MRSA with MIC value of 4 g/mL, which was 2-fold more potent than Chloromycin, and it also displayed 3-fold stronger antifungal activity (MIC = 4 g/mL) than fluconazole (MIC = 16 g/mL) against Beer yeast. The preliminary interactive investigations of compound 6f with calf thymus DNA revealed that compound 6f could effectively intercalate into DNA to form compound 6f–DNA complex which might block DNA replication to exert antimicrobial activities. Molecular docking experiments suggested that compound 6f projected into base-pairs of DNA hexamer duplex forming two hydrogen bonds with guanine of DNA. The theoretical calculations were in accordance with the experimental results.

  13. Mixed Compound of DCPTA and CCC Increases Maize Yield by Improving Plant Morphology and Up-Regulating Photosynthetic Capacity and Antioxidants.

    Directory of Open Access Journals (Sweden)

    Yongchao Wang

    Full Text Available DCPTA (2-diethylaminoethyl-3, 4-dichlorophenylether and CCC (2-chloroethyltrimethyl- ammonium chloride have a great effect on maize growth, but applying DCPTA individually can promote the increase of plant height, resulting in the rise of lodging percent. Plant height and lodging percent decrease in CCC-treated plants, but the accumulation of biomass reduce, resulting in yield decrease. Based on the former experiments, the performance of a mixture which contained 40 mg DCPTA and 20 mg CCC as active ingredients per liter of solution, called PCH was tested with applying 40mg/L DCPTA and 20mg/L CCC individually. Grain yield, yield components, internode characters, leaf area per plant, plant height and lodging percent as well as chlorophyll content, chlorophyll fluorescence, enzymatic antioxidants, membranous peroxide and organic osmolyte were analyzed in two years (2011 and 2012, using maize hybrid, Zhengdan 958 (ZD 958 at density of 6.75 plants m-2. CCC, DCPTA and PCH were sprayed on the whole plant leaves at 7 expanded leaves stage and water was used as control. Compared to control, PCH significantly increased grain yield (by 9.53% and 6.68% from 2011 to 2012. CCC significantly decreased kernel number per ear (by 6.78% and 5.69% and thousand kernel weight (TKW (by 8.57% and 6.55% from 2011 to 2012. Kernel number per ear and TKW increased in DCPTA-treated and PCH-treated plants, but showed no significant difference between them. In CCC-treated and PCH-treated plants, internode length and plant height decreased, internode diameter increased, resulting in the significant decline of lodging percent. With DCPTA application, internode diameter increased, but internode length and plant height increased at the same time, resulting in the augment of lodging percent. Bending strength and puncture strength were increased by applying different plant growth regulators (PGRs. In PCH-treated plants, bending strength and puncture strength were greater than other

  14. Mixed Compound of DCPTA and CCC Increases Maize Yield by Improving Plant Morphology and Up-Regulating Photosynthetic Capacity and Antioxidants.

    Science.gov (United States)

    Wang, Yongchao; Gu, Wanrong; Xie, Tenglong; Li, Lijie; Sun, Yang; Zhang, He; Li, Jing; Wei, Shi

    2016-01-01

    DCPTA (2-diethylaminoethyl-3, 4-dichlorophenylether) and CCC (2-chloroethyltrimethyl- ammonium chloride) have a great effect on maize growth, but applying DCPTA individually can promote the increase of plant height, resulting in the rise of lodging percent. Plant height and lodging percent decrease in CCC-treated plants, but the accumulation of biomass reduce, resulting in yield decrease. Based on the former experiments, the performance of a mixture which contained 40 mg DCPTA and 20 mg CCC as active ingredients per liter of solution, called PCH was tested with applying 40mg/L DCPTA and 20mg/L CCC individually. Grain yield, yield components, internode characters, leaf area per plant, plant height and lodging percent as well as chlorophyll content, chlorophyll fluorescence, enzymatic antioxidants, membranous peroxide and organic osmolyte were analyzed in two years (2011 and 2012), using maize hybrid, Zhengdan 958 (ZD 958) at density of 6.75 plants m-2. CCC, DCPTA and PCH were sprayed on the whole plant leaves at 7 expanded leaves stage and water was used as control. Compared to control, PCH significantly increased grain yield (by 9.53% and 6.68%) from 2011 to 2012. CCC significantly decreased kernel number per ear (by 6.78% and 5.69%) and thousand kernel weight (TKW) (by 8.57% and 6.55%) from 2011 to 2012. Kernel number per ear and TKW increased in DCPTA-treated and PCH-treated plants, but showed no significant difference between them. In CCC-treated and PCH-treated plants, internode length and plant height decreased, internode diameter increased, resulting in the significant decline of lodging percent. With DCPTA application, internode diameter increased, but internode length and plant height increased at the same time, resulting in the augment of lodging percent. Bending strength and puncture strength were increased by applying different plant growth regulators (PGRs). In PCH-treated plants, bending strength and puncture strength were greater than other treatments

  15. Error-Prone Translesion DNA Synthesis by Escherichia coli DNA Polymerase IV (DinB on Templates Containing 1,2-dihydro-2-oxoadenine

    Directory of Open Access Journals (Sweden)

    Masaki Hori

    2010-01-01

    Full Text Available Escherichia coli DNA polymerase IV (Pol IV is involved in bypass replication of damaged bases in DNA. Reactive oxygen species (ROS are generated continuously during normal metabolism and as a result of exogenous stress such as ionizing radiation. ROS induce various kinds of base damage in DNA. It is important to examine whether Pol IV is able to bypass oxidatively damaged bases. In this study, recombinant Pol IV was incubated with oligonucleotides containing thymine glycol (dTg, 5-formyluracil (5-fodU, 5-hydroxymethyluracil (5-hmdU, 7,8-dihydro-8-oxoguanine (8-oxodG and 1,2-dihydro-2-oxoadenine (2-oxodA. Primer extension assays revealed that Pol IV preferred to insert dATP opposite 5-fodU and 5-hmdU, while it inefficiently inserted nucleotides opposite dTg. Pol IV inserted dCTP and dATP opposite 8-oxodG, while the ability was low. It inserted dCTP more effectively than dTTP opposite 2-oxodA. Pol IV's ability to bypass these lesions decreased in the order: 2-oxodA > 5-fodU~5-hmdU > 8-oxodG > dTg. The fact that Pol IV preferred to insert dCTP opposite 2-oxodA suggests the mutagenic potential of 2-oxodA leading to A:T→G:C transitions. Hydrogen peroxide caused an ~2-fold increase in A:T→G:C mutations in E. coli, while the increase was significantly greater in E. coli overexpressing Pol IV. These results indicate that Pol IV may be involved in ROS-enhanced A:T→G:C mutations.

  16. Synthesis of oligonucleotide phosphorodithioates

    DEFF Research Database (Denmark)

    Beaton, G.; Brill, W. K D; Grandas, A.;

    1991-01-01

    The synthesis of DNA containing sulfur at the two nonbonding internucleotide valencies is reported. Several different routes using either tervalent or pentavalent mononucleotide synthons are described. © 1991.......The synthesis of DNA containing sulfur at the two nonbonding internucleotide valencies is reported. Several different routes using either tervalent or pentavalent mononucleotide synthons are described. © 1991....

  17. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease

    OpenAIRE

    Kennedy, Edward M.; Bassit, Leda C.; Mueller, Henrik; Kornepati, Anand V. R.; Bogerd, Hal P.; Nie, Ting; Chatterjee, Payel; Javanbakht, Hassan; Schinazi, Raymond F.; Cullen, Bryan R.

    2014-01-01

    Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of bot...

  18. Synthesis and structural characterization of dioxomolybdenum and dioxotungsten hydroxamato complexes and their function in the protection of radiation induced DNA damage.

    Science.gov (United States)

    Paul, Shiv Shankar; Selim, Md; Saha, Abhijit; Mukherjea, Kalyan K

    2014-02-21

    The synthesis and structural characterization of two novel dioxomolybdenum(VI) (1) and dioxotungsten(VI) (2) complexes with 2-phenylacetylhydroxamic acid (PAHH) [M(O)2(PAH)2] [M = Mo, W] have been accomplished. The dioxomolybdenum(VI) and dioxotungsten(VI) moiety is coordinated by the hydroxamate group (-CONHO(-)) of the 2-phenylacetylhydroxamate (PAH) ligand in a bi-dentate fashion. In both the complexes the PAHH ligand is coordinated through oxygen atoms forming a five membered chelate. The hydrogen atom of N-H of the hydroxamate group is engaged in intermolecular H-bonding with the carbonyl oxygen of another coordinated hydroxamate ligand, thereby forming an extended 1D chain. The ligand as well as both the complexes exhibit the ability to protect from radiation induced damage both in CTDNA as well as in pUC19 plasmid DNA. As the damage to DNA is caused by the radicals generated during radiolysis, its scavenging imparts protection from the damage to DNA. To understand the mechanism of protection, binding affinities of the ligand and the complex with DNA were determined using absorption and emission spectral studies and viscosity measurements, whereby the results indicate that both the complexes and the hydroxamate ligand interact with calf thymus DNA in the minor groove. The intrinsic binding constants, obtained from UV-vis studies, are 7.2 × 10(3) M(-1), 5.2 × 10(4) M(-1) and 1.2 × 10(4) M(-1) for the ligand and complexes 1 and 2 respectively. The Stern-Volmer quenching constants obtained from a luminescence study for both the complexes are 5.6 × 10(4) M(-1) and 1.6 × 10(4) M(-1) respectively. The dioxomolybdenum(VI) complex is found to be a more potent radioprotector compared to the dioxotungsten(VI) complex and the ligand. Radical scavenging chemical studies suggest that the complexes have a greater ability to scavenge both the hydroxyl as well as the superoxide radicals compared to the ligand. The free radical scavenging ability of the ligand and the

  19. Synthesis of a Novel Organic Molecule for DNA Cleavage%新型有机小分子DNA切割试剂的合成

    Institute of Scientific and Technical Information of China (English)

    安东; 赵晓辉; 周密; 叶志文

    2014-01-01

    transphosphorylation pathway through oxidation-reduction reaction. Thus, this compound may be useful as artificial nucleic acid cleaving agent and the study may be usefully applied to achieve a more effective DNA cleavage for optimizing the structure and the distance of func-tional group to synergistic catalytic cleavage of the phosphodiester bond. In conclusion, design and synthesis of a novel phosphodiester receptor compound 4 containing guanidinoethyl and hydroxyethyl side arms was achieved successfully. We propose to introduce more such compounds as cleaving agents of nucleic acids to be widely investigated and found to be quite efficient.%根据活性基团的协同催化原理,设计合成了有机小分子核酸切割剂1-(N-胍乙基)-4-(N-羟乙基)哌嗪盐酸盐(4),并通过核磁共振和液相色谱-质谱联用技术对其结构进行了表征.利用琼糖凝胶电泳研究了pH值对其切割pUC 19 DNA 效率的影响,通过自由基猝灭实验研究其切割DNA的反应类型.运用密度泛函理论,利用 Gaussian 软件进行了理论计算,研究其裂解DNA的反应方式.研究结果表明,在pH=7.2时化合物4的裂解效率最高,且能通过非氧化还原反应以磷酯转移的方式裂解DNA的磷酸二酯键.

  20. Synthesis of C-5, C-2' and C-4'-neomycin-conjugated triplex forming oligonucleotides and their affinity to DNA-duplexes.

    Science.gov (United States)

    Tähtinen, Ville; Granqvist, Lotta; Virta, Pasi

    2015-08-01

    Neomycin-conjugated homopyrimidine oligo 2'-deoxyribonucleotides have been synthesized on a solid phase and their potential as triplex forming oligonucleotides (TFOs) with DNA-duplexes has been studied. For the synthesis of the conjugates, C-5, C-2' and C-4'-tethered alkyne-modified nucleoside derivatives were used as an integral part of the standard automated oligonucleotide chain elongation. An azide-derived neomycin was then conjugated to the incorporated terminal alkynes by Cu(I)-catalyzed 1,3-dipolar cycloaddition (the click chemistry). Concentrated ammonia released the desired conjugates in acceptable purity and yields. The site of conjugation was expectedly important for the Hoogsteen-face recognition: C-5-conjugation showed a notable positive effect, whereas the influence of the C-2' and C-4'-modification remained marginal. In addition to conventional characterization methods (UV- and CD-spectroscopy), (19)F NMR spectroscopy was applied for the monitoring of triplex/duplex/single strand-conversions. PMID:26118338