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Sample records for ccc dna synthesis

  1. Mechanism for CCC DNA synthesis in hepadnaviruses.

    Directory of Open Access Journals (Sweden)

    Ji A Sohn

    Full Text Available Hepadnavirus replication requires the synthesis of a covalently closed circular (CCC DNA from the relaxed circular (RC viral genome by an unknown mechanism. CCC DNA formation could require enzymatic activities of the viral reverse transcriptase (RT, or cellular DNA repair enzymes, or both. Physical mapping of the 5' and 3' ends of RC DNA and sequence analysis of CCC DNA revealed that CCC DNA synthesis requires the removal of the RT and an RNA oligomer from the 5' ends of minus and plus strand DNA, respectively, removal of sequences from the terminally redundant minus strand, completion of the less than full-length plus strand, and ligation of the ends. Two models have been proposed that could explain CCC DNA formation. The first (model 1 invokes a role for the RT to catalyze a cleavage-ligation reaction leading to the formation of a unit length minus strand in CCC DNA and a DNA repair reaction for the completion and ligation of plus strand DNA; the second (model 2 predicts that CCC DNA formation depends entirely on cellular DNA repair enzymes. To determine which mechanism is utilized, we developed cell lines expressing duck hepatitis B virus genomes carrying mutations permitting us to follow the fate of viral DNA sequences during their conversion from RC to CCC DNA. Our results demonstrated that the oligomer at the 5' end of minus strand DNA is completely or at least partially removed prior to CCC DNA synthesis. The results indicated that both RC DNA strands undergo DNA repair reactions carried out by the cellular DNA repair machinery as predicted by model 2. Thus, our study provided the basis for the identification of the cellular components required for CCC DNA formation.

  2. Alpha-interferon suppresses hepadnavirus transcription by altering epigenetic modification of cccDNA minichromosomes.

    Directory of Open Access Journals (Sweden)

    Fei Liu

    2013-09-01

    Full Text Available Covalently closed circular DNA (cccDNA of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the transcriptional template for viral mRNA synthesis. Elimination of cccDNA is the prerequisite for either a therapeutic cure or immunological resolution of HBV infection. Although accumulating evidence suggests that inflammatory cytokines-mediated cure of virally infected hepatocytes does occur and plays an essential role in the resolution of an acute HBV infection, the molecular mechanism by which the cytokines eliminate cccDNA and/or suppress its transcription remains elusive. This is largely due to the lack of convenient cell culture systems supporting efficient HBV infection and cccDNA formation to allow detailed molecular analyses. In this study, we took the advantage of a chicken hepatoma cell line that supports tetracycline-inducible duck hepatitis B virus (DHBV replication and established an experimental condition mimicking the virally infected hepatocytes in which DHBV pregenomic (pg RNA transcription and DNA replication are solely dependent on cccDNA. This cell culture system allowed us to demonstrate that cccDNA transcription required histone deacetylase activity and IFN-α induced a profound and long-lasting suppression of cccDNA transcription, which required protein synthesis and was associated with the reduction of acetylated histone H3 lysine 9 (H3K9 and 27 (H3K27 in cccDNA minichromosomes. Moreover, IFN-α treatment also induced a delayed response that appeared to accelerate the decay of cccDNA. Our studies have thus shed light on the molecular mechanism by which IFN-α noncytolytically controls hepadnavirus infection.

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

    Science.gov (United States)

    Seeger, Christoph; Sohn, Ji A

    2016-01-01

    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

  4. HBV cccDNA in patients′ sera as an indicator for HBV reactivation and an early signal of liver damage

    Institute of Scientific and Technical Information of China (English)

    Ying Chen; Johnny Sze; Ming-Liang He

    2004-01-01

    AIM: To evaluate the covalently closed circle DNA (cccDNA)level of hepatitis B virus (HBV) in patients′ liver and sera.METHODS: HBV DNA was isolated from patients′liver biopsies and sera. A sensitive real-time PCR method, which is capable of differentiation of HBV viral genomic DNA and cccDNA, was used to quantify the total HBV cccDNA. The total HBV viral DNA was quantitated by real-time PCR using a HBV diagnostic kit (PG Biotech, LTD, Shenzhen, China)described previously.RESULTS: For the first time, we measured the level of HBV DNA and cccDNA isolated from ten HBV patients′liver biopsies and sera. In the liver biopsies, cccDNA was detected from all the biopsy samples. The copy number of cccDNA ranged from from 0.03 to 173.1 per cell, the copy number of total HBV DNA ranged from 0.08 to 3 717 per cell. The ratio of total HBV DNA to cccDNA ranged from 1 to 3 406. In the sera,cccDNA was only detected from six samples whereas HBV viral DNA was detected from all ten samples. The ratio of cccDNA to total HBV DNA ranged from 0 to 1.77%. To further investigate the reason why cccDNA could only be detected in some patients′sera, we performed longitudinal studies. The cccDNA was detected from the patients′sera with HBV reactivation but not from the patients′sera without HBV reactivation. The level of cccDNA in the sera was correlated with ALT and viral load in the HBV reactivation patients.CONCLUSION: HBV cccDNA is actively transcribed and replicated in some patients′hepatocytes, which is reflected by a high ratio of HBV total DNA vs cccDNA. Detection of cccDNA in the liver biopsy will provide an end-point for the anti-HBV therapy. The occurrence of cccDNA in the sera is an early signal of liver damage, which may be another important clinical parameter.

  5. A T7 Endonuclease I Assay to Detect Talen-Mediated Targeted Mutation of HBV cccDNA.

    Science.gov (United States)

    Bloom, Kristie; Ely, Abdullah; Arbuthnot, Patrick

    2017-01-01

    Gene editing using designer nucleases is now widely used in many fields of molecular biology. The technology is being developed for the treatment of viral infections such as persistant hepatitis B virus (HBV). The replication intermediate of HBV comprising covalently closed circular DNA (cccDNA) is stable and resistant to available licensed antiviral agents. Advancing gene editing as a means of introducing targeted mutations into cccDNA thus potentially offers the means to cure infection by the virus. Essentially, targeted mutations are initiated by intracellular DNA cleavage, then error-prone nonhomologous end joining results in insertions and deletions (indels) at intended sites. Characterization of these mutations is crucial to confirm activity of potentially therapeutic nucleases. A convenient tool for evaluation of the efficiency of target cleavage is the single strand-specific endonuclease, T7EI. Assays employing this enzyme entail initial amplification of DNA encompassing the targeted region. Thereafter the amplicons are denatured and reannealed to allow hybridization between indel-containing and wild-type sequences. Heteroduplexes that contain mismatched regions are susceptible to action by T7EI and cleavage of the hybrid amplicons may be used as an indicator of efficiency of designer nucleases. The protocol described here provides a method of isolating cccDNA from transfected HepG2.2.15 cells and evaluation of the efficiency of mutation by a transcription activator-like effector nuclease that targets the surface open reading frame of HBV.

  6. 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.

  7. 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.

  8. 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.

  9. Hairpin DNA probe with 5'-TCC/CCC-3' overhangs for the creation of silver nanoclusters and miRNA assay.

    Science.gov (United States)

    Xia, Xiaodong; Hao, Yuanqiang; Hu, Shengqiang; Wang, Jianxiu

    2014-01-15

    A facile strategy for the assay of target miRNA using fluorescent silver nanoclusters (AgNCs) has been described. Due to the preferable interaction between cytosine residues and Ag(+), a short cytosine-rich oligonucleotide (ODN) with only six bases 5'-TCCCCC-3' served as an efficient scaffold for the creation of the AgNCs. The AgNCs displayed a bright red emission when excited at 545nm. Such ODN base-stabilized AgNCs have been exploited for miRNA sensing. Overhangs of TCC at the 5' end (5'-TCC) and CCC at the 3' end (CCC-3') (denoted as 5'-TCC/CCC-3') appended to the hairpin ODN probe which also contains recognition sequences for target miRNA were included. Interestingly, the AgNCs/hairpin ODN probe showed similar spectral properties as that templated by 5'-TCCCCC-3'. The formation of the hairpin ODN probe/miRNA duplex separated the 5'-TCC/CCC-3' overhangs, thus disturbing the optical property or structure of the AgNCs. As a result, fluorescence quenching of the AgNCs/hairpin ODN probe was obtained, which allows for facile determination of target miRNA. The proposed method is simple and cost-effective, holding great promise for clinical applications.

  10. 乙肝病毒相关肝病患者肝移植后外周血单个核细胞及肝组织中乙型肝炎病毒cccDNA的检测%Detection of hepatitis B virus cccDNA in peripheral blood monuclear cells and liver tissues in hepatitis B virus-related disease patients after liver transplantation

    Institute of Scientific and Technical Information of China (English)

    张荣; 吴忠均; 曹辰; 陈映

    2011-01-01

    背景:肝移植后受者体内绝大部分病毒负荷被清除,植入新肝后其复发性肝炎病原体从肝外进入肝内的途径及其复制规律目前尚无定论.目的:检测肝移植前后外周血单个核细胞和肝组织中乙型肝炎病毒cccDNA及血清中乙型肝炎病毒DNA的表达.方法:采用淋巴细胞分离液从乙肝病毒相关终末期肝病37例患者外周血中分离出单个核细胞,采用荧光定量PCR检测肝移植前后及移植后乙肝复发3个时期外周血单个核细胞和肝组织中cccDNA及血清乙型肝炎病毒DNA表达.结果与结论:肝移植前,单个核细胞cccDNA阳性12例,肝组织cccDNA阳性6例,检出率分别为32%和16%,单个核细胞、肝组织中cccDNA拷贝范围分别为(3.028~6.508)×104,(4.158~6.234)×104 拷贝/mL.肝移植后,单个核细胞cccDNA阳性1例,无血清乙型肝炎病毒DNA检测阳性病例.6例肝移植后乙肝复发病例中外周血单个核细胞cccDNA阳性4例,肝组织活检cccDNA阳性1例,6例血清乙型肝炎病毒 DNA均为阳性.提示乙肝病毒相关终末期肝病患者肝移植后乙肝复发途径可能是残留乙肝病毒在外周单个核细胞中以cccDNA为模板复制,然后再迁移到肝脏.%BACKGROUND: Life-Iong prophylaxis against hepatitis B virus (HBV) recurrence is recommended in patients transplanted for HBV-related disease. But exact mechanism of HBV recurrence and its copy law is unknown.OBJECTIVE: By detection of HBV cccDNA and HBV DNA in peripheral blood mononuclear cells (PBMCs) and liver tissues and serum from HBV-related end-stage liver disease patients before and after liver transplantation.METHODS: PBMCs were isolated from HBV-related end-stage liver disease patients by lymphocyte separation medium.HBVcccDNA in PBMCs and liver tissue and the serum HBVDNA at three periods of before, after liver transplantation and postoperative recurrence were detected by fluorescent quantitative PCR.RESULTS AND CONCLUSION: Before liver

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

    Science.gov (United States)

    Reaiche-Miller, Georget Y; Thorpe, Michael; Low, Huey Chi; Qiao, Qiao; Scougall, Catherine A; Mason, William S; Litwin, Samuel; Jilbert, Allison R

    2013-11-01

    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(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.

  12. Towards the Batch Synthesis of Long DNA

    Science.gov (United States)

    2002-10-01

    Laplacian on a Riemannian Manifold, Cambridge, Cambridge (1997). 131 Arfken , G., Mathematical Methods for Physicists. Academic Press, Orlando (1985...typical phosphoramidite chemical synthesis method .1 On the other hand, two ss (single-stranded) DNAs can be joined or ligated into a single ds (double...the preferred method for the de novo laboratory synthesis of long DNA.3 More generally there are undoubtedly profound clinical (e.g., gene therapeutic

  13. 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...

  14. 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...

  15. Thymidine analogues for tracking DNA synthesis.

    Science.gov (United States)

    Cavanagh, Brenton L; Walker, Tom; Norazit, Anwar; Meedeniya, Adrian C B

    2011-09-15

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

  16. Chemically-enzymatic synthesis of photosensitive DNA.

    Science.gov (United States)

    Westphal, Kinga; Zdrowowicz, Magdalena; Zylicz-Stachula, Agnieszka; Rak, Janusz

    2017-02-01

    The sensitizing propensity of radio-/photosensitizing nucleoside depends on DNA sequence surrounding a sensitizer. Therefore, in order to compare sensitizers with regard to their ability to induce a DNA damage one has to study the sequence dependence of damage yield. However, chemical synthesis of oligonucleotides labeled with sensitizing nucleosides is hindered due to the fact that a limited number of such nucleoside phosphoramidites are accessible. Here, we report on a chemically-enzymatic method, employing a DNA polymerase and ligase, that enables a modified nucleoside, in the form of its 5'-triphosphate, to be incorporated into DNA fragment in a pre-determined site. Using such a protocol two double-stranded DNA fragments - a long one, 75 base pairs (bp), and a short one, 30bp in length - were pin-point labeled with 5-bromodeoxyuridine. Four DNA polymerases together with DHPLC for the inspection of reaction progress were used to optimize the process under consideration. As an ultimate test showing that the product possessing an assumed nucleotide sequence was actually obtained, we irradiated the synthesized oligonucleotide with UVB photons and analyzed its photoreactivity with the LC-MS method. Our results prove that a general approach enabling precise labeling of DNA with any nucleoside modification processed by DNA polymerase and ligase has been worked out.

  17. 7 CFR 1494.601 - Acceptance of offers by CCC.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Acceptance of offers by CCC. 1494.601 Section 1494... Program Operations § 1494.601 Acceptance of offers by CCC. (a) Establishment of acceptable sales prices... that becomes available to CCC. (b) Acceptance of offers for a CCC bonus on a competitive basis....

  18. Thymidine Analogues for Tracking DNA Synthesis

    Directory of Open Access Journals (Sweden)

    Brenton L. Cavanagh

    2011-09-01

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

  19. DNA Compatible Multistep Synthesis and Applications to DNA Encoded Libraries.

    Science.gov (United States)

    Satz, Alexander Lee; Cai, Jianping; Chen, Yi; Goodnow, Robert; Gruber, Felix; Kowalczyk, Agnieszka; Petersen, Ann; Naderi-Oboodi, Goli; Orzechowski, Lucja; Strebel, Quentin

    2015-08-19

    Complex mixtures of DNA encoded small molecules may be readily interrogated via high-throughput sequencing. These DNA encoded libraries (DELs) are commonly used to discover molecules that interact with pharmaceutically relevant proteins. The chemical diversity displayed by the library is key to successful discovery of potent, novel, and drug-like chemical matter. The small molecule moieties of DELs are generally synthesized though a multistep process, and each chemical step is accomplished while it is simultaneously attached to an encoding DNA oligomer. Hence, library chemical diversity is often limited to DNA compatible synthetic reactions. Herein, protocols for 24 reactions are provided that have been optimized for high-throughput production of DELs. These protocols detail the multistep synthesis of benzimidazoles, imidazolidinones, quinazolinones, isoindolinones, thiazoles, and imidazopyridines. Additionally, protocols are provided for a diverse range of useful chemical reactions including BOC deprotection (under pH neutral conditions), carbamylation, and Sonogashira coupling. Last, step-by-step protocols for synthesizing functionalized DELs from trichloronitropyrimidine and trichloropyrimidine scaffolds are detailed.

  20. DNA Nanoparticles for Improved Protein Synthesis In Vitro.

    Science.gov (United States)

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

    2016-02-24

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

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    Seki,Shuji

    1984-06-01

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

  3. 7 CFR 1494.501 - Submission of offers to CCC.

    Science.gov (United States)

    2010-01-01

    ... before the time the offer is to be considered by CCC, unless otherwise required by law; (viii) No attempt... required certifications, unless CCC determines that acceptance of the offer would be in the best interests... 7 Agriculture 10 2010-01-01 2010-01-01 false Submission of offers to CCC. 1494.501 Section...

  4. 7 CFR 17.9 - CCC payment to suppliers.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false CCC payment to suppliers. 17.9 Section 17.9... to suppliers. (a) General. (1) The supplier shall request payment from CCC for the amount of the commodity price or the ocean freight or ocean freight differential to be financed by CCC. (2) The...

  5. Specific functions of the Rep and Rep׳ proteins of porcine circovirus during copy-release and rolling-circle DNA replication.

    Science.gov (United States)

    Cheung, Andrew K

    2015-07-01

    The roles of two porcine circovirus replication initiator proteins, Rep and Rep׳, in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replication) and generates the single-stranded circular (ssc) genome from the displaced DNA strand. In the process, a minus-genome primer (MGP) necessary for complementary-strand synthesis, from ssc to ccc, is synthesized. Rep׳ cleaves the growing nascent-strand to regenerate the parent ccc molecule. In the process, a Rep׳-DNA hybrid containing the right palindromic sequence (at the origin of DNA replication) is generated. Analysis of the virus particle showed that it is composed of four components: ssc, MGP, capsid protein and a novel Rep-related protein (designated Protein-3).

  6. RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV

    Science.gov (United States)

    Tashjian, Tommy F.; Lin, Ida; Belt, Verena; Cafarelli, Tiziana M.; Godoy, Veronica G.

    2017-01-01

    In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB’s fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability.

  7. Spinach thioredoxin m inhibits DNA synthesis in fertilized Xenopus eggs.

    OpenAIRE

    Hartman, H; Wu, M.; Buchanan, B.B.; Gerhart, J C

    1993-01-01

    A role for thioredoxin in metazoan DNA synthesis has been assessed by injecting rapidly dividing Xenopus eggs with purified heterologous thioredoxins, which might act as inhibitors if they were to replace resident thioredoxins in some but not all reaction steps. Of 10 tested proteins, spinach chloroplast thioredoxin m is the most potent inhibitor. Eggs cleave and produce cells lacking nuclei. DNA synthesis is severely reduced. Development arrests before gastrulation. In egg extracts, thioredo...

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

    Directory of Open Access Journals (Sweden)

    Akanksha Srivastava

    Full Text Available 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 anticancer potential using human colon adenocarcinoma (HT29 and human kidney adenocarcinoma (A498 cancer cell lines. Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 were the most potent as determined by examination of morphological features and by inhibition of growth by graded concentrations of crude extracts and thin-layer chromatography (TLC eluates. Cell cycle analysis and multiplex assays using cancer biomarkers also confirmed Geitlerinema sp. CCC728 and Arthrospira sp. CCC729 as cancer drug resources. Apoptotic studies in the cells of A498 (cancer and MCF-10A (normal human epithelial exposed to crude extracts and TLC fractions revealed no significant impact on MCF-10A cells emphasizing its importance in the development of anticancer drug. Identification of biomolecules from these extracts are in progress.

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Effects of CCC on Water- logging Resistance in Pepper

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Four concentrations of CCC were used to treat pepper seedlings. The results indicated that 50 - 150mg@ L-1 CCC decreased the content of malonaldehydic acid (MDA) and increased the content of soluble protein, ascorbic acid (AsA) and glutathion(GSH), and activity of superoxade dismutase (SOD) and catalase (CAT), whereas 200mg@ L -1 CCC caused the soluble protein content and the CAT activity of Xiangyan 1 and the AsA content of Xiangyan 10 to descend. The comprehensive evaluation manifested that 100 - 150mg@ L-1 CCC had obvious effects on water - logging resistance of pepper. Different varietes required different concentrations, eg, 100mg@ L 1 CCC suited Xiangan l.while 150 mg@L -1 CCC suited Xiangyan 10.

  11. 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...

  12. Design and Synthesis of Biaryl DNA-Encoded Libraries.

    Science.gov (United States)

    Ding, Yun; Franklin, G Joseph; DeLorey, Jennifer L; Centrella, Paolo A; Mataruse, Sibongile; Clark, Matthew A; Skinner, Steven R; Belyanskaya, Svetlana

    2016-10-10

    DNA-encoded library technology (ELT) is a powerful tool for the discovery of new small-molecule ligands to various protein targets. Here we report the design and synthesis of biaryl DNA-encoded libraries based on the scaffold of 5-formyl 3-iodobenzoic acid. Three reactions on DNA template, acylation, Suzuki-Miyaura coupling and reductive amination, were applied in the library synthesis. The three cycle library of 3.5 million diversity has delivered potent hits for phosphoinositide 3-kinase α (PI3Kα).

  13. RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress

    DEFF Research Database (Denmark)

    Bhowmick, Rahul; Minocherhomji, Sheroy; Hickson, Ian D

    2016-01-01

    Homologous recombination (HR) is necessary to counteract DNA replication stress. Common fragile site (CFS) loci are particularly sensitive to replication stress and undergo pathological rearrangements in tumors. At these loci, replication stress frequently activates DNA repair synthesis in mitosis....... This mitotic DNA synthesis, termed MiDAS, requires the MUS81-EME1 endonuclease and a non-catalytic subunit of the Pol-delta complex, POLD3. Here, we examine the contribution of HR factors in promoting MiDAS in human cells. We report that RAD51 and BRCA2 are dispensable for MiDAS but are required to counteract...

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

    Directory of Open Access Journals (Sweden)

    David R Halpin

    2004-07-01

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

  15. Programme DNA Lattices: Design, Synthesis and Applications

    Science.gov (United States)

    2006-02-01

    the Nick of Space: Generalized Nucleic Acid Complementarity and the Development of DNA Nanotechnology, Synlett 2000, 1536-1548, (2000) [See00c] N.C...Generalized Nucleic Acid Complementarity and the Development of DNA Nanotechnology, Synlett 2000, 1536-1548, (2000) 8. N.C. Seeman, DNA Nicks and Nodes

  16. Carbon-Carbon Composites (CCC) - A Historical Perspective.

    Science.gov (United States)

    1996-09-01

    for both the vehicle nosecap and leading edges. Full-scale protected CCC hot structures for the hypersonic glider have been fabricated and tested in...forebody of a military hypersonic glider . This article had a complex aerodynamic configuration. It was composed of silicon carbide-coated CCC substrate

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

    CERN Document Server

    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.

  18. 76 FR 57940 - CCC Export Credit Guarantee (GSM-102) Program

    Science.gov (United States)

    2011-09-19

    ... Guarantee (GSM-102) Program AGENCY: Foreign Agricultural Service and Commodity Credit Corporation, USDA... administer the Export Credit Guarantee (GSM-102) Program. Changes in this proposed rule incorporate program...: Background On July 27, 2011, CCC published a proposed rule titled ``CCC Export Credit Guarantee...

  19. Isolation of RNA and DNA from leukocytes and cDNA synthesis.

    NARCIS (Netherlands)

    Jansen, J.H.; Reijden, B.A. van der

    2006-01-01

    In this chapter, methods to isolate RNA and DNA from human leukocytes for the subsequent use in molecular diagnostic tests are described. In addition, protocols for cDNA synthesis are given, both for the use in conventional reverse transcription (RT)-polymerase chain reaction (PCR), and for the use

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

    Science.gov (United States)

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

    2016-01-01

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

  1. 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.

  2. 40 CFR Table 1 to Subpart Ccc of... - Applicability of General Provisions (40 CFR Part 63, Subpart A) to Subpart CCC

    Science.gov (United States)

    2010-07-01

    ... (40 CFR Part 63, Subpart A) to Subpart CCC 1 Table 1 to Subpart CCC of Part 63 Protection of... Hazardous Air Pollutants for Steel Pickling-HCl Process Facilities and Hydrochloric Acid Regeneration Plants Pt. 63, Subpt. CCC, Table 1 Table 1 to Subpart CCC of Part 63—Applicability of General Provisions...

  3. 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.

  4. Specific functions of the Rep and Rep' proteins of porcine circovirus during copy-release and rolling-circle DNA replication

    Science.gov (United States)

    The roles of two porcine circovirus replication initiator proteins, Rep and Rep', in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replica...

  5. Residues of chlormequat (CCC in fruits and other parts of tomato plants after treating the seedlings with 14C-CCC

    Directory of Open Access Journals (Sweden)

    Joanna Ostrzycka

    2013-12-01

    Full Text Available CCC remaining in tomato plants after treating the seedlings with a solution of 125 mg/l CCC to prevent their excessive growth has been studied in experiment conducted for two years. When seedling had been treated twice with CCC, the tomatoes of the first crop from these plants contained 0.09 mg CCC/kg fresh fruit. The amount of residual CCC decreased with each further crop. The last crop contained only 0.02 mg CCC/kg fresh fruit. The amount of CCC in the remains of leaves and stems at the end of the vegetation period was similar to that in the fruit of the first crop, however, the amount of CCC in the remains of the roots was several times larger than in the fruit. CCC which had been added directly to compost soil was quickly degraded.

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

    Science.gov (United States)

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

    2016-01-01

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

  7. Translesion synthesis past acrolein-derived DNA adducts by human mitochondrial DNA polymerase γ.

    Science.gov (United States)

    Kasiviswanathan, Rajesh; Minko, Irina G; Lloyd, R Stephen; Copeland, William C

    2013-05-17

    Acrolein, a mutagenic aldehyde, is produced endogenously by lipid peroxidation and exogenously by combustion of organic materials, including tobacco products. Acrolein reacts with DNA bases forming exocyclic DNA adducts, such as γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine (γ-HOPdG) and γ-hydroxy-1,N(6)-propano-2'-deoxyadenosine (γ-HOPdA). The bulky γ-HOPdG adduct blocks DNA synthesis by replicative polymerases but can be bypassed by translesion synthesis polymerases in the nucleus. Although acrolein-induced adducts are likely to be formed and persist in mitochondrial DNA, animal cell mitochondria lack specialized translesion DNA synthesis polymerases to tolerate these lesions. Thus, it is important to understand how pol γ, the sole mitochondrial DNA polymerase in human cells, acts on acrolein-adducted DNA. To address this question, we investigated the ability of pol γ to bypass the minor groove γ-HOPdG and major groove γ-HOPdA adducts using single nucleotide incorporation and primer extension analyses. The efficiency of pol γ-catalyzed bypass of γ-HOPdG was low, and surprisingly, pol γ preferred to incorporate purine nucleotides opposite the adduct. Pol γ also exhibited ∼2-fold lower rates of excision of the misincorporated purine nucleotides opposite γ-HOPdG compared with the corresponding nucleotides opposite dG. Extension of primers from the termini opposite γ-HOPdG was accomplished only following error-prone purine nucleotide incorporation. However, pol γ preferentially incorporated dT opposite the γ-HOPdA adduct and efficiently extended primers from the correctly paired terminus, indicating that γ-HOPdA is probably nonmutagenic. In summary, our data suggest that acrolein-induced exocyclic DNA lesions can be bypassed by mitochondrial DNA polymerase but, in the case of the minor groove γ-HOPdG adduct, at the cost of unprecedented high mutation rates.

  8. 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.

  9. 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

  10. 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.

  11. Species-specific kinetics and zonation of hepatic DNA synthesis induced by ligands of PPARalpha.

    Science.gov (United States)

    Al Kholaifi, Abdullah; Amer, Abeer; Jeffery, Brett; Gray, Tim J B; Roberts, Ruth A; Bell, David R

    2008-07-01

    Peroxisome proliferator-activated receptor alpha (PPARalpha) ligands evoke a profound mitogenic response in rodent liver, and the aim of this study was to characterize the kinetics of induction of DNA synthesis. The CAR ligand, 1,4-bis[2-(3,5-dichoropyridyloxy)]benzene, caused induction of hepatocyte DNA synthesis within 48 h in 129S4/SvJae mice, but the potent PPARalpha ligand, ciprofibrate, induced hepatocyte DNA synthesis only after 3 or 4 days dosing; higher or lower doses did not hasten the DNA synthesis response. This contrasted with the rapid induction (24 h) reported by Styles et al., 1988, Carcinogenesis 9, 1647-1655. C57BL/6 and DBA/2J mice showed significant induction of DNA synthesis after 4, but not 2, days ciprofibrate treatment. Alderley Park and 129S4/SvJae mice dosed with methylclofenapate induced hepatocyte DNA synthesis at 4, but not 2, days after dosing and proved that inconsistency with prior work was not due to a difference in mouse strain or PPARalpha ligand. Ciprofibrate-induced liver DNA synthesis and growth was absent in PPARalpha-null mice and are PPARalpha dependent. In the Fisher344 rat, hepatocyte DNA synthesis was induced at 24 h after dosing, with a second peak at 48 h. Lobular localization of hepatocyte DNA synthesis showed preferential periportal induction of DNA synthesis in rat but panlobular zonation of hepatocyte DNA synthesis in mouse. These results characterize a markedly later hepatic induction of panlobular DNA synthesis by PPARalpha ligands in mouse, compared to rapid induction of periportal DNA synthesis in rat.

  12. Replication stress activates DNA repair synthesis in mitosis.

    Science.gov (United States)

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

    2015-12-10

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

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

    Science.gov (United States)

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

    2010-05-04

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

  14. Swelling and Replicative DNA Synthesis of Detergent-treated Mouse Ascites Sarcoma Cells

    Directory of Open Access Journals (Sweden)

    Seki,Shuji

    1978-04-01

    Full Text Available Previous investigation showed that mouse ascites sarcoma cells permeabilized with appropriate concentrations of detergents (Triton X-100, Nonidet P-40 and Brij 58 had high replicative DNA synthesis in the presence of the four deoxyribonucleoside triphosphates, ATP, Mg2+ and proper ionic environment. The present study showed the optimum detergent concentration for DNA synthesis coincided closely with the minimum detergent concentration for inducing cell swelling. Phase contrast microscopy and electron microscopy of Triton-permeabilized cells showed the characteristic swollen cytoplasms and nucleus. Autoradiographic study showed that the DNA synthesis in permeable cells was confined to the nucleus. Cell viability and [3H] deoxythymidine uptake were impaired at much lower concentrations of Triton X-100 than the optimum concentration for in vitro DNA synthesis. In Triton-permeabilized cells, the minimum Triton concentration that produced cell swelling also seemed to produce high repliative DNA synthesis, which reflects the in vivo state of DNA synthesis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Unrau, P.

    1977-01-01

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

  16. 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.

  17. 76 FR 44836 - CCC Export Credit Guarantee (GSM-102) Program

    Science.gov (United States)

    2011-07-27

    ... exporter's assignee and the foreign financial institution or the amount calculated using the Treasury bill....100(h) has been modified to permit CCC to charge a fee for amendments over and above the normal... regulations (5 CFR Part 1315), only requires, unless otherwise specified, Federal agencies to pay their...

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

    Science.gov (United States)

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

    2016-09-01

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

  19. 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.

  20. 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.

  1. 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.

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

    Science.gov (United States)

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

    2016-07-15

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

  3. 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.

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

    Institute of Scientific and Technical Information of China (English)

    陈晓光; 韩锐

    1995-01-01

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

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

    Science.gov (United States)

    Confort, C; Charrasse, S; Clos, J

    1991-10-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1982-12-01

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

  7. A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A

    Directory of Open Access Journals (Sweden)

    Shunsuke Takahashi

    2014-03-01

    Full Text Available Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP. Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow and random coiled ssλDNA (−flow via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (−flow was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s. This suggested that the random coiled form of DNA (−flow reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow was approximately 75% higher than that of random coiled ssλDNA (−flow (91 vs. 52 bases/s. The DNA synthesis reaction rate of the Klenow fragment (3’-5’exo– was promoted by DNA stretching with buffer flow.

  8. Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?

    Science.gov (United States)

    Boregowda, Rajeev; Sohn, Ji A.; Ledesma, Felipe Cortes; Caldecott, Keith W.; Seeger, Christoph; Hu, Jianming

    2015-01-01

    Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5’ end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5’ DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5’ DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5’ end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo. PMID:26079492

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

    Science.gov (United States)

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

    2016-01-01

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

  10. One-step synthesis of silver nanoparticles, nanorods, and nanowires on the surface of DNA network.

    Science.gov (United States)

    Wei, Gang; Zhou, Hualan; Liu, Zhiguo; Song, Yonghai; Wang, Li; Sun, Lanlan; Li, Zhuang

    2005-05-12

    Here, we describe a one-step synthesis of silver nanoparticles, nanorods, and nanowires on DNA network surface in the absence of surfactant. Silver ions were first adsorbed onto the DNA network and then reduced in sodium borohydride solution. Silver nanoparticles, nanorods, and nanowires were formed by controlling the size of pores of the DNA network. The diameter of the silver nanoparticles and the aspect ratio of the silver nanorods and nanowires can be controlled by adjusting the DNA concentration and reduction time.

  11. DNA and RNA Synthesis in Animal Cells in Culture--Methods for Use in Schools

    Science.gov (United States)

    Godsell, P. M.; Balls, M.

    1973-01-01

    Describes the experimental procedures used for detecting DNA and RNA synthesis in xenopus cells by autoradiography. The method described is suitable for senior high school laboratory classes or biology projects, if supervised by a teacher qualified to handle radioisotopes. (JR)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-01-09

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

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

    Science.gov (United States)

    Kaltschmidt, E; Kahan, L; Nomura, M

    1974-02-01

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

  14. 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.

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

    Science.gov (United States)

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

    2010-12-01

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

  16. Inhibition of mouse peritoneal macrophage DNA synthesis by infection with the Arenavirus Pichinde. Interim report

    Energy Technology Data Exchange (ETDEWEB)

    Friedlander, A.M.; Jahrling, P.B.; Merrill, P.; Tobery, S.

    1983-01-19

    Macrophage DNA synthesis and proliferation occur during the development of cell-mediated immunity and in the early non-specific reaction to infection. Arenaviruses have a predilection for infection of cells of the reticuloendothelial system and in this study we have examined the effect of the arenavirus Pichinde on macrophage DNA synthesis. We have found that infection of mouse peritoneal macrophages with Pichinde caused a profound dose dependent inhibition of the DNA synthesis induced by macrophage growth factor/colony stimulating factor. At a multiplicity of inoculum of five there is a 75-95% inhibition of DNA synthesis. Viable virus is necessary for inhibition since Pichinde inactivated by heat or cobalt irradiation had no effect. Similarly, virus pre-treated with an antiserum to Pichinde was without inhibitory effect. Inhibition was demonstrated by measuring DNA synthesis spectrofluorometrically as well as by 3H-thymidine incorporation. The inhibition of DNA synthesis was not associated with any cytopathology. There was no evidence that the inhibition was due to soluble factors, such as prostaglandins or interferon, released by infected cells. These studies demonstrate, for the first time in vitro, a significant alteration in macrophage function caused by infection with an arenavirus. It is possible that inhibition of macrophage proliferation represents a mechanism by which some microorganisms interfere with host resistance.

  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. RecG Directs DNA Synthesis during Double-Strand Break Repair.

    Directory of Open Access Journals (Sweden)

    Benura Azeroglu

    2016-02-01

    Full Text Available Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300, arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability.

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

    Directory of Open Access Journals (Sweden)

    Pablo Darscht

    2011-05-01

    Full Text Available 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 en el área de ingeniería para dispositivos médicos para diferentes empresas de investigación biomédica a nivel internacional.AbstractCase study of the Centro de Cardioestimuladores del Uruguay - CCC Medical Devices prepared on behalf of Ingenio within the project financed by de Incubator Initiative of InfoDev-World Bank Group. This study refers to the steps followed by a highly innovative local company and to the changes in its business environment. The start up of a small pacemakers company that after going through different stages is presently increasing its market share in the area of engineering of medical devices for biomedic research companies worldwide.

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

    Science.gov (United States)

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

    1988-04-01

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

  1. Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

    Science.gov (United States)

    Christen, Matthias; Deutsch, Samuel; Christen, Beat

    2015-08-21

    Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-09-01

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

  3. 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.

  4. The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ

    OpenAIRE

    Hirota, Kouji; Yoshikiyo, Kazunori; Guilbaud, Guillaume; Tsurimoto, Toshiki; Murai, Junko; Tsuda, Masataka; Phillips, Lara G.; Narita, Takeo; Nishihara, Kana; Kobayashi, Kaori; Yamada, Kouich; Nakamura, Jun; Pommier, Yves; Lehmann, Alan; Sale, Julian E.

    2015-01-01

    The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with ...

  5. Gap-directed translesion DNA synthesis of an abasic site on circular DNA templates by a human replication complex.

    Directory of Open Access Journals (Sweden)

    Giuseppe Villani

    Full Text Available DNA polymerase ε (pol ε is believed to be the leading strand replicase in eukaryotes whereas pols λ and β are thought to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP site. We have previously reported that human pols λ, β and η can perform translesion synthesis (TLS of an AP site in the presence of pol ε. In the case of pol λ and β, this TLS requires the presence of a gap downstream from the product synthetized by the ε replicase. However, since these studies were conducted exclusively with a linear DNA template, we decided to test whether the structure of the template could influence the capacity of the pols ε, λ, β and η to perform TLS of an AP site. Therefore, we have investigated the replication of damaged "minicircle" DNA templates. In addition, replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC, and the accessory proteins replication protein A (RPA. Finally we have compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ and η on a circular template. Our results indicate that in vitro gap-directed TLS synthesis by pols λ and β in the presence of pol ε, RPA and PCNA is unaffected by the structure of the DNA template. Moreover, monoubiquitination of PCNA does not affect TLS by pol λ while it appears to slightly stimulate TLS by pol η.

  6. 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...

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

    Science.gov (United States)

    Levring, Trine B; Kongsbak, Martin; Rode, Anna K O; Woetmann, Anders; Ødum, Niels; Bonefeld, Charlotte Menné; Geisler, Carsten

    2015-09-08

    Adaptive immune responses require activation and expansion of antigen-specific T cells. Whereas early T cell activation is independent of exogenous cystine (Cys2), T cell proliferation is dependent of Cys2. However, the exact roles of Cys2 in T cell proliferation still need to be determined. The 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 Cys2. Vice-versa, the GSH synthesis inhibitor L-buthionine-sulfoximine (BSO) and inhibition of Cys2 uptake with glutamate inhibited GSH and DNA synthesis in parallel. We further found that thioredoxin (Trx) can partly substitute for GSH during DNA synthesis. Finally, we show that GSH or Trx is required for the activity of ribonucleotide reductase (RNR), the enzyme responsible for generation of the deoxyribonucleotide DNA building blocks. In conclusion, we show that activated human T cells require exogenous Cys2 to proliferate and that this is partly explained by the fact that Cys2 is required for production of GSH, which in turn is required for optimal RNR-mediated deoxyribonucleotide synthesis and DNA replication.

  8. DNA synthesis in mouse brown adipose tissue is under. beta. -adrenergic control

    Energy Technology Data Exchange (ETDEWEB)

    Rehnmark, S.; Nedergaard, J. (Univ. of Stockholm (Sweden))

    1989-02-01

    The rate of DNA synthesis in mouse brown adipose tissue was followed with injections of ({sup 3}H)thymidine. Cold exposure led to a large increase in the rate of ({sup 3}H)thymidine incorporation, reaching a maximum after 8 days, after which the activity abruptly ceased. A series of norepinephrine injections was in itself able to increase ({sup 3}H)thymidine incorporation. When norepinephrine was injected in combination with the {alpha}-adrenergic antagonist phentolamine or with the {beta}-adrenergic antagonist propranolol, the stimulation was fully blocked by propranolol. It is suggested that stimulation of DNA synthesis in brown adipose tissue is a {beta}-adrenergically mediated process and that the tissue is an interesting model for studies of physiological control of DNA synthesis.

  9. Role of mucosal prostaglandins and DNA synthesis in gastric cytoprotection by luminal epidermal growth factor.

    Science.gov (United States)

    Konturek, S J; Brzozowski, T; Piastucki, I; Dembinski, A; Radecki, T; Dembinska-Kiec, A; Zmuda, A; Gregory, H

    1981-01-01

    This study compares the effect of epidermal growth factor and prostaglandins (PGE2 or PGI2), applied topically to gastric mucosa, on gastric secretion and formation of ASA-induced gastric ulcerations in rats. Epidermal growth factor given topically in non-antisecretory doses prevented dose-dependently the formation of ASA-induced ulcers without affecting prostaglandin generation but with a significant rise in DNA synthesis in the oxyntic mucosa. The anti-ulcer effect of topical prostaglandins was also accompanied by an increase in DNA synthesis. This study indicates that topical epidermal growth factor, like PGE2 or PGI2, is cytoprotective and that this cytoprotection is not mediated by the inhibition of gastric secretion or prostaglandin formation but related to the increase in DNA synthesis in oxyntic mucosa. PMID:7030877

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1985-01-01

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

  11. 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.

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

    Science.gov (United States)

    Gao, Yang; Yang, Wei

    2016-06-10

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

  13. Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique.

    Science.gov (United States)

    Knobloch, Jürgen; Kunz, Werner; Grevelding, Christoph G

    2002-12-01

    The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4',6-diamidino-2-phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.

  14. 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.

  15. DNA-mediated silver nanoclusters: synthesis, properties and applications.

    Science.gov (United States)

    Latorre, Alfonso; Somoza, Álvaro

    2012-05-07

    Fluorescent DNA-AgNCs have emerged as an alternative to standard emitters because of their unique properties: high fluorescent quantum yield, photostability, a broad pallet of colors (blue to near-IR), and the fact that their properties are easily modulated by the DNA sequence and environment. Applications as gene, ion, or small-molecule sensors have been reported.

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

    Science.gov (United States)

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

    1994-01-01

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

  17. Effect of ethidium bromide on transmission of mitochondrial genomes and DNA synthesis in the petite negative yeast Schizosaccharomyces pomhe.

    Science.gov (United States)

    Wolf, K; Del Giudice, L

    1980-04-01

    Treatment of haploid strains of the petite negative yeast Schizosaccharomyces pomhe with ethidium bromide prior to mating with untreated cells reduces transmission of mitochondrial markers from the treated strains. This effect is fully reversible after 20 generations of growth in drug free medium before mating. In contrast to the petite positive yeast Saccharomyces cerevisiae, where nuclear DNA synthesis is not affected but mitochondrial DNA is degraded in the presence of 20 μg/ml ethidium bromide, the same concentration decreases both nuclear and mitochondrial DNA synthesis in Schizosaccharomyces pomhe. After removal of the drug, nuclear DNA synthesis increases faster than its mitochondrial counterpart in Schizosaccharomyces pomhe.

  18. A euryarchaeal histone modulates strand displacement synthesis by replicative DNA polymerases.

    Science.gov (United States)

    Sun, Fei; Huang, Li

    2016-07-01

    Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase (PolB), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of PolB and PolD from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPfA1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both PolB and PolD were efficient in strand displacement. HPfA1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPfA1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.

  19. 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.

  20. Critical Coalescence Concetration (CCC as a parameter for evaluation of selected quaternary ammonium compounds

    Directory of Open Access Journals (Sweden)

    Danuta Szyszka

    2013-10-01

    Full Text Available The objective of this paper was to determine the Critical Coalescence Concentration (CCC of surfactants such as N(dodecyloxycarboxymethyl N,N,N-(trimethylammonium bromide (DMGM- 12, N-[2-(dodecyoxycarboxyethyl] N,N,N-(trimethylammonium bromide (DMALM-12 and N-[3- (dodecanoyloxycarboxyprophyl] N,N,N-(trimethylammonium bromide (DMPM-11. The surfactants used represent quaternary ammonium compounds containing a hydrophobic moiety with an ester group (commonly known as “esterquats”. The CCC value was determined by analysis of the relationship between concentration of surfactant and average air bubble diameter. The values of the critical coalescence concentration (CCC were estimated using a graphical method.

  1. 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.

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

    NARCIS (Netherlands)

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

    1986-01-01

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

  3. 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...

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

    DEFF Research Database (Denmark)

    Nielsen, Jens Høiriis

    1985-01-01

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

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

    NARCIS (Netherlands)

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

    2011-01-01

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

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

    Science.gov (United States)

    Taylor, John-Stephen

    2015-12-01

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

  7. 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

    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 greater impact in the narrow and deep minor groove of a B-type dsDNA duplex than in the wide and shallow minor groove of an A-type DNA:RNA hybrid and (ii) the B-type dsDNA duplex allowing the pyrene to intercalate and bury its apolar surface....

  8. Synthesis and characterization of DNA nano-meso-microspheres as drug delivery carriers for intratumoral chemotherapy

    Science.gov (United States)

    Enriquez Schumacher, Iris Vanessa

    Conventional cancer chemotherapy results in systemic toxicity which severely limits effectiveness and often adversely affects patient quality of life. There is a need to find new drugs and delivery methods for less toxic therapy. Previous studies concerning DNA complexing with chemotherapy drugs suggest unique opportunities for DNA as a mesosphere drug carrier. The overall objective of this research was devoted to the synthesis and evaluation of novel DNA-drug nano-mesospheres designed for localized chemotherapy via intratumoral injection. My research presents DNA nano-meso-microspheres (DNA-MS) that were prepared using a modified steric stabilization method originally developed in this lab for the preparation of albumin MS. DNA-MS were prepared with glutaraldehyde covalent crosslinking (genipin crosslinking was attempted) through the DNA base pairs. In addition, novel crosslinking of DNA-MS was demonstrated using chromium, gadolinium, or iron cations through the DNA phosphate groups. Covalent and ionic crosslinked DNA-MS syntheses yielded smooth and spherical particle morphologies with multimodal size distributions. Optimized DNA-MS syntheses produced particles with narrow and normal size distributions in the 50nm to 5mum diameter size range. In aqueous dispersions approximately 200% swelling was observed with dispersion stability for more than 48 hours. Typical process conditions included a 1550rpm initial mixing speed and particle filtration through 20mum filters to facilitate preparation. DNA-MS were in situ loaded during synthesis for the first time with mitoxantrone, 5-fluorouracil, and methotrexate. DNA-MS drug incorporation was 12%(w/w) for mitoxantrone, 9%(w/w) for methotrexate, and 5%(w/w) for 5-fluorouracil. In vitro drug release into phosphate buffered saline was observed for over 35 days by minimum sink release testing. The effect of gadolinium crosslink concentration on mitoxantrone release was evaluated at molar equivalences in the range of 20% to

  9. Synthesis of the Tellurium-Derivatized Phosphoramidites and their Incorporation into DNA Oligonucleotides

    Science.gov (United States)

    Jiang, Sibo; Sheng, Jia

    2015-01-01

    Introduction In this unit, an efficient method for the synthesis of 2’-tellerium modified phosphoramidite and its incorporation into oligonucleotide are presented. We choose 5’-O-DMTr-2,2’-anhydro-uridine and -thymidine nucleosides (S.1, S.2) as starting materials due to their easy preparation. The 5’-O-DMTr-2,2’-anhydro-uridine and -thymidine can be converted to corresponding the 2’-tellerium-derivatized nucleosides by treating with the telluride nucleophiles. Subsequently, the 2’-Te-nucleosides can be transformed into 3’-phosphoramidites, which are the building blocks for DNA/RNA synthesis. The DNA synthesis, purification and applications of oligonucleotides containing 2’-Te-U or 2’-Te-T are described in this protocol. PMID:22147418

  10. 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.

  11. 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.

  12. Mitochondrial DNA synthesis studied autoradiographically in various cell types in vivo

    Directory of Open Access Journals (Sweden)

    H. Korr

    1998-02-01

    Full Text Available It is generally accepted that mitochondria are able to proliferate even in postmitotic cells due to their natural turnover and also to satisfy increased cell energy requirements. However, no detailed studies are available, particularly with respect to specific cell types. Since [3H]-thymidine is incorporated not only into nuclear (n DNA but also into the DNA of cytoplasmic mitochondria, an autoradiographic approach was developed at the light microscopy level in order to study basic questions of mitochondrial (mt proliferation in organs of rodents in situ via the cytoplasmic incorporation of [3H]-thymidine injected into the animals 1 h before sacrifice. Experiments carried out on mice after X-irradiation showed that cytoplasmic labeling was not due to a process such as unscheduled nuclear DNA synthesis (nUDS. Furthermore, half-lives of mitochondria between 8-23 days were deduced specifically in relation to cell types. The phase of mtDNA synthesis was about 75 min. Finally, mt proliferation was measured in brain cells of mice as a function of age. While all neurons showed a decreasing extent of mtDNA synthesis during old age, nUDS decreased only in distinct cell types of the cortex and hippocampus. We conclude that the leading theories explaining the phenomenon of aging are closely related, i.e., aging is due to a decreasing capacity of nDNA repair, which leads to unrepaired nDNA damage, or to an accumulation of mitochondria with damaged mtDNA, which leads to a deficit of cellular energy production

  13. 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.

  14. 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.

  15. 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.

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

    Science.gov (United States)

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

    2010-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Agbavwe Christy

    2011-12-01

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

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

    Science.gov (United States)

    Wickramaratne, Susith; Seiler, Christopher L.

    2016-01-01

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

  19. The nexus of vitamin homeostasis and DNA synthesis and modification in mammalian brain.

    Science.gov (United States)

    Spector, Reynold; Johanson, Conrad E

    2014-01-10

    The purpose of this review is to discuss the implications of the 2009 discovery of the sixth deoxyribonucleoside (dN) [5-hydroxymethyldeoxycytidine (hmdC)] in DNA which is the most abundant in neurons. The concurrent discovery of the three ten-eleven translocation enzymes (TET) which not only synthesize but also oxidize hmdC in DNA, prior to glycosylase removal and base excision repair, helps explain many heretofore unexplained phenomena in brain including: 1) the high concentration of ascorbic acid (AA) in neurons since AA is a cofactor for the TET enzymes, 2) the requirement for reduced folates and the dN synthetic enzymes in brain, 3) continued DNA synthesis in non-dividing neurons to repair the dynamic formation/removal of hmdC, and 4) the heretofore unexplained mechanism to remove 5-methyldeoxycytidine, the fifth nucleoside, from DNA. In these processes, we also describe the important role of choroid plexus and CSF in supporting vitamin homeostasis in brain: especially for AA and folates, for hmdC synthesis and removal, and methylated deoxycytidine (mdC) removal from DNA in brain. The nexus linking AA and folates to methylation, hydroxymethylation, and demethylation of DNA is pivotal to understanding not only brain development but also the subsequent function.

  20. Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution.

    Science.gov (United States)

    Christian, Thomas D; Romano, Louis J; Rueda, David

    2009-12-15

    The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3'-hydroxyl of the growing DNA chain. Here we report a single-molecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule Förster resonance energy transfer intensity drops in discrete steps to values consistent with single-nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer/template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.

  1. 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.

  2. 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.

  3. Design, synthesis and selection of DNA-encoded small-molecule libraries.

    Science.gov (United States)

    Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

    2009-09-01

    Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

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

    Science.gov (United States)

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

    2011-01-18

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

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

    Directory of Open Access Journals (Sweden)

    Madhavan H N

    2007-01-01

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

  6. DNA Origami Rotaxanes: Tailored Synthesis and Controlled Structure Switching.

    Science.gov (United States)

    Powell, John T; Akhuetie-Oni, Benjamin O; Zhang, Zhao; Lin, Chenxiang

    2016-09-12

    Mechanically interlocked supramolecular assemblies are appealing building blocks for creating functional nanodevices. Herein, we describe the multistep assembly of large DNA origami rotaxanes that are capable of programmable structural switching. We validated the topology and structural integrity of these rotaxanes by analyzing the intermediate and final products of various assembly routes by electrophoresis and electron microscopy. We further analyzed two structure-switching behaviors of our rotaxanes, which are both mediated by DNA hybridization. In the first mechanism, the translational motion of the macrocycle can be triggered or halted at either terminus. In the second mechanism, the macrocycle can be elongated after completion of the rotaxane assembly, giving rise to a unique structure that is otherwise difficult to access.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sheik Dawood Shahida Parveen

    2014-01-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

  10. 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.

  11. 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.

  12. 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).

  13. 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

    2016-05-06

    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.

  14. 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.

  15. Recent Advances in the Synthesis and Functions of Reconfigurable Interlocked DNA Nanostructures.

    Science.gov (United States)

    Lu, Chun-Hua; Cecconello, Alessandro; Willner, Itamar

    2016-04-27

    Interlocked circular DNA nanostructures, e.g., catenanes or rotaxanes, provide functional materials within the area of DNA nanotechnology. Specifically, the triggered reversible reconfiguration of the catenane or rotaxane structures provides a means to yield new DNA switches and to use them as dynamic scaffolds for controlling chemical functions and positioning functional cargoes. The synthesis of two-ring catenanes and their switchable reconfiguration by pH, metal ions, or fuel/anti-fuel stimuli are presented, and the functions of these systems, as pendulum or rotor devices or as switchable catalysts, are described. Also, the synthesis of three-, five-, and seven-ring catenanes is presented, and their switchable reconfiguration using fuel/anti-fuel strands is addressed. Implementation of the dynamically reconfigured catenane structures for the programmed organization of Au nanoparticle (NP) assemblies, which allows the plasmonic control of the fluorescence properties of Au NP/fluorophore loads associated with the scaffold, and for the operation of logic gates is discussed. Interlocked DNA rotaxanes and their different synthetic approaches are presented, and their switchable reconfiguration by means of fuel/anti-fuel strands or photonic stimuli is described. Specifically, the use of the rotaxane as a scaffold to organize Au NP assemblies, and the control of the fluorescence properties with Au NP/fluorophore hybrids loaded on the rotaxane scaffold, are introduced. The future prospectives and challenges in the field of interlocked DNA nanostructures and the possible applications are discussed.

  16. Effects of beta interferon on human fibroblasts at different population doubling levels. Proliferation, cell volume, thymidine uptake, and DNA synthesis

    OpenAIRE

    1984-01-01

    Cellular aging had no effect on the ability of beta interferon to increase cell volume and population doubling time in 76-109 cells, a line of human skin fibroblasts. However, DNA synthesis in cells at high population doubling levels (PDL 55-70) was inhibited after 72 h of beta interferon treatment (1,000 U/ml) while no inhibition of DNA synthesis was observed in cells at middle population doubling levels (PDL 30-40).

  17. Developing Inhibitors of Translesion DNA Synthesis as Therapeutic Agents Against Lung Cancer

    Science.gov (United States)

    2014-10-01

    nuclear magnetic resonance ( NMR spectroscopy (Figure 3). Figure 2. Mass spectroscopy to verify the molecular weight of 3-ethynyl-5-nitroindolyl-2...8217 -deoxynucleoside. 0 20 25 mia Figure 3. NMR spectrum of 3-ethynyl-5-nitroindolyl- 2’ -deoxynucleoside. ~~. A-::.~, C-*!&t:.,. I As described...provided in Figure 2D . The kinetic parameters for pol  during normal and translesion DNA synthesis are summarized in Table 1. These data indicate

  18. Iron reverses impermeable chelator inhibition of DNA synthesis in CCl 39 cells.

    OpenAIRE

    Alcain, F J; Löw, H; Crane, F. L.

    1994-01-01

    Treatment of Chinese hamster lung fibroblasts (CCl 39 cells) with the impermeable iron(II) chelator bathophenanthroline disulfonate (BPS) inhibits DNA synthesis when cell growth is initiated with growth factors including epidermal growth factor plus insulin, thrombin, or ceruloplasmin, but not with 10% fetal calf serum. The BPS treatment inhibits transplasma membrane electron transport. The treatment leads to release of iron from the cells as determined by BPS iron(II) complex formation over ...

  19. Quercetin-Iron Complex: Synthesis, Characterization, Antioxidant, DNA Binding, DNA Cleavage, and Antibacterial Activity Studies.

    Science.gov (United States)

    Raza, Aun; Xu, Xiuquan; Xia, Li; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-11-01

    Quercetin-iron (II) complex was synthesized and characterized by elemental analysis, ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, mass spectrometry, proton nuclear magnetic resonance spectroscopy, thermogravimetry and differential scanning calorimetry, scanning electron micrography and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal:ligand) of the complex. Antioxidant study of the quercetin and its metal complex against 2, 2-di-phenyl-1-picryl hydrazyl radical showed that the complex has much more radical scavenging activity than free quercetin. The interaction of quercetin-iron (II) complex with DNA was determined using ultraviolet visible spectra, fluorescence spectra and agarose gel electrophoresis. The results showed that quercetin-iron (II) complex can intercalate moderately with DNA, quench a strong intercalator ethidium bromide and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form to nicked circular form and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was an oxidative cleavage pathway. These results revealed the potential nuclease activity of complex to cleave DNA. In addition, antibacterial activity of complex on E.coli and S. aureus was also investigated. The results showed that complex has higher antibacterial activity than ligand.

  20. Rutin-Nickel Complex: Synthesis, Characterization, Antioxidant, DNA Binding, and DNA Cleavage Activities.

    Science.gov (United States)

    Raza, Aun; Bano, Shumaila; Xu, Xiuquan; Zhang, Rong Xian; Khalid, Haider; Iqbal, Furqan Muhammad; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-12-17

    The rutin-nickel (II) complex (RN) was synthesized and characterized by elemental analysis, UV-visible spectroscopy, IR, mass spectrometry, (1)H NMR, TG-DSC, SEM, and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal/ligand) of the complex. An antioxidant study of rutin and its metal complex against DPPH radical showed that the complex has more radical scavenging activity than free rutin. The interaction of complex RN with DNA was determined using fluorescence spectra and agarose gel electrophoresis. The results showed that RN can intercalate moderately with DNA, quench a strong intercalator ethidium bromide (EB), and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form (SC) to nicked circular form (NC), and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was a hydrolytic cleavage pathway. These results revealed the potential nuclease activity of the complex to cleave DNA.

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

    Science.gov (United States)

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

    2009-11-06

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

  2. Integration of CCC-HVDC and VSC-HVDC Systems to Supply an Island Network

    Directory of Open Access Journals (Sweden)

    G.B. Gharehpetian

    2013-05-01

    Full Text Available Acombination of Capacitor Commutated Converter (CCC HVDC and voltage source converter (VSC HVDC is proposed to supply an island system without any local generation. The key point of this integration is the flat characteristic of dc voltage of CCC-HVDC, which provides the condition for VSC to connect to CCC dc link via a current regulator. The advantages of proposed combined in feeding system are requiring only one dc line and having better dynamic responses. The structure of the proposed in feeding system as well as its control system is shown in this study. The simulation results are presented to confirm the effectiveness of the proposed system. Two other schemes for in feeding the passive island systems are studied to demonstrate the advantages of the proposed system.

  3. Green synthesis of gold nanoparticles for staining human cervical cancer cells and DNA binding assay.

    Science.gov (United States)

    De, Swati; Kundu, Rikta; Ghorai, Atanu; Mandal, Ranju Prasad; Ghosh, Utpal

    2014-11-01

    Gold nanoparticles have been functionalized by non-ionic surfactants (polysorbates) used in pharmaceutical formulations. This results in the formation of more well-dispersed gold nanoparticles (GNPs) than the GNPs formed in neat water. The synthesized GNPs show good temporal stability. The synthesis conditions are mild and environmentally benign. The GNPs can bind to ct-DNA and displace bound dye molecules. The DNA-binding assay is significant as it preliminarily indicated that DNA-GNP conjugates can be formed. Such conjugates are extremely promising for applications in nanobiotechnology. The GNPs can also stain the human cervical cancer (HeLa) cells over a wide concentration range while remaining non-cytotoxic, thus providing a non invasive cell staining method. This result is very promising as we observe staining of HeLa cells at very low GNP concentrations (1 μM) while the cell viability is retained even at 10-fold higher GNP concentrations.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-01

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

  5. The histone variant H2A.Bbd is enriched at sites of DNA synthesis.

    Science.gov (United States)

    Sansoni, Viola; Casas-Delucchi, Corella S; Rajan, Malini; Schmidt, Andreas; Bönisch, Clemens; Thomae, Andreas W; Staege, Martin S; Hake, Sandra B; Cardoso, M Cristina; Imhof, Axel

    2014-06-01

    Histone variants play an important role in shaping the mammalian epigenome and their aberrant expression is frequently observed in several types of cancer. However, the mechanisms that mediate their function and the composition of the variant-containing chromatin are still largely unknown. A proteomic interrogation of chromatin containing the different H2A variants macroH2A.1.2, H2A.Bbd and H2A revealed a strikingly different protein composition. Gene ontology analysis reveals a strong enrichment of splicing factors as well as components of the mammalian replisome in H2A.Bbd-containing chromatin. We find H2A.Bbd localizing transiently to sites of DNA synthesis during S-phase and during DNA repair. Cells that express H2A.Bbd have a shortened S-phase and are more susceptible to DNA damage, two phenotypes that are also observed in human Hodgkin's lymphoma cells that aberrantly express this variant. Based on our experiments we conclude that H2A.Bbd is targeted to newly synthesized DNA during replication and DNA repair. The transient incorporation of H2A.Bbd may be due to the intrinsic instability of nucleosomes carrying this variant or a faster chromatin loading. This potentially leads to a disturbance of the existing chromatin structure, which may have effects on cell cycle regulation and DNA damage sensitivity.

  6. Mechanisms of assembly of the enzyme-ssDNA complexes required for recombination-dependent DNA synthesis and repair in bacteriophage T4

    Energy Technology Data Exchange (ETDEWEB)

    Morrical, S.; Hempstead, K.; Morrical, M. [Univ. of Vermont College of Medicine, Burlington, VT (United States)

    1994-12-31

    During late stages of bacteriophage T4 infection in E. coli, the initiation of phage DNA replication is dependent on the homologous recombination activity of the T4 uvsX protein. In vitro, uvsX protein initiates DNA synthesis on a duplex template by inserting the 3{prime} end of a homologous ssDNA molecule into the duplex. The resulting D-loop structure serves as a primer-template junction for the assembly of the T4 replication fork. Two key steps in this initiation process are (A) the assembly of uvsX-ssDNA complexes necessary for recombination activity and for the priming of lead-strand DNA synthesis, and (B) the assembly of the T4 primosome (gp41 helicase/gp61 primase complex) onto the single-stranded template for lagging-strand synthesis. Our laboratory is focusing on the mechanisms of these two different but related enzyme-ssDNA assembly processes. In this extended abstract, we describe recent efforts in our laboratory to elucidate the mechanism by which the gp41 helicase enzyme is assembled onto gp32-covered ssDNA, a process requiring the activity of a special helicase assembly factor, the T4 gp59 protein.

  7. 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.

  8. Final Corrective Action Study for the Former CCC/USDA Facility in Hanover, Kansas

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, Lorraine M. [Argonne National Lab. (ANL), Argonne, IL (United States)

    2013-11-01

    Low concentrations of carbon tetrachloride in groundwater and vapor intrusion into a limited number of residences (attributable to the contaminant concentrations in groundwater) have been identified in Hanover, Kansas, at and near a grain storage facility formerly leased and operated by the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA). At the request of the Kansas Department of Health and Environment (KDHE 2009h), the CCC/USDA has prepared this Corrective Action Study (CAS) for the facility. The CAS examines corrective actions to address the contamination in groundwater and soil vapor.

  9. 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.

  10. Biochemical analysis of six genetic variants of error-prone human DNA polymerase ι involved in translesion DNA synthesis.

    Science.gov (United States)

    Kim, Jinsook; Song, Insil; Jo, Ara; Shin, Joo-Ho; Cho, Hana; Eoff, Robert L; Guengerich, F Peter; Choi, Jeong-Yun

    2014-10-20

    DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N(2)-ethyl(Et)G, O(6)-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1-445) proteins and DNA templates containing a G, N(2)-EtG, O(6)-MeG, 8-oxoG, or abasic site. The Δ1-25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg(2+) (but not with Mn(2+)), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg(2+)). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg(2+) or Mn(2+), except for that opposite N(2)-EtG with Mn(2+) (showing a 9-fold increase for dCTP incorporation). The Δ1-25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg(2+)), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1-25 variant, was ∼7-fold stronger with 0.15 mM Mn(2+) than with Mg(2+). The results indicate that the R96G variation severely impairs most of the Mg(2+)- and Mn(2+)-dependent TLS abilities of pol ι, whereas the Δ1-25 variation selectively and substantially enhances the Mg(2+)-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences

  11. Phase II Investigation at the Former CCC/USDA Grain Storage Facility in Savannah, Missouri

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, Lorraine M. [Argonne National Lab. (ANL), Argonne, IL (United States). Environmental Science Division. Applied Geosciences and Environmental Management Section

    2012-05-01

    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. 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 statewide 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 (MDNR) confirmed the presence of carbon tetrachloride in the Morgan well, as well as in a second well on property currently owned by the Missouri Department of Transportation (MoDOT), directly east of the former CCC/USDA facility. The identified concentrations in these two wells were above the EPA maximum contaminant level (MCL) and the Missouri risk-based corrective action default target level (DTL) values of 5.0 μg/L for carbon tetrachloride in water used for domestic purposes (EPA 1999; MDNR 2000a,b, 2006). 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 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 an Intergovernmental Agreement established in 2007 between the Farm Service Agency of the USDA and the MDNR, to address carbon tetrachloride

  12. Conceptual "Heat-Driven" approach to the synthesis of DNA oligonucleotides on microarrays.

    Science.gov (United States)

    Grajkowski, A; Cieślak, J; Chmielewski, M K; Marchán, V; Phillips, L R; Wilk, A; Beaucage, S L

    2003-12-01

    The discovery of deoxyribonucleoside cyclic N-acylphosphoramidites, a novel class of phosphoramidite monomers for solid-phase oligonucleotide synthesis, has led to the development of a number of phosphate protecting groups that can be cleaved from DNA oligonucleotides under thermolytic neutral conditions. These include the 2-(N-formyl-N-methyl)aminoethyl, 4-oxopentyl, 3-(N-tert-butyl)carboxamido-1-propyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, and 4-methythiobutyl groups. When used for 5'-hydroxyl protection of nucleosides, the analogous 1-phenyl-2-[N-methyl-N-(2-pyridyl)]aminoethyloxycarbonyl group exhibited excellent thermolytic properties, which may permit an iterative "heat-driven" synthesis of DNA oligonucleotides on microarrays. In this regard, progress has been made toward the use of deoxyribonucleoside cyclic N-acylphosphoramidites in solid-phase oligonucleotide syntheses without nucleobase protection. Given that deoxyribonucleoside cyclic N-acylphosphoramidites produce oligonucleotides with heat-sensitive phosphate protecting groups, blocking the 5'-hydroxyl of these monomers with, for example, the thermolabile 1-phenyl-2-[N-methyl-N-(2-pyridyl)]aminoethyloxycarbonyl group may provide a convenient thermo-controlled method for the synthesis of oligonucleotides on microarrays.

  13. DNA Binding and Recognition of a CC Mismatch in a DNA Duplex by Water-Soluble Peptidocalix[4]arenes: Synthesis and Applications.

    Science.gov (United States)

    Alavijeh, Nahid S; Zadmard, Reza; Balalaie, Saeed; Alavijeh, Mohammad S; Soltani, Nima

    2016-10-07

    Water-soluble peptidocalix[4]arenes were synthesized by the introduction of arginine-rich narrow groove-binding residues at lower rims through solid-phase synthesis. The study of binding of these water-soluble bidentate ligands to well-matched and mismatched DNA duplexes by fluorescent titrations, ethidium bromide (EB) displacement assays, DNA-melting experiments, and circular dichroism (CD) analysis revealed a sequence-dependent groove-binding mechanism.

  14. Timing of initiation of macronuclear DNA synthesis is set during the preceding cell cycle in Paramecium tetraurelia: analysis of the effects of abrupt changes in nutrient level

    Energy Technology Data Exchange (ETDEWEB)

    Ching, A.S.L.; Berger, J.D.

    1986-11-01

    In many eukaryotic organisms, initiation of DNA synthesis is associated with a major control point within the cell cycle and reflects the commitment of the cell to the DNA replication-division portion of the cell cycle. In paramecium, the timing of DNA synthesis initiation is established prior to fission during the preceding cell cycle. DNA synthesis normally starts at 0.25 in the cell cycle. When dividing cells are subjected to abrupt nutrient shift-up by transfer from a chemostat culture to medium with excess food, or shift-down from a well-fed culture to exhausted medium, DNA synthesis initiation in the post-shift cell cycle occurs at 0.25 of the parental cell cycle and not at either 0.25 in the post-shift cell cycle or at 0.25 in the equilibrium cell cycle produced under the post-shift conditions. The long delay prior to initiation of DNA synthesis following nutritional shift-up is not a consequence of continued slow growth because the rate of protein synthesis increases rapidly to the normal level after shift-up. Analysis of the relation between increase in cell mass and initiation of DNA synthesis following nutritional shifts indicates that increase in cell mass, per se, is neither a necessary nor a sufficient condition for initiation of DNA synthesis, in spite of the strong association between accumulation of cell mass and initiation of DNA synthesis in cells growing under steady-state conditions.

  15. Small molecule inhibitors of PCNA/PIP-box interaction suppress translesion DNA synthesis.

    Science.gov (United States)

    Actis, Marcelo; Inoue, Akira; Evison, Benjamin; Perry, Scott; Punchihewa, Chandanamali; Fujii, Naoaki

    2013-04-01

    Proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and DNA damage response. Numerous proteins interact with PCNA through their short sequence called the PIP-box to be promoted to their respective functions. PCNA supports translesion DNA synthesis (TLS) by interacting with TLS polymerases through PIP-box interaction. Previously, we found a novel small molecule inhibitor of the PCNA/PIP-box interaction, T2AA, which inhibits DNA replication in cells. In this study, we created T2AA analogues and characterized them extensively for TLS inhibition. Compounds that inhibited biochemical PCNA/PIP-box interaction at an IC50 <5 μM inhibited cellular DNA replication at 10 μM as measured by BrdU incorporation. In cells lacking nucleotide-excision repair activity, PCNA inhibitors inhibited reactivation of a reporter plasmid that was globally damaged by cisplatin, suggesting that the inhibitors blocked the TLS that allows replication of the plasmid. PCNA inhibitors increased γH2AX induction and cell viability reduction mediated by cisplatin. Taken together, these findings suggest that inhibitors of PCNA/PIP-box interaction could chemosensitize cells to cisplatin by inhibiting TLS.

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

    Institute of Scientific and Technical Information of China (English)

    ShenJiayao; XiaoPengfeng; HouPeng; JiMeiju; SunXiao; HeNongyue

    2003-01-01

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

  17. 78 FR 79253 - CCC Export Credit Guarantee (GSM-102) Program and Facility Guarantee Program (FGP)

    Science.gov (United States)

    2013-12-27

    ... Credit Guarantee GSM-102 Program and Facility Guarantee Program FGP ; Proposed Rule #0;#0;Federal... Commodity Credit Corporation 7 CFR Part 1493 RIN 0551-AA74 CCC Export Credit Guarantee (GSM-102) Program and... administer the Export Credit Guarantee (GSM-102) Program. Changes in this proposed rule incorporate...

  18. 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.

  19. Synthesis of biodegradable polymer-mesoporous silica composite microspheres for DNA prime-protein boost vaccination.

    Science.gov (United States)

    Ho, Jenny; Huang, Yi; Danquah, Michael K; Wang, Huanting; Forde, Gareth M

    2010-03-18

    DNA vaccines or proteins are capable of inducing specific immunity; however, the translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein we demonstrate a composite microsphere formulation, composed of mesoporous silica spheres (MPS) and poly(D,L-lactide-co-glycolide) (PLGA), enables the controlled delivery of a prime-boost vaccine via the encapsulation of plasmid DNA (pDNA) and protein in different compartments. Method with modified dual-concentric-feeding needles attached to a 40 kHz ultrasonic atomizer was studied. These needles focus the flow of two different solutions, which passed through the ultrasonic atomizer. The process synthesis parameters, which are important to the scale-up of composite microspheres, were also studied. These parameters include polymer concentration, feed flowrate, and volumetric ratio of polymer and pDNA-PEI/MPS-BSA. This fabrication technique produced composite microspheres with mean D[4,3] ranging from 6 to 34 microm, depending upon the microsphere preparation. The resultant physical morphology of composite microspheres was largely influenced by the volumetric ratio of pDNA-PEI/MPS-BSA to polymer, and this was due to the precipitation of MPS at the surface of the microspheres. The encapsulation efficiencies were predominantly in the range of 93-98% for pDNA and 46-68% for MPS. In the in vitro studies, the pDNA and protein showed different release kinetics in a 40 day time frame. The dual-concentric-feeding in ultrasonic atomization was shown to have excellent reproducibility. It was concluded that this fabrication technique is an effective method to prepare formulations containing a heterologous prime-boost vaccine in a single delivery system.

  20. Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA

    Institute of Scientific and Technical Information of China (English)

    XU Chun-hai; LI Zhao-shen; DAI Jun-ying; ZHU Hai-yang; YU Jian-wu; L(U) Shu-Ian

    2011-01-01

    peripheral blood mononuclear cells; marrow mononuclear cells Background Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).Methods Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard.Results The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0x102 copies/ml to 3.9x108 copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative.Conclusion The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.

  1. Radio Search for H2CCC toward HD 183143 as a Candidate for a Diffuse Interstellar Band Carrier

    CERN Document Server

    Araki, Mitsunori; Yamabe, Hiromichi; Tsukiyama, Koichi; Kuze, Nobuhiko

    2012-01-01

    To clarify the authenticity of a recently proposed identification of H2CCC (linear-C3H2) as a diffuse interstellar band carrier, we searched for the rotational transition of H2CCC at a frequency of 103 GHz toward HD 183143 using a 45-m telescope at the Nobeyama Radio Observatory. Although rms noise levels of 32 mK in the antenna temperature were achieved, detection of H2CCC was unsuccessful, producing a 3 sigma upper limit corresponding to a column density of 2.0 \\times 1013 cm-2. The upper limit indicates that the contribution of H2CCC to the diffuse interstellar band at 5450 {\\AA} is less than 1/25; thus, it is unlikely that the laboratory bands of the B1B1-X1A1 transition of H2CCC and the diffuse interstellar bands at 5450 {\\AA} (and also 4881 {\\AA}) toward HD 183143 are related.

  2. Radio Search for H2CCC toward HD 183143 as a Candidate for a Diffuse Interstellar Band Carrier

    Science.gov (United States)

    Araki, Mitsunori; Takano, Shuro; Yamabe, Hiromichi; Tsukiyama, Koichi; Kuze, Nobuhiko

    2012-07-01

    To clarify the authenticity of a recently proposed identification of H2CCC (linear-C3H2) as a diffuse interstellar band (DIB) carrier, we searched for the rotational transition of H2CCC 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 H2CCC was unsuccessful, producing a 3σ upper limit corresponding to a column density of 2.0 × 1013 cm-2. The upper limit indicates that the contribution of H2CCC to the DIB at 5450 Å is less than 1/25; thus, it is unlikely that the laboratory bands of the B 1 B 1-X 1 A 1 transition of H2CCC and the DIBs at 5450 Å (and also 4881 Å) toward HD 183143 are related.

  3. 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.

  4. A chemical method for fast and sensitive detection of DNA synthesis in vivo

    OpenAIRE

    Salic, Adrian; Mitchison, Timothy J.

    2008-01-01

    We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction (“click” chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount ...

  5. Induction of DNA repair synthesis by ultraviolet radiation and methylmethanesulphonate in cultured mouse lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bianchi, V.; Zantedeschi, A.; Levis, A.G. (Padua Univ. (Italy). Ist. di Zoologia e Anatomia Comparata); Ronchese, F. (Inst. of Pathological Anatomy, Padua (Italy))

    1983-02-01

    The induction of DNA repair synthesis by UV-radiation and methylmethanesulphonate (MMS) was studied in mouse lymphocytes and leukemic cells by means of autoradiography and scintillation counting, after labelling in vitro with tritiated thymidine ((/sup 3/H)dThd). Repair stimulation was detected by both procedures in LSTRA AND YC8 leukemic cell lines as well as in primary fibroblasts of BALB/c and BALB/Mo mice. No stimulation was observed in primary cultures of lymphocytes from the spleen, thymus and lymph-nodes of the same mice. In primary lymphocytes neither stimulation with concanavalin A (Con A) nor pre-incubation with 5-bromodeoxyuridine (BUdR) were effective in making evident DNA repair. The data put into question the reliability of the repair test for the prediction of carcinogenic potential of chemicals.

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

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

  7. A chemical method for fast and sensitive detection of DNA synthesis in vivo.

    Science.gov (United States)

    Salic, Adrian; Mitchison, Timothy J

    2008-02-19

    We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction ("click" chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.

  8. 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...

  9. Synthesis, characterization, and photoactivated DNA cleavage by copper (II)/cobalt (II) mediated macrocyclic complexes.

    Science.gov (United States)

    Naik, H R Prakash; Naik, H S Bhojya; Aravinda, T; Lamani, D S

    2010-01-01

    We report the synthesis of new photonuclease consisting of two Co(II)/Cu(II) complexes of macrocyclic fused quinoline. Metal complexes are [MLX(2)], type where M = Co(II) (5), Cu(II) (6), and X = Cl, and are well characterized by elemental analysis, Fourier transform infrared spectroscopy, (1)H-NMR and electronic spectra. We have shown that photocleavage of plasmid DNA is markedly enhanced when this ligand is irradiated in the presence of Cu(II), and more so than that of cobalt. The chemistry of ternary and binary Co(II) complexes showing efficient light induced (360 nm) DNA cleavage activity is summarized. The role of the metal in photoinduced DNA cleavage reactions is explored by designing complex molecules having macrocyclic structure. The mechanistic pathways are found to be concentration dependent on Co(II)/Cu(II) complexes and the photoexcitation energy photoredox chemistry. Highly effective DNA cleavage ability of 6 is attributed to the effective cooperation of the metal moiety.

  10. Requirement of Rad5 for DNA Polymerase ζ-Dependent Translesion Synthesis in Saccharomyces cerevisiae

    Science.gov (United States)

    Pagès, Vincent; Bresson, Anne; Acharya, Narottam; Prakash, Satya; Fuchs, Robert P.; Prakash, Louise

    2008-01-01

    In yeast, Rad6–Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases η or ζ or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities—a ubiquitin ligase that promotes Mms2–Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polζ. Using duplex plasmids carrying different site-specific DNA lesions—an abasic site, a cis–syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct—we show that Rad5 is needed for Polζ-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS. PMID:18757916

  11. Design, synthesis and DNA-binding study of some novel morpholine linked thiazolidinone derivatives

    Science.gov (United States)

    War, Javeed Ahmad; Srivastava, Santosh Kumar; Srivastava, Savitri Devi

    2017-02-01

    The emergence of multiple drug resistance amongst bacterial strains resulted in many clinical drugs to be ineffective. Being vulnerable to bacterial infections any lack in the development of new antimicrobial drugs could pose a serious threat to public health. Here we report design and synthesis of a novel class of morpholine linked thiazolidinone hybrid molecules. The compounds were characterized by FT-IR, NMR and HRMS techniques. Susceptibility tests showed that most of the synthesized molecules were highly active against multiple bacterial strains. Compound 3f displayed MIC values which were better than the standard drug for most of the tested strains. DNA being a well defined target for many antimicrobial drugs was probed as possible target for these synthetic molecules. DNA-binding study of 3f with sm-DNA was probed through UV-vis absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. The studies revealed that compound 3f has strong affinity towards DNA and binds at the minor groove. The docking studies revealed that the compound 3f shows preferential binding towards A/T residues.

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

    Science.gov (United States)

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

    2016-03-29

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

  13. PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Ayal Hendel

    2011-09-01

    Full Text Available Translesion DNA synthesis (TLS is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+ cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.

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

    Institute of Scientific and Technical Information of China (English)

    Xie Zuofu; Lin Xiandong; Zhou Dongmei; Lin Sheng

    1998-01-01

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

  15. Error-prone translesion synthesis past DNA-peptide cross-links conjugated to the major groove of DNA via C5 of thymidine.

    Science.gov (United States)

    Wickramaratne, Susith; Boldry, Emily J; Buehler, Charles; Wang, Yen-Chih; Distefano, Mark D; Tretyakova, Natalia Y

    2015-01-01

    DNA-protein cross-links (DPCs) are exceptionally bulky, structurally diverse DNA adducts formed in cells upon exposure to endogenous and exogenous bis-electrophiles, reactive oxygen species, and ionizing radiation. If not repaired, DPCs can induce toxicity and mutations. It has been proposed that the protein component of a DPC is proteolytically degraded, giving rise to smaller DNA-peptide conjugates, which can be subject to nucleotide excision repair and replication bypass. In this study, polymerase bypass of model DNA-peptide conjugates structurally analogous to the lesions induced by reactive oxygen species and DNA methyltransferase inhibitors was examined. DNA oligomers containing site-specific DNA-peptide conjugates were generated by copper-catalyzed [3 + 2] Huisgen cyclo-addition between an alkyne-functionalized C5-thymidine in DNA and an azide-containing 10-mer peptide. The resulting DNA-peptide conjugates were subjected to steady-state kinetic experiments in the presence of recombinant human lesion bypass polymerases κ and η, followed by PAGE-based assays to determine the catalytic efficiency and the misinsertion frequency opposite the lesion. We found that human polymerase κ and η can incorporate A, G, C, or T opposite the C5-dT-conjugated DNA-peptide conjugates, whereas human polymerase η preferentially inserts G opposite the lesion. Furthermore, HPLC-ESI(-)-MS/MS sequencing of the extension products has revealed that post-lesion synthesis was highly error-prone, resulting in mutations opposite the adducted site or at the +1 position from the adduct and multiple deletions. Collectively, our results indicate that replication bypass of peptides conjugated to the C5 position of thymine by human translesion synthesis polymerases leads to large numbers of base substitution and frameshift mutations.

  16. DNA synthesis index: higher for human gallbladders with cholesterol gallstones than with pigment gallstones

    Energy Technology Data Exchange (ETDEWEB)

    Lamote, J.; Putz, P.; Francois, M.; Willems, G.

    1983-09-01

    (/sup 3/H)dThd uptake by the gallbladder epithelium was estimated in 33 patients with cholesterol stones, in 13 patients with pigment stones, and in 12 gallbladders without stones. Proliferative parameters were estimated by autoradiography after in vitro incubation with (/sup 3/H)-dThd. Stones were identified by quantitative infrared spectroscopy. The degree of inflammation of the gallbladder wall was estimated by a histologic scoring method. In the gallbladders containing cholesterol stones the DNA synthesis index (1.39 +/- 0.28%) was higher (P less than .01) than in the gallbladders without stones (0.19 +/- 0.04%). No significant increase in proliferative parameters was found in the gallbladders with pigment stones (0.24 +/- 0.06%). No correlation was found between total stone number, weight or volume, and the DNA synthesis index. No evidence was observed that inflammation could influence the epithelial cell proliferation. Something in the bile of patients with cholesterol stones rather than the physical presence of stones may be the cause of the variations observed.

  17. Adaptation and validation of DNA synthesis detection by fluorescent dye derivatization for high-throughput screening.

    Science.gov (United States)

    Ranall, Max V; Gabrielli, Brian G; Gonda, Thomas J

    2010-05-01

    Cellular proliferation is fundamental to organism development, tissue renewal, and diverse disease states such as cancer. In vitro measurement of proliferation by high-throughput screening allows rapid characterization of the effects of small-molecule or genetic treatments on primary and established cell lines. Current assays that directly measure the cell cycle are not amenable to high-throughput processing and analysis. Here we report the adaptation of the chemical method for detecting DNA synthesis by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into both high-throughput liquid handling and high-content imaging analysis. We demonstrate that chemical detection of EdU incorporation is effective for high-resolution analysis and quantitation of DNA synthesis by high-content imaging. To validate this assay platform we used treatments of MCF10A cells with media supplements and pharmacological inhibitors that are known to affect cell proliferation. Treatments with specific kinase inhibitors indicate that EGF and serum stimulation employs both the mitogen extracellular kinase (MEK)/extracellular-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K)/AKT signaling networks. As described here, this method is fast, reliable, and inexpensive and yields robust data that can be easily interpreted.

  18. 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....

  19. A bichaperone (Hsp70-Hsp78) system restores mitochondrial DNA synthesis following thermal inactivation of Mip1p polymerase.

    Science.gov (United States)

    Germaniuk, Aleksandra; Liberek, Krzysztof; Marszalek, Jaroslaw

    2002-08-02

    Mitochondrial DNA synthesis is a thermosensitive process in the yeast Saccharomyces cerevisiae. We found that restoration of mtDNA synthesis following heat treatment of cells is dependent on reactivation of the mtDNA polymerase Mip1p through the action of a mitochondrial bichaperone system consisting of the Hsp70 system and the Hsp78 oligomeric protein. mtDNA synthesis was inefficiently restored after heat shock in yeast lacking either functional component of the bichaperone system. Furthermore, the activity of purified Mip1p was also thermosensitive; however, the purified components of the mitochondrial bichaperone system (Ssc1p, Mdj1p, Mge1p, and Hsp78p) were able to protect its activity under moderate heat shock conditions as well as to reactivate thermally inactivated Mip1p. Interestingly, the reactivation of endogenous Mip1p contributed more significantly to the restoration of mtDNA synthesis than did import of newly synthesized Mip1p from the cytosol. These observations suggest an important link between function of mitochondrial chaperones and the propagation of mitochondrial genomes under ever-changing environmental conditions.

  20. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

    DEFF Research Database (Denmark)

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia

    2016-01-01

    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...

  1. A comprehensive strategy to discover inhibitors of the translesion synthesis DNA polymerase κ.

    Directory of Open Access Journals (Sweden)

    Kinrin Yamanaka

    Full Text Available Human DNA polymerase kappa (pol κ is a translesion synthesis (TLS polymerase that catalyzes TLS past various minor groove lesions including N(2-dG linked acrolein- and polycyclic aromatic hydrocarbon-derived adducts, as well as N(2-dG DNA-DNA interstrand cross-links introduced by the chemotherapeutic agent mitomycin C. It also processes ultraviolet light-induced DNA lesions. Since pol κ TLS activity can reduce the cellular toxicity of chemotherapeutic agents and since gliomas overexpress pol κ, small molecule library screens targeting pol κ were conducted to initiate the first step in the development of new adjunct cancer therapeutics. A high-throughput, fluorescence-based DNA strand displacement assay was utilized to screen ∼16,000 bioactive compounds, and the 60 top hits were validated by primer extension assays using non-damaged DNAs. Candesartan cilexetil, manoalide, and MK-886 were selected as proof-of-principle compounds and further characterized for their specificity toward pol κ by primer extension assays using DNAs containing a site-specific acrolein-derived, ring-opened reduced form of γ-HOPdG. Furthermore, candesartan cilexetil could enhance ultraviolet light-induced cytotoxicity in xeroderma pigmentosum variant cells, suggesting its inhibitory effect against intracellular pol κ. In summary, this investigation represents the first high-throughput screening designed to identify inhibitors of pol κ, with the characterization of biochemical and biologically relevant endpoints as a consequence of pol κ inhibition. These approaches lay the foundation for the future discovery of compounds that can be applied to combination chemotherapy.

  2. Synthesis of a multibranched porphyrin-oligonucleotide scaffold for the construction of DNA-based nano-architectures.

    Science.gov (United States)

    Clavé, Guillaume; Chatelain, Grégory; Filoramo, Arianna; Gasparutto, Didier; Saint-Pierre, Christine; Le Cam, Eric; Piétrement, Olivier; Guérineau, Vincent; Campidelli, Stéphane

    2014-05-01

    The interest in the functionalization of oligonucleotides with organic molecules has grown considerably over the last decade. In this work, we report on the synthesis and characterization of porphyrin-oligonucleotide hybrids containing one to four DNA strands (P1-P4). The hybrid P4, which inserts one porphyrin and four DNA fragments, was combined with gold nanoparticles and imaged by transmission electron microscopy.

  3. Phospholipase C-delta1 expression is linked to proliferation, DNA synthesis, and cyclin E levels.

    Science.gov (United States)

    Stallings, Jonathan D; Zeng, Yue X; Narvaez, Francisco; Rebecchi, Mario J

    2008-05-16

    We previously reported that phospholipase C-delta1 (PLC-delta1) accumulates in the nucleus at the G1/S transition, which is largely dependent on its binding to phosphatidylinositol 4,5-bisphosphate ( Stallings, J. D., Tall, E. G., Pentyala, S., and Rebecchi, M. J. (2005) J. Biol. Chem. 280, 22060-22069 ). Here, using small interfering RNA (siRNA) that specifically targets rat PLC-delta1, we investigated whether this enzyme plays a role in cell cycle control. Inhibiting expression of PLC-delta1 significantly decreased proliferation of rat C6 glioma cells and altered S phase progression. [3H]Thymidine labeling and fluorescence-activated cell sorting analysis indicated that the rates of G1/S transition and DNA synthesis were enhanced. On the other hand, knockdown cultures released from the G1/S boundary were slower to reach full G2/M DNA content, consistent with a delay in S phase. The levels of cyclin E, a key regulator of the G1/S transition and DNA synthesis, were elevated in asynchronous cultures as well as those blocked at the G1/S boundary. Epifluorescence imaging showed that transient expression of human phospholipase C-delta1, resistant to these siRNA, suppressed expression of cyclin E at the G1/S boundary despite treatment of cultures with rat-specific siRNA. Although whole cell levels of phosphatidylinositol 4,5-bisphosphate were unchanged, suppression of PLC-delta1 led to a significant rise in the nuclear levels of this phospholipid at the G1/S boundary. These results support a role for PLC-delta1 and nuclear phospholipid metabolism in regulating cell cycle progression.

  4. Phenyl 1,2,3-triazole-thymidine ligands stabilize G-quadruplex DNA, inhibit DNA synthesis and potentially reduce tumor cell proliferation over 3'-azido deoxythymidine.

    Science.gov (United States)

    Mahesh Kumar, Jerald; Idris, Mohammed M; Srinivas, Gunda; Vinay Kumar, Pallerla; Meghah, Vuppalapaty; Kavitha, Mitta; Reddy, Chada Raji; Mainkar, Prathama S; Pal, Biswajit; Chandrasekar, Srivari; Nagesh, Narayana

    2013-01-01

    Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3) and non-nucleoside 1,2,3-triazoles (L4-L14) were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3'-Phenyl-1,2,3- triazole-thymidine (L2) and 3'-4-Chlorophenyl-1,2,3-triazole-thymidine (L3), demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC₅₀ value for L3 was lowest (50 µM) among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3) cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA) than non quadruplex forming DNA (NQF DNA). T(m) of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA). From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3) interact with quadruplex DNA with significantly higher affinity (K(d)≈10⁻⁷ M). Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC₅₀ values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands are

  5. Effect of chlormequat (CCC on the accumulation of ethephon in tomatoes and on ethephon-stimulated ripening

    Directory of Open Access Journals (Sweden)

    Janusz Czapski

    2013-12-01

    Full Text Available In greenhouse experiment, tomato seedlings were treated with CCC (250 mg·l-1 twice before transplanting. When about 10% of fruits were showing signs of ripening (pink fruits, ethephon solution (960 mg·l-1 was applied either to leaves only or to fruits only, in order to make ripening more uniform. CCC treatment delayed the process of fruit ripening and lowered the ethephon accumulation in ripe fruits as compared to the control (CCC untreated plants. The results were similar when ethephon was applied to leaves only or to fruits only.

  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.

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

    Science.gov (United States)

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

    2014-03-14

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

  9. Optimal design for inspection and maintenance policy based on the CCC chart

    OpenAIRE

    Chan, Ling-Yau; Wu, Shaomin

    2009-01-01

    In this paper, the concept of cumulative count of conforming chart (CCC chart) is applied in inspection and maintenance planning for systems where minor inspection, major inspection, minor maintenance and major maintenance are available. Several inspection and maintenance plans are defined and studied quantitatively. Analytic expressions of relevant statistics and their expectations are derived. These inspection and maintenance plans are optimized from an economic consideration.

  10. 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 ...

  11. cis-acting sequences that control the level of viral DNA synthesis in the polyomavirus late region.

    Science.gov (United States)

    Melucci-Vigo, G; Ciotta, C; Risuleo, G

    1989-01-01

    A deletion in the polyomavirus late region results in a drastic reduction of viral replication, as shown after transfection of viral DNA into 3T6 cells. This mutation is cis acting, since cotransfection with wild-type DNA did not restore the normal phenotype. Viral DNA synthesis returned to normal levels only after reintroduction of the authentic sequences in either orientation. The data presented here suggest that these sequences are involved in the binding of a factor(s) that controls the level of viral replication. Images PMID:2552181

  12. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    OpenAIRE

    DeWyngaert, M A; Hinkle, D C

    1980-01-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (...

  13. ENHANCEMENT OF DNA SYNTHESIS IN CULTURED ADULT RAT HEPATOCYTES BY 5-HT THROUGH STIMULATION OF 5-HT2 RECEPTOR

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Hepatocytes were isolated from livers of adult male Sprague-Dawley rats and cultured in Williams'E Medium with [3 H] thymidine. The effect of 5-hydroxytryptamine (5-HT) was investigated through adding various concentrations (10-8~10-3 mol/L) of 5-HT to the hepatocyte cultures in the presence or absence of epidermal growth factor (EGF) and insulin. The involvement of 5-HT2 receptor was examined by adding a 5-HT2 receptor antagonist, ketanserin (10-6 mol/L), to some of the cultures containing 5-HT. The increment of DNA synthesis was measured by [3 H] thymidine incorporation. The results showed that 5-HT2 (≥10-6 mol/L) significantly (P<0.05) increased the amount of DNA synthesis induced by EGF and insulin in the cultured adult rat hepaptocytes. The effect of 5-HT in enhancing DNA synthesis began to appear at a concentration between 10-7 and 10-6 mol/L and reached maximum at concentrations of ≥10-4 mol/L. The enhancement of DNA synthesis by 5-HT was significantly (P<0.05) antagonized by ketanserin, suggesting that this effect of 5-HT was mediated by 5-HT2 receptor subtype.

  14. Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases α, β, η, ι, κ, REV1 and Klenow Fragment exo-, but not by DNA polymerase ζ.

    Science.gov (United States)

    Suzuki, Masayo; Kino, Katsuhito; Kawada, Taishu; Oyoshi, Takanori; Morikawa, Masayuki; Kobayashi, Takanobu; Miyazawa, Hiroshi

    2016-03-01

    Guanine is the most easily oxidized of the four DNA bases, and contiguous guanines (GG) in a sequence are more readily oxidized than a single guanine in a sequence. Continued oxidation of GGs results in a contiguous oxidized guanine lesion. Two contiguous 2,5-diamino-4H-imidazol-4-ones, an oxidized form of guanine that hydrolyses to 2,2,4-triamino-5(2H)-oxazolone (Oz), are detected following the oxidation of GG. In this study, we analysed translesion synthesis (TLS) across two contiguous Oz molecules (OzOz) using Klenow Fragment exo(-) (KF exo(-)) and DNA polymerases (Pols) α, β, ζ, η, ι, κ and REV1. We found that KF exo(-) and Pols α, β, ι and REV1 inserted one nucleotide opposite the 3' Oz of OzOz and stalled at the subsequent extension, and that Pol κ incorporated no nucleotide. Pol η only inefficiently elongated the primer up to full-length across OzOz; the synthesis of most DNA strands stalled at the 3' or 5' Oz of OzOz. Surprisingly, however, Pol ζ efficiently extended the primer up to full-length across OzOz, unlike the other DNA polymerases, but catalysed error-prone nucleotide incorporation. We therefore believe that Pol ζ is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ζ.

  15. Synthesis and spectroscopic studies of the aminoglycoside (neomycin)--perylene conjugate binding to human telomeric DNA.

    Science.gov (United States)

    Xue, Liang; Ranjan, Nihar; Arya, Dev P

    2011-04-12

    Synthesis of a novel perylene-neomycin conjugate (3) and the properties of its binding to human telomeric G-quadruplex DNA, 5'-d[AG3(T2AG3)3] (4), are reported. Various spectroscopic techniques were employed to characterize the binding of conjugate 3 to 4. A competition dialysis assay revealed that 3 preferentially binds to 4, in the presence of other nucleic acids, including DNA, RNA, DNA-RNA hybrids, and other higher-order structures (single strands, duplexes, triplexes, other G-quadruplexes, and the i-motif). UV thermal denaturation studies showed that thermal stabilization of 4 increases as a function of the increasing concentration of 3. The fluorescence intercalator displacement (FID) assay displayed a significantly tighter binding of 3 with 4 as compared to its parent constituents [220-fold stronger than neomycin (1) and 4.5-fold stronger than perylene diamine (2), respectively]. The binding of 3 with 4 resulted in pronounced changes in the molar ellipticity of the DNA absorption region as confirmed by circular dichroism. The UV-vis absorption studies of the binding of 3 to 4 resulted in a red shift in the spectrum of 3 as well as a marked hypochromic change in the perylene absorption region, suggesting that the ligand-quadruplex interaction involves stacking of the perylene moiety. Docking studies suggest that the perylene moiety serves as a bridge that end stacks on 4, making contacts with two thymine bases in the loop, while the two neomycin moieties branch into the grooves of 4.

  16. 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

  17. 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

  18. Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA.

    Science.gov (United States)

    Furukohri, Asako; Nishikawa, Yoshito; Akiyama, Masahiro Tatsumi; Maki, Hisaji

    2012-07-01

    DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.

  19. Allele-specific polymerase chain reaction for detection of a mutation in the relax circular DNA and the covalently closed circular DNA of hepatitis B virus.

    Science.gov (United States)

    Pan, Wan-Long; Hu, Jie-Li; Fang, Yan; Luo, Qiang; Xu, Ge; Xu, Lei; Jing, Zhou-Hong; Shan, Xue-Feng; Zhu, Yan-Ling; Huang, Ai-Long

    2013-12-01

    The relax circle DNA (rcDNA) sequence and the covalently closed circle DNA (cccDNA) sequence in hepatitis B virus (HBV) are crucial regions for HBV infections. To analyze mutations in rcDNA and cccDNA, DNA sequencing is often used, although it is time-consuming and expensive. Herein, we report a simple, economic, albeit accurate allele-specific polymerase chain reaction (AS-PCR) to detect mutations in these regions of HBV. This method can be extensively used to screen for mutations at specific positions of HBV genome.

  20. 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.

  1. Final report : results of the 2006 investigation of potential contamination at the former CCC/USDA facility in Ramona, Kansas.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M.; Environmental Science Division

    2007-10-18

    The investigation reported here was conducted by the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) in 2006. The investigation addressed carbon tetrachloride contamination on the former CCC/USDA grain storage facility at Ramona, Kansas. The results clearly demonstrate that only minimal contamination is associated with the past use of carbon tetrachloride on the former CCC/USDA property. No soil contamination was detected at concentrations above Kansas Department of Health and Environment (KDHE) risk-based screening level (RBSL) Tier 2 standard of 200 {micro}g/kg for the soil-to-groundwater protection pathway. Carbon tetrachloride concentrations in groundwater above the RBSL and maximum contaminant level (MCL) value of 5.0 {micro}g/L were detected in only two samples, collected at adjacent locations on the southeast part of the property. The relatively low concentrations detected and the limited areal extent of the contamination demonstrate that no imminent threat exists on the former CCC/USDA property to warrant remediation. The soil and groundwater contamination detected on the former CCC/USDA property is clearly separate from contamination detected at off-site locations. The carbon tetrachloride and chloroform contamination in groundwater (at concentrations above the RBSL and MCL value) associated with past activities on the former CCC/USDA property is contained within the property boundaries. Data collected independently by the KDHE in 2006 validate these findings and, furthermore, provide additional evidence that the sources identified on the Co-op property (west of the former CCC/USDA property) are separate from the comparatively minor results of past activities on the former CCC/USDA property. The KDHE concluded in its 2006 report that the sources are separate and that the Co-op is the principally responsible party for the carbon tetrachloride contamination detected during its 2006 investigation.

  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. Growth pattern of single fission yeast cells is bilinear and depends on temperature and DNA synthesis.

    Science.gov (United States)

    Baumgärtner, Stephan; Tolić-Nørrelykke, Iva M

    2009-05-20

    Cell growth and division have to be tightly coordinated to keep the cell size constant over generations. Changes in cell size can be easily studied in the fission yeast Schizosaccharomyces pombe because these cells have a cylindrical shape and grow only at the cell ends. However, the growth pattern of single cells is currently unclear. Linear, exponential, and bilinear growth models have been proposed. Here we measured the length of single fission yeast cells with high spatial precision and temporal resolution over the whole cell cycle by using time-lapse confocal microscopy of cells with green fluorescent protein-labeled plasma membrane. We show that the growth profile between cell separation and the subsequent mitosis is bilinear, consisting of two linear segments separated by a rate-change point (RCP). The change in growth rate occurred at the same relative time during the cell cycle and at the same relative extension for different temperatures. The growth rate before the RCP was independent of temperature, whereas the growth rate after the RCP increased with an increase in temperature, leading to clear bilinear growth profiles at higher temperatures. The RCP was not directly related to the initiation of growth at the new end (new end take-off). When DNA synthesis was inhibited by hydroxyurea, the RCP was not detected. This result suggests that completion of DNA synthesis is required for the increase in growth rate. We conclude that the growth of fission yeast cells is not a simple exponential growth, but a complex process with precise rates regulated by the events during the cell cycle.

  4. The effect of human milk on DNA synthesis of neonatal rat hepatocytes in primary culture.

    Science.gov (United States)

    Kohno, Y; Shiraki, K; Mura, T

    1991-03-01

    We studied the effect of human milk on DNA synthesis of neonatal hepatocytes to elucidate the physiologic role of human milk in growth of the liver. Neonatal hepatocytes were isolated from 5-d-old rats and cultured in serum-free medium. Human milk stimulated DNA synthesis of these hepatocytes in a concentration-dependent manner. The stimulatory activity of 7.5% (vol/vol) human milk plus 0.1 mumol/L insulin was five times that of control and was almost the same as that of 20 micrograms/L human epidermal growth factor (hEGF) plus insulin. The effect of human milk was additive with treatment with hEGF and insulin. The milk associated with prolonged jaundice of infants was significantly more active than the milk that was not associated with jaundice, although the concentration of hEGF was not different between the two types of milk. The mitogenic activity of milk was heat-labile, inactivated by DTT and stable after treatment with trypsin. Three peaks of the activity were detected in milk by gel filtration and the fraction containing proteins of molecular weight between 36,000 and 76,000 showed the highest activity. Anti-hEGF antibody did not inhibit this activity completely. These results suggested the presence of mitogens other than hEGF or a more active form of hEGF in human milk. The milk associated with breast-milk jaundice exerts a different influence on cell growth and may affect maturation of the liver function related to bilirubin metabolism. The mitogenic activity of milk might be important for growth and development of the liver in infants.

  5. Synthesis of G-N2-(CH2)3-N2-G Trimethylene DNA interstrand cross-links

    Science.gov (United States)

    Gruppi, Francesca; Salyard, Tracy L. Johnson; Rizzo, Carmelo J.

    2014-01-01

    The synthesis of G-N2-(CH2)3-N2-G trimethylene DNA interstrand cross-links (ICLs) in a 5′-CG-3′ and 5′-GC-3′ sequence from oligodeoxynucleotides containing N2-(3-aminopropyl)-2′-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine is presented. Automated solid-phase DNA synthesis was used for unmodified bases and modified nucleotides were incorporated via their corresponding phosphoramidite reagent by a manual coupling protocol. The preparation of the phosphoramidite reagents for incorporation of N2-(3-aminopropyl)-2′-deoxyguanosine is reported. The high-purity trimethylene DNA interstrand cross-link product is obtained through a nucleophilic aromatic substitution reaction between the N2-(3-aminopropyl)-2′-deoxyguanosine and 2-fluoro-O6-(trimethylsilylethyl)inosine containing oligodeoxynucleotides. PMID:25431636

  6. Induction of DNA synthesis and apoptosis are separable functions of E2F-1

    DEFF Research Database (Denmark)

    Phillips, A C; Bates, S; Ryan, K M;

    1997-01-01

    The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic...... response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant...... apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E...

  7. Delay in maturation of the submandibular gland in Chagas disease correlates with lower DNA synthesis

    Directory of Open Access Journals (Sweden)

    José B Alves

    2008-09-01

    Full Text Available It has been demonstrated that the acute phase of Trypanosoma cruzi infection promotes several changes in the oral glands. The present study examined whether T. cruzi modulates the expression of host cell apoptotic or mitotic pathway genes. Rats were infected with T. cruzi then sacrificed after 18, 32, 64 or 97 days, after which the submandibular glands were analyzed by immunohistochemistry. Immunohistochemical analyses using an anti-bromodeoxyuridine antibody showed that, during acute T. cruzi infection, DNA synthesizing cells in rat submandibular glands were lower than in non-infected animals (p < 0.05. However, after 64 days of infection (chronic phase, the number of immunolabeled cells are similar in both groups. However, immunohistochemical analysis of Fas and Bcl-2 expression did not find any difference between infected and non-infected animals in both the acute and chronic stages. These findings suggest that the delay in ductal maturation observed at the acute phase of Chagas disease is correlated with lower expression of DNA synthesis genes, but not apoptotic genes.

  8. Epidermal DNA synthesis in organ culture explants. A study of hairless mouse ear epidermis.

    Science.gov (United States)

    Hansteen, I L; Iversen, O H; Refsum, S B

    1979-10-01

    Explants of split mouse ear were incubated in organ culture for up to 48 h, and the cell proliferation was studied by the addition of Thymidine-methyl-3-H (3HTdR) to the medium during different time periods, mainly for the first 14 h of incubation. Cultures were started at 0900, 2130 and 2300. In all cases the labelling index remained stable for 6-8 h, and then increased. The mean grain count, however, was falling and so was the epidermal DNA-specific uptake of 3HTdR. Based on the experimental results, calculations can be made of the flux of cells through S. It is concluded that the increasing LI is not due to inherent diurnal variation in cell proliferation, and is not a sign of real growth but caused instead by a complete block of the cell exit from S, probably combined with periods of an increased entrance rate into S. Other methodological factors, however, may also contribute to the increasing LI. Hence, this system is not suited for the measurement of factors that influence epidermal DNA synthesis.

  9. Bacterial Obg proteins: GTPases at the nexus of protein and DNA synthesis.

    Science.gov (United States)

    Kint, Cyrielle; Verstraeten, Natalie; Hofkens, Johan; Fauvart, Maarten; Michiels, Jan

    2014-08-01

    Obg proteins (also known as ObgE, YhbZ and CgtA) are conserved P-loop GTPases, essential for growth in bacteria. Like other GTPases, Obg proteins cycle between a GTP-bound ON and a GDP-bound OFF state, thereby controlling cellular processes. Interestingly, the in vitro biochemical properties of Obg proteins suggest that they act as sensors for the cellular GDP/GTP pools and adjust their activity according to the cellular energy status. Obg proteins have been attributed a host of cellular functions, including roles in essential cellular processes (DNA replication, ribosome maturation) and roles in different stress adaptation pathways (stringent response, sporulation, general stress response). This review summarizes the current knowledge on Obg activity and function. Furthermore, we present a model that integrates the different functions of Obg by assigning it a fundamental role in cellular physiology, at the hub of protein and DNA synthesis. In particular, we believe that Obg proteins might provide a connection between different global pathways in order to fine-tune cellular processes in response to a given energy status.

  10. Relationship between unscheduled DNA synthesis and mutation induction in male mice

    Energy Technology Data Exchange (ETDEWEB)

    Sega, G. A.

    1979-01-01

    Unscheduled DNA synthesis (UDS) induced in the germ cells of male mice by chemical and physical agents can be studied in vivo by making use of the timing of spermatogenesis and spermiogenesis. In meiotic and post-meiotic germ-cell stages UDS occurs from leptotene through mid-spermatid stages but is not detected in later stages. No consistent correlation has been seen between the occurrence of UDS in the germ cells and reduced dominant-lethal frequencies or reduced specific-locus mutation frequencies. It is suggested that the UDS observed in the germ cells may be principally involved in the removal of DNA lesions which, if left, could give rise to subtle genetic damage that current mammalian genetic tests may not be able to detect. Characterization of mouse stocks with reduced UDS capability in their germ cells plus the development of biochemical genetic markers that can measure single amino acid substitutions will likely be necessary before the relationship between UDS in mammalian germ cells and repair of genetic damage can be clearly established.

  11. 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

  12. 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

  13. 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.

  14. DNA polymerase delta, RFC and PCNA are required for repair synthesis of large looped heteroduplexes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Corrette-Bennett, Stephanie E; Borgeson, Claudia; Sommer, Debbie; Burgers, Peter M J; Lahue, Robert S

    2004-01-01

    Small looped mispairs are corrected by DNA mismatch repair (MMR). In addition, a distinct process called large loop repair (LLR) corrects loops up to several hundred nucleotides in extracts of bacteria, yeast or human cells. Although LLR activity can be readily demonstrated, there has been little progress in identifying its protein components. This study identified some of the yeast proteins responsible for DNA repair synthesis during LLR. Polyclonal antisera to either Pol31 or Pol32 subunits of polymerase delta efficiently inhibited LLR in extracts by blocking repair just prior to gap filling. Gap filling was inhibited regardless of whether the loop was retained or removed. These experiments suggest polymerase delta is uniquely required in yeast extracts for LLR-associated synthesis. Similar results were obtained with antisera to the clamp loader proteins Rfc3 and Rfc4, and to PCNA, i.e. LLR was inhibited just prior to gap filling for both loop removal and loop retention. Thus PCNA and RFC seem to act in LLR only during repair synthesis, in contrast to their roles at both pre- and post-excision steps of MMR. These biochemical experiments support the idea that yeast polymerase delta, RFC and PCNA are required for large loop DNA repair synthesis.

  15. Protein-DNA chimeras: synthesis of two-arm chimeras and non-mechanical effects of the DNA spring

    Energy Technology Data Exchange (ETDEWEB)

    Wang Yong; Wang, Andrew; Qu Hao; Zocchi, Giovanni, E-mail: zocchi@physics.ucla.ed [Department of Physics and Astronomy, University of California Los Angeles, Los Angeles, CA 90095-1547 (United States)

    2009-08-19

    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.

  16. Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX.

    Science.gov (United States)

    Mounce, Bryan C; Tsan, Fei Chin; Droit, Lindsay; Kohler, Sarah; Reitsma, Justin M; Cirillo, Lisa A; Tarakanova, Vera L

    2011-11-25

    Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

  17. Synthesis and DNA-binding properties of novel DNA cyclo-intercalators containing purine-glucuronic acid hybrids.

    Science.gov (United States)

    Zhang, Renshuai; Chen, Shaopeng; Wang, Xueting; Yu, Rilei; Li, Mingjing; Ren, Sumei; Jiang, Tao

    2016-06-24

    Novel DNA cyclo-intercalators, which incorporated two intercalator subunits linked by two bridges, were synthesized. Binding of the compounds to calf-thymus DNA was studied by fluorescence spectroscopy, and docking simulations were used to predict the binding modes of these cyclic compounds. The spectral data demonstrated that all of these compounds can interact with CT-DNA. The sugar moiety played an important role in the process of binding between the intercalators containing glucuronic acid and DNA. The length and flexibility of the connecting bridges affected the binding affinity of the resultant cyclo-intercalators. Docking simulations showed that compounds 7 and 8 interact with DNA as mono-intercalators.

  18. 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 ...

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

    Science.gov (United States)

    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 showed the presence of two subunit α (16 kD) and β (17 kD).

  20. UV-assisted photocatalytic synthesis of highly dispersed Ag nanoparticles supported on DNA decorated graphene for quantitative iodide analysis.

    Science.gov (United States)

    Kong, Fen-Ying; Li, Wei-Wei; Wang, Jing-Yi; Wang, Wei

    2015-07-15

    Herein, we report, for the first time, the synthesis of reduced graphene oxide-DNA-Ag (RGO-DNA-Ag) nanohybrids by ultraviolet (UV) irradiation of aqueous solutions of GO and Ag ions in the presence of DNA. The morphology and microstructure characterizations of the resultant nanohybrids reveal that the proposed method leads to the simultaneous reduction of GO and Ag ions together with efficient dispersion of Ag nanoparticles on the surface of RGO sheets. This simple and fast synthesis route is carried out at ambient conditions without using any additional chemical reducing agents, which has the potential to provide new avenues for the green fabrication of various RGO-based nanomaterials. Additionally, the RGO-DNA-Ag nanohybrids can be utilized as a novel sensing interfacial for direct determination of iodide by simple differential pulse voltammetry (DPV), without requiring any preceding preconcentration of the analyte. Based on the RGO-DNA-Ag nanohybrids modified electrode, a wide linear range of 1μM-1mM and a low detection limit of 0.2μM were obtained. This sensitive and direct method of analysis can be applied successfully to the determination of iodide in real samples.

  1. Synthesis of deoxynucleoside triphosphates that include proline, urea, or sulfonamide groups and their polymerase incorporation into DNA.

    Science.gov (United States)

    Hollenstein, Marcel

    2012-10-15

    To expand the chemical array available for DNA sequences in the context of in vitro selection, I present herein the synthesis of five nucleoside triphosphate analogues containing side chains capable of organocatalysis. The synthesis involved the coupling of L-proline-containing residues (dU(tP)TP and dU(cP)TP), a dipeptide (dU(FP)TP), a urea derivative (dU(Bpu)TP), and a sulfamide residue (dU(Bs)TP) to a suitably protected common intermediate, followed by triphosphorylation. These modified dNTPs were shown to be excellent substrates for the Vent (exo(-)) and Pwo DNA polymerases, as well as the Klenow fragment of E. coli DNA polymerase I, although they were only acceptable substrates for the 9°N(m) polymerase. All of the modified dNTPs, with the exception of dU(Bpu)TP, were readily incorporated into DNA by the polymerase chain reaction (PCR). Modified oligonucleotides efficiently served as templates for PCR for the regeneration of unmodified DNA. Thermal denaturation experiments showed that these modifications are tolerated in the major groove. Overall, these heavily modified dNTPs are excellent candidates for SELEX.

  2. Phosphorylation of the PCNA binding domain of the large subunit of replication factor C by Ca2+/calmodulin-dependent protein kinase II inhibits DNA synthesis

    DEFF Research Database (Denmark)

    Maga, G; Mossi, R; Fischer, R

    1997-01-01

    that the PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF......Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and DNA repair. It is a molecular matchmaker required for loading of the proliferating cell nuclear antigen (PCNA) sliding clamp onto double-strand DNA and for PCNA-dependent DNA synthesis by DNA polymerases...

  3. Certificate Completion And Compliance (CCC For Building Certification In Malaysia: Literature Review

    Directory of Open Access Journals (Sweden)

    Zakaria R.

    2014-01-01

    Full Text Available Certification of buildings is one of the conditions set by the government to ensure that buildings are constructed to achieve the required standards. This intended to safeguard the interests of the parties involved, especially the buyers. To improve public service delivery while not ignoring the interests of those involved, government held a new system for new building to obtain theirbuilding certification. The new system used called Certificate Completion and Compliance (CCC where the appointed professionals were commonly known as Prinsiple Submitting Person (PSP, which will issue the certificate of the building. This study is to look at the existing literature related to building certification using PSP services under this CCC method. Literature from both local and overseas were revied in this paper. The authors intend to collect data from respondentsthat is the architects of 337 people and covers 12 local authorities in Selangor (first developed state in Malaysia. All types of buildings involved in this study. This study uses quantitative methods and qualitative questionnaires through interviews with respondents. All data were analysed by descriptive and inferential statisticsapproach.

  4. 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...

  5. 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 synthesis of H2O2 showed a similar exponential rise (P synthesis of H2O2 but not to sperm viability and synthesis of other reactive oxygen species.

  6. Interacting RNA polymerase motors on a DNA track: effects of traffic congestion and intrinsic noise on RNA synthesis.

    Science.gov (United States)

    Tripathi, Tripti; Chowdhury, Debashish

    2008-01-01

    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 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 mechanochemical 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 different measures of fluctuations in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis depends on the concentrations of the RNAPs as well as on those of some of the reactants and the products of the enzymatic reactions catalyzed by RNAP. We suggest appropriate experimental systems and techniques for testing our theoretical predictions.

  7. 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...

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

    Science.gov (United States)

    Brière, N; Chailler, P

    1993-05-01

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

  9. Synthesis and investigation of the specific activity of the DNA-doxorubicin conjugates

    Science.gov (United States)

    Kokorev, A. V.; Zaborovskiy, A. V.; Kotlyarov, A. A.; Balykova, L. A.; Malkina, M. A.; Kargina, I. V.; Gromova, E. V.; Medvezhonkov, V. Yu; Gurevich, K. G.; Shchukin, S. A.; Pyataev, N. A.

    2017-01-01

    In the present work, the method of obtaining the conjugate of the anticancer chemotherapeutic agent doxorubicin to the exogenous double-stranded DNA of the sturgeons is proposed (the source: commercial drug “Derinat”). The optimal conditions for synthesis of conjugate (pH, temperature and the mass ratio of the components), ensuring the highest degree of binding the chemotherapeutic agent to a carrier, were picked out. Clearing the conjugate from the non-encapsulated chemotherapeutic agent was being made by ultrafiltration method. The investigation of the toxicity and specific antineoplastic activity of the synthesized complex was conducted. The performance of the drug toxicity were established on the intact mice in compliance with the accepted standards. The antineoplastic activity was evaluated upon the Tumor Growth Inhibition Index and Metastasis Inhibition Index on mice with the transplanted Lewis lung carcinoma (LLC). It was demonstrated that the conjugate toxicity is approximately lower that the one of the unconjugated doxorubicin (LD 50 was equal 14.6 mg/kg and 9.9 mg/kg for the conjugate and doxorubicin, respectively). The specific antineoplastic activity was investigated in equitoxic doses of the drug. It was established that the conjugate being administered in equitoxic doses possesses a stronger antineoplastic activity, than the water-soluble drug (maximum 35% more as to the tumor volume and 51% more as to the Tumor Growth Inhibition index).

  10. Synthesis of core-shell gold coated magnetic nanoparticles and their interaction with thiolated DNA.

    Science.gov (United States)

    Robinson, Ian; Tung, Le D; Maenosono, Shinya; Wälti, Christoph; Thanh, Nguyen T K

    2010-12-01

    Core-shell magnetic nanoparticles have received significant attention recently and are actively investigated owing to their large potential for a variety of applications. Here, the synthesis and characterization of bimetallic nanoparticles containing a magnetic core and a gold shell are discussed. The gold shell facilitates, for example, the conjugation of thiolated biological molecules to the surface of the nanoparticles. The composite nanoparticles were produced by the reduction of a gold salt on the surface of pre-formed cobalt or magnetite nanoparticles. The synthesized nanoparticles were characterized using ultraviolet-visible absorption spectroscopy, transmission electron microscopy, energy dispersion X-ray spectroscopy, X-ray diffraction and super-conducting quantum interference device magnetometry. The spectrographic data revealed the simultaneous presence of cobalt and gold in 5.6±0.8 nm alloy nanoparticles, and demonstrated the presence of distinct magnetite and gold phases in 9.2±1.3 nm core-shell magnetic nanoparticles. The cobalt-gold nanoparticles were of similar size to the cobalt seed, while the magnetite-gold nanoparticles were significantly larger than the magnetic seeds, indicating that different processes are responsible for the addition of the gold shell. The effect on the magnetic properties by adding a layer of gold to the cobalt and magnetite nanoparticles was studied. The functionalization of the magnetic nanoparticles is demonstrated through the conjugation of thiolated DNA to the gold shell.

  11. Regulation of translesion DNA synthesis: Posttranslational modification of lysine residues in key proteins.

    Science.gov (United States)

    McIntyre, Justyna; Woodgate, Roger

    2015-05-01

    Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research have revealed that many proteins involved in TLS are, in fact, subject to a previously underappreciated number of lysine modifications. In this review, we will summarize the known lysine modifications of several key proteins involved in TLS; PCNA and Y-family polymerases η, ι, κ and Rev1 and we will discuss the potential regulatory effects of such modification in controlling TLS in vivo.

  12. Harnessing DNA Synthesis to Develop Rapid Responses to Emerging and Pandemic Pathogens

    Directory of Open Access Journals (Sweden)

    Lisa M. Runco

    2011-01-01

    Full Text Available Given the interconnected nature of our world today, emerging pathogens and pandemic outbreaks are an ever-growing threat to the health and economic stability of the global community. This is evident by the recent 2009 Influenza A (H1N1 pandemic, the SARS outbreak, as well as the ever-present threat of global bioterrorism. Fortunately, the biomedical community has been able to rapidly generate sequence data so these pathogens can be readily identified. To date, however, the utilization of this sequence data to rapidly produce relevant experimental results or actionable treatments is lagging in spite of obtained sequence data. Thus, a pathogenic threat that has emerged and/or developed into a pandemic can be rapidly identified; however, translating this identification into a targeted therapeutic or treatment that is rapidly available has not yet materialized. This commentary suggests that the growing technology of DNA synthesis should be fully implemented as a means to rapidly generate in vivo data and possibly actionable therapeutics soon after sequence data becomes available.

  13. 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.

  14. 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.

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

    Science.gov (United States)

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

    2016-04-26

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

  16. Effects of 8-halo-7-deaza-2'-deoxyguanosine triphosphate on DNA synthesis by DNA polymerases and cell proliferation.

    Science.gov (United States)

    Yin, Yizhen; Sasaki, Shigeki; Taniguchi, Yosuke

    2016-08-15

    8-OxodG (8-oxo-2'-deoxyguanosine) is representative of nucleoside damage and shows a genotoxicity. To significantly reveal the contributions of 7-NH and C8-oxygen to the mutagenic effect of 8-oxodG by DNA polymerases, we evaluated the effects of the 8-halo-7-deaza-dG (8-halogenated 7-deaza-2'-deoxyguanosine) derivatives by DNA polymerases. 8-Halo-7-deaza-dGTPs were poorly incorporated by both KF(exo(-)) and human DNA polymerase β opposite dC or dA into the template DNA. Furthermore, it was found that KF(exo(-)) was very sensitive to the introduction of the C8-halogen, while polymerase β can accommodate the C8-halogen resulting in an efficient dCTP insertion opposite the 8-halo-7-deaza-dG in the template DNA. These results indicate that strong hydrogen bonding between 7-NH in the 8-oxo-G nucleobase and 1-N in the adenine at the active site of the DNA polymerase is required for the mutagenic effects. Whereas, I-deaza-dGTP shows an antiproliferative effect for the HeLa cells, suggesting that it could become a candidate as a new antitumor agent.

  17. The influence of beryllium on cell survival rates in theIn-vitro culture system, on intracellular DNA synthesis and on SRBC-IgM antibody production responses

    OpenAIRE

    Yoshida, Tsutomu; Shima , Syogo; Kurita , Hideki; Nagaoka, Kaoru; Taniwaki, Hiroshige; Asada, Yasuki; Shai , Kai-ping; Koike, Mitsumasa; Morita, Kunihiko

    1997-01-01

    Immunocytotoxicity of beryllium (Be) was evaluated by studying cell viability, intracellular DNA synthesis and SRBC-IgM response in an in-vitro culture system using non-sensitized spleen cells of a C57BL mouse. Be addition showed a suppressive effect on cell viability, an enhancing effect on DNA synthesis and on IgM antibody production. The suppressive effect on cell viability manifested itself markedly as the concentration of Be was increased or the culture time was prolonged. The DNA synthe...

  18. 换流站无功功率补偿新概念—CCC

    Institute of Scientific and Technical Information of China (English)

    郭天兴; 孙振权; 邱毓昌

    2003-01-01

    本文对直流输电用传统的补偿方式滤波器(filter)加并联电容器组(shunt capacitor bank)、晶闸管控制串联补偿技术(Thyriator controlled Series Capacitor简称TCSC)、以及一种新的补偿技术即换流电容器装置(Ca-pacitor commutated converters,简称CCC)进行了分析。并着重对CCC技术的性能进行了理论分析。应用CCC技术可使经过逆变的交流电网有较高的抗干扰能力,并可增强交流电网的传输能力,提高系统的稳定性。

  19. 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

  20. Unscheduled DNA synthesis in rat pleural mesothelial cells treated with mineral fibres.

    Science.gov (United States)

    Renier, A; Lévy, F; Pillière, F; Jaurand, M C

    1990-08-01

    Unscheduled DNA synthesis (UDS) was studied in confluent rat pleural mesothelial cells (RPMCs) arrested in G0/G1 with hydroxyurea (HU) and treated with various fibre types, i.e., chrysotile, crocidolite or attapulgite. In addition, the effects of UV light and of benzo[a]pyrene were determined as references. Using autoradiography after [3H]thymidine incorporation ([3H]dThd), RPMCs treated with 4 micrograms/cm2 of chrysotile fibres exhibited a low but significant enhancement of net grains compared to untreated cells. Treatment with higher doses of chrysotile was not possible because of the impairment of microscopic observation due to the presence of the fibres. Using liquid scintillation counting, RPMCs treated with chrysotile or crocidolite showed a significant dose-dependent increase in [3H]dThd incorporation compared to untreated cells. In contrast, attapulgite did not enhance [3H]dThd incorporation compared to untreated cells. Treatment of RPMCs with 1, 2 or 4 micrograms/ml of benzo[a]pyrene resulted in a significant increase in [3H]dThd incorporation. In order to discount a possible role of S cells in the augmentation of [3H]dThd incorporation, despite the presence of 5 mM HU, S cells were counted by autoradiography. Results indicated that the percentage of S cells was similar in asbestos-treated and untreated cultures. Stimulation of the S phase also seems unlikely because treatment of RPMCs with asbestos fibres in the absence of HU resulted in a reduction of [3H]dThd incorporation attributed to an impairment of the S phase by the fibres. 1-4 micrograms/ml benzo[a]pyrene or 10-50 J/m2 UV light resulted in an approximate doubling of [3H]dThd incorporation. The effects of inhibitors of DNA repair were determined in chrysotile-treated RPMCs. [3H]dThd incorporation was inhibited by cytosine arabinoside and nalidixic acid. These results show that asbestos produces UDS in RPMCs.

  1. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    Science.gov (United States)

    DeWyngaert, M A; Hinkle, D C

    1980-02-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein.

  2. [Overgrowth and DNA synthesis of neuroepithelium in embryonic stages of induced Long-Evans rat myeloschisis].

    Science.gov (United States)

    Chono, Y

    1993-01-01

    Overgrowth of the myeloschisis, namely the excessive amount of the neural plate tissue, has been reported in the human myeloschisis. However, it is still debatable how the overgrowth develops and whether the overgrowth is the cause, or the secondary effect of spinal dysraphism. The author induced myeloschisis in the fetuses of Long-Evans rats by the administration of ethylenethiourea (ETU) to pregnant rats on day 10 of gestation. The fetuses were removed 1 hour after the treatment with bromodeoxyuridine (BrdU) to the dams on day 14 and 21. The fetuses were fixed in alcohol and embedded in paraffin. H-E staining and the immunohistologic examination were performed on the staining patterns to anti-neurofilament (NFP), anti-glial fibrillary acidic protein (GFAP) and anti-BrdU antibody by ABC method. On day 14, the lateral portion of everted neural plate showed a loose arrangement of cells and there was rosette formation in the mesoderm. On day 21, cell necrosis was observed at the dorsolateral portion of myeloschisis, although the ventral portion showed almost normal cytoarchitecture and was positive to NFP and GFAP. The cause of myeloschisis in this model is supposed to be the local and direct cytotoxic effect of ETU to neuro-ectodermal junction. On day 14, control animals contained few BrdU-incorporated cells at the basal plate of neural tube. In contrast, everted neural plate showed an active uptake of BrdU diffusely in the subependymal matrix layer cells. Overgrowth was not yet identified. On day 21, overgrowth of myeloschisis was found in spite of a few positive cells to BrdU which was identical to the control animals. These findings seem to suggest that cells in the myeloschisis retain their ability of DNA synthesis for longer periods of development and overgrowth found on day 21 is possibly a secondary effect of spinal dysraphism in this model.

  3. A Novel Cobalt(Ⅲ) Mixed-polypyridyl Complex: Synthesis,Characterization and DNA Binding

    Institute of Scientific and Technical Information of China (English)

    CHEN,Hui-Li(陈绘丽); YANG,Pin(杨频)

    2002-01-01

    A novel complex[Co(phen)2HPIP]Cl3[phen=phenanethroline,HPIP=2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanethroline]has been synthesized and structurally characterized by elemental analysis,UV,IR and 1H NMR spectroscopies. The interaction of the complex with calf thymus DNA(CT DNA)has been studied using absorption and emission spectroscopy, DNA melting techniques and cyclic voltammetry. The compound shows absorption hypochromicity, fluorescence enhancement and DNA melting temperature increment when binding to CT DNA. CV measurement shows a shift in reduction potential and a change in peak current with addition of DNA.These results prove that the compound inserts into DNA base pairs. The shift of peak potential indicates the ion interaction mode between the complex and DNA. The binding constant of the compound to DNA is 4.37×104. The complex also seems to be an efficient photocleavage reagent.

  4. 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.

  5. 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...

  6. DNA polymerase kappa from Trypanosoma cruzi localizes to the mitochondria, bypasses 8-oxoguanine lesions and performs DNA synthesis in a recombination intermediate.

    Science.gov (United States)

    Rajão, M A; Passos-Silva, D G; DaRocha, W D; Franco, G R; Macedo, A M; Pena, S D J; Teixeira, S M; Machado, C R

    2009-01-01

    DNA polymerase kappa (Pol kappa) is a low-fidelity polymerase that has the ability to bypass several types of lesions. The biological role of this enzyme, a member of the DinB subfamily of Y-family DNA polymerases, has remained elusive. In this report, we studied one of the two copies of Pol kappa from the protozoan Trypanosoma cruzi (TcPol kappa). The role of this TcPol kappa copy was investigated by analysing its subcellular localization, its activities in vitro, and performing experiments with parasites that overexpress this polymerase. The TcPOLK sequence has the N-terminal extension which is present only in eukaryotic DinB members, but its C-terminal region is more similar to prokaryotic and archaeal counterparts since it lacks C(2)HC motifs and PCNA interaction domain. Our results indicate that in contrast to its previously described orthologues, this polymerase is localized to mitochondria. The overexpression of TcPOLK increases T. cruzi resistance to hydrogen peroxide, and in vitro polymerization assays revealed that TcPol kappa efficiently bypasses 8-oxoguanine lesions. Remarkably, our results also demonstrate that the DinB subfamily of polymerases can participate in homologous recombination, based on our findings that TcPol kappa increases T. cruzi resistance to high doses of gamma irradiation and zeocin and can catalyse DNA synthesis within recombination intermediates.

  7. Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.

    Science.gov (United States)

    Fuller, Carl W; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Bibillo, Arek; Stranges, P Benjamin; Dorwart, Michael; Tao, Chuanjuan; Li, Zengmin; Guo, Wenjing; Shi, Shundi; Korenblum, Daniel; Trans, Andrew; Aguirre, Anne; Liu, Edward; Harada, Eric T; Pollard, James; Bhat, Ashwini; Cech, Cynthia; Yang, Alexander; Arnold, Cleoma; Palla, Mirkó; Hovis, Jennifer; Chen, Roger; Morozova, Irina; Kalachikov, Sergey; Russo, James J; Kasianowicz, John J; Davis, Randy; Roever, Stefan; Church, George M; Ju, Jingyue

    2016-05-10

    DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

  8. Effect of Chlorocholine Chloride (CCC on the Plants’ Height and Inulin Content in Jerusalem Artichoke (Helianthus tuberosus L.

    Directory of Open Access Journals (Sweden)

    Mikołaj Wawrzyniak

    2016-11-01

    Full Text Available Jerusalem artichoke (Helianthus tuberosus L. is herbaceous perennial plant rich in inulin and useful source of biomass. Due to its low agricultural requirements and high adaptability, it can provide very high biomass yields even on low quality sites. The plant is used in food industry, bio-fuel production, forage, pharmacy and nutrition. Its tubers accumulate approx. 10-20% of inulin in fresh weight. Currently, the use of the Helianthius tuberosus L. as a potential dietary strategy in patients affected by type 2 Diabetes is challenge. Moreover, deep understanding of the relationship between diet and composition of gut microbiota can bring the new insight in the treatment of inflammatory dependent diseases. The aim of this study was to examine an effect of plant growth retardant Chlorocholine Chloride (CCC on the plants’ height of H. tuberosus and inulin content in the tubers. We examined in the field a procedure for its shoots reduction. Material for the experiment were bought in a Polish commercial company and 528 tubers were planted in field in the middle of April 2014. Then, half of them were sprayed with 0.75% retardant of CCC . Furthermore, every week for 12 following weeks, the plants’ heights were measured. After the vegetation was over, 6 tubers for each treatment were dug out and chemically analyzed for inulin content using High Pressure Size Exclusion Chromatography. After first week of CCC use, 16% decrease of the heights plants was observed. Height of plants sprayed with CCC were significantly different comparing to Control. Weekly growth was significantly  slower in plants sprayed with CCC on first three weeks after applying retardant. Differences in plants height sustain to the end of measurements. Used retardant and its concentration did not affect the inulin content of the tubers.

  9. Mixed lineage kinase 3 inhibits phorbol myristoyl acetate-induced DNA synthesis but not osteopontin expression in rat mesangial cells.

    Science.gov (United States)

    Parameswaran, Narayanan; Hall, Carolyn S; Bock, Barbara C; Sparks, Harvey V; Gallo, Kathleen A; Spielman, William S

    2002-12-01

    Mixed lineage kinase 3 (MLK 3) (also called SPRK or PTK-1) is a recently described member of the family of the mixed lineage kinase subfamily of Ser/Thr protein kinases that interacts with mitogen-activated protein kinase pathways. In order to test the biological relevance and potential interaction of MLK 3 with protein kinase C-mediated signaling pathways, human MLK 3 was stably expressed in rat glomerular mesangial cells using a retroviral vector (LXSN) and the effects of phorbol myristoyl acetate (PMA) on DNA synthesis and osteopontin mRNA expression were examined. In control (vector-transfected) mesangial cells PMA increased [3H]-thymidine incorporation in a concentration-dependent manner. In mesangial cells stably expressing MLK 3, the PMA-induced increase in [3H]-thymidine incorporation was significantly reduced (> 50%). However, the PMA-induced increase in osteopontin mRNA was not affected by MLK 3 expression. To determine the mechanisms of these effects, activation of ERK2, JNK1 and p38 in response to PMA was examined in both vector and MLK 3 transfected cells. ERK2 activation was increased several fold by PMA in control cells but was attenuated significantly in MLK 3 expressing cells, suggesting that MLK 3 expression in mesangial cells can negatively regulate the ERK pathway. PMA had no significant effect on JNK and P38 activation, in either vector- or MLK 3-expressing cells. PD98059, a MEK inhibitor blocked PMA-induced DNA synthesis without affecting osteopontin expression. These results suggest that while protein kinase C activation increases cellular proliferation and osteopontin mRNA expression, over-expression of MLK 3 affects only the PKC-induced DNA synthesis, probably through inhibition of ERK. These results also indicate a novel mechanism of growth regulation by a member of the mixed-lineage kinase family that might have significant therapeutic implications in proliferative glomerulonephritis.

  10. Recent advances in small organic molecules as DNA intercalating agents: synthesis, activity, and modeling.

    Science.gov (United States)

    Rescifina, Antonio; Zagni, Chiara; Varrica, Maria Giulia; Pistarà, Venerando; Corsaro, Antonino

    2014-03-03

    The interaction of small molecules with DNA plays an essential role in many biological processes. As DNA is often the target for majority of anticancer and antibiotic drugs, study about the interaction of drug and DNA has a key role in pharmacology. Moreover, understanding the interactions of small molecules with DNA is of prime significance in the rational design of more powerful and selective anticancer agents. Two of the most important and promising targets in cancer chemotherapy include DNA alkylating agents and DNA intercalators. For these last the DNA recognition is a critical step in their anti-tumor action and the intercalation is not only one kind of the interactions in DNA recognition but also a pivotal step of several clinically used anti-tumor drugs such as anthracyclines, acridines and anthraquinones. To push clinical cancer therapy, the discovery of new DNA intercalators has been considered a practical approach and a number of intercalators have been recently reported. The intercalative binding properties of such molecules can also be harnessed as diagnostic probes for DNA structure in addition to DNA-directed therapeutics. Moreover, the problem of intercalation site formation in the undistorted B-DNA of different length and sequence is matter of tremendous importance in molecular modeling studies and, nowadays, three models of DNA intercalation targets have been proposed that account for the binding features of intercalators. Finally, despite DNA being an important target for several drugs, most of the docking programs are validated only for proteins and their ligands. Therefore, a default protocol to identify DNA binding modes which uses a modified canonical DNA as receptor is needed.

  11. 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

  12. Induction of maturation of human B-cell lymphomas in vitro. Morphologic changes in relation to immunoglobulin and DNA synthesis.

    Science.gov (United States)

    Beiske, K.; Ruud, E.; Drack, A.; Marton, P. F.; Godal, T.

    1984-01-01

    In vitro stimulation of cells from 8 non-Hodgkin's lymphomas comprising several histologic types with a tumor promotor (TPA) and with or without anti-immunoglobulins directed against the surface immunoglobulin of the tumor cells is reported. Morphologic transformation to immunoblastic and plasmablastic cells, but not to plasma cells, and induction of Ig and DNA synthesis were observed. A comparative analysis, including flow cytofluorometry, light microscopy combined with immunocytochemistry, and electron microscopy, suggests that the three events may not always be associated phenomena at the single-cell level even in monoclonal cell populations. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:6375389

  13. Computational study of putative residues involved in DNA synthesis fidelity checking in Thermus aquaticus DNA polymerase I.

    Science.gov (United States)

    Elias, Angela A; Cisneros, G Andrés

    2014-01-01

    A fidelity-checking site for DNA polymerase I has been proposed based on recent single-molecule Förster resonance energy transfer studies. The checking site is believed to ensure proper base pairing of the newly inserted nucleotide. Computational studies have been utilized to predict residues involved in this putative checking site on the Klenow and Bacillus fragments. Here, we employ energy decomposition analysis, electrostatic free energy response, and noncovalent interaction plots to identify the residues involved in the hypothesized checking site in the homologous Klenow fragment from Thermus aquaticus (Klentaq). Our results indicate multiple protein residues that show altered interactions for three mispairs compared to the correctly paired DNA dimer. Many of these residues are also conserved along A family polymerases.

  14. 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

  15. Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms.

    Science.gov (United States)

    Evans, Geraint W; Hohlbein, Johannes; Craggs, Timothy; Aigrain, Louise; Kapanidis, Achillefs N

    2015-07-13

    DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intra-protein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s(-1), much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.

  16. DNA microarray synthesis by using PDMS molecular stamp (II) -- Oligonucleotide on-chip synthesis using PDMS stamp

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5′-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleotide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the conventional synthesis methods.

  17. 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.

  18. Association of Hepatitis B Virus Covalently Closed Circular DNA and Human APOBEC3B in Hepatitis B Virus-Related Hepatocellular Carcinoma.

    Directory of Open Access Journals (Sweden)

    Xuan Luo

    Full Text Available Chronic Hepatitis B Virus (HBV infections can progresses to liver cirrhosis and hepatocellular carcinoma (HCC. The HBV covalently-closed circular DNA cccDNA is a key to HBV persistence, and its degradation can be induced by the cellular deaminase APOBEC3. This study aimed to measure the distribution of intrahepatic cccDNA levels and evaluate the association between levels of cccDNA and APOBEC3 in HCC patients. Among 49 HCC patients, 35 matched cancerous and contiguous noncancerous liver tissues had detectable cccDNA, and the median intrahepatic cccDNA in the cancerous tissues (CT was significantly lower than in the contiguous noncancerous tissues (CNCT (p = 0.0033. RCA (rolling circle amplification, followed by 3D-PCR identified positive amplification in 27 matched HCC patients. Sequence analysis indicated G to A mutations accumulated to higher levels in CT samples compared to CNCT samples, and the dinucleotide context showed preferred editing in the GpA context. Among 7 APOBEC3 genes, APOBEC3B was the only one up-regulated in cancerous tissues both at the transcriptional and protein levels (p < 0.05. This implies APOBEC3B may contribute to cccDNA editing and subsequent degradation in cancerous tissues.

  19. 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.

  20. Urinary tract infection drives genome instability in uropathogenic Escherichia coli and necessitates translesion synthesis DNA polymerase IV for virulence.

    Science.gov (United States)

    Gawel, Damian; Seed, Patrick C

    2011-01-01

    Uropathogenic Escherichia coli (UPEC) produces ~80% of community-acquired UTI, the second most common infection in humans. During UTI, UPEC has a complex life cycle, replicating and persisting in intracellular and extracellular niches. Host and environmental stresses may affect the integrity of the UPEC genome and threaten its viability. We determined how the host inflammatory response during UTI drives UPEC genome instability and evaluated the role of multiple factors of genome replication and repair for their roles in the maintenance of genome integrity and thus virulence during UTI. The urinary tract environment enhanced the mutation frequency of UPEC ~100-fold relative to in vitro levels. Abrogation of inflammation through a host TLR4-signaling defect significantly reduced the mutation frequency, demonstrating in the importance of the host response as a driver of UPEC genome instability. Inflammation induces the bacterial SOS response, leading to the hypothesis that the UPEC SOS-inducible translesion synthesis (TLS) DNA polymerases would be key factors in UPEC genome instability during UTI. However, while the TLS DNA polymerases enhanced in vitro, they did not increase in vivo mutagenesis. Although it is not a source of enhanced mutagenesis in vivo, the TLS DNA polymerase IV was critical for the survival of UPEC during UTI during an active inflammatory assault. Overall, this study provides the first evidence of a TLS DNA polymerase being critical for UPEC survival during urinary tract infection and points to independent mechanisms for genome instability and the maintenance of genome replication of UPEC under host inflammatory stress.

  1. 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

  2. Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase.

    Science.gov (United States)

    Kupperman, E; Wen, W; Meinkoth, J L

    1993-08-01

    Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.

  3. DNA synthesis and microtubule assembly-related events in fertilized Paracentrotus lividus eggs: reversible inhibition by 10 mM procaine.

    Science.gov (United States)

    Raymond, M N; Foucault, G; Coffe, G; Pudles, J

    1986-04-01

    This report describes the effects of 10 mM procaine on microtubule assembly and on DNA synthesis, as followed by [3H]colchicine binding assays and [3H]thymidine incorporation respectively, in fertilized Paracentrotus lividus eggs. In the absence of microtubule assembly inhibitors, about 25% of the total egg tubulin is submitted to two cycles of polymerization prior to the first cell division, this polymerization process precedes DNA synthesis. If the zygotes are treated with 10 mM procaine in the course of the cell cycle, tubulin polymerization is inhibited or microtubules are disassembled. DNA synthesis is inhibited when procaine treatment is performed 10 min, before the initiation of the S-period. However, when the drug is applied in the course of this synthetic period, the process is normally accomplished, but the next S-period becomes inhibited. Moreover, procaine treatment increases the cytoplasmic pH of the fertilized eggs by about 0.6 to 0.8 pH units. This pH increase precedes microtubule disassembly and inhibition of DNA synthesis. Washing out the drug induces a decrease of the intracellular pH which returns to about the same value as that of the fertilized egg controls. This pH change is then followed by the reinitiation of microtubule assembly, DNA synthesis and cell division. Our results show that the inhibition of both tubulin polymerization and DNA synthesis in fertilized eggs treated with 10 mM procaine, appears to be related to the drug-induced increase in cytoplasmic pH.

  4. Effects of cadmium on estrogen receptor mediated signaling and estrogen induced DNA synthesis in T47D human breast cancer cells.

    Science.gov (United States)

    Zang, Yu; Odwin-Dacosta, Shelly; Yager, James D

    2009-01-30

    Cadmium (Cd) has been shown to bind to the human estrogen receptor (ER), yet studies on Cd's estrogenic effects have yielded inconsistent results. In this study, we investigated the effects of Cd on DNA synthesis and its simultaneous effects on both genomic (mediated by nuclear ER (nER)) and non-genomic (mediated by membrane-bound ER (mER)) signaling in human breast cancer derived T47D cells. No effects on DNA synthesis were observed for non-cytotoxic concentrations of CdCl(2) (0.1-1000 nM), and Cd did not increase progesterone receptor (PgR) or pS2 mRNA levels. However, Cd stimulated phosphorylation of ERK1/2 MAPK, detectable following 10 min and 18 h of treatment. The sustained Cd-induced ERK1/2 phosphorylation was inhibited by the ER antagonist ICI 182,780, suggesting the involvement of ER. In addition, Cd enhanced DNA synthesis and pS2 mRNA levels in estrogen (10 pM estradiol) treated T47D cells. The MEK1/2 specific inhibitor U0126 blocked DNA synthesis stimulated by estradiol (E2) and the E2-Cd mixtures. These findings indicate that the ERK1/2 signaling is critical in E2-related DNA synthesis. The sustained ERK1/2 phosphorylation may contribute to the Cd-induced enhancement of DNA synthesis and pS2 mRNA in mixture with low-concentration E2.

  5. Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia

    Science.gov (United States)

    Pearce, Kaycey; Cai, Diancai; Roberts, Adam C; Glanzman, David L

    2017-01-01

    Previously, we reported that long-term memory (LTM) in Aplysia can be reinstated by truncated (partial) training following its disruption by reconsolidation blockade and inhibition of PKM (Chen et al., 2014). Here, we report that LTM can be induced by partial training after disruption of original consolidation by protein synthesis inhibition (PSI) begun shortly after training. But when PSI occurs during training, partial training cannot subsequently establish LTM. Furthermore, we find that inhibition of DNA methyltransferase (DNMT), whether during training or shortly afterwards, blocks consolidation of LTM and prevents its subsequent induction by truncated training; moreover, later inhibition of DNMT eliminates consolidated LTM. Thus, the consolidation of LTM depends on two functionally distinct phases of protein synthesis: an early phase that appears to prime LTM; and a later phase whose successful completion is necessary for the normal expression of LTM. Both the consolidation and maintenance of LTM depend on DNA methylation. DOI: http://dx.doi.org/10.7554/eLife.18299.001 PMID:28067617

  6. Abnormal levels of UV-induced unscheduled DNA synthesis in ataxia telangiectasia cells after exposure to ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Jaspers, N.G.J. (Erasmus Universiteit, Rotterdam (Netherlands). Dept. of Cell Biology and Genetics; Nederlandse Centrale Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek, Rijswijk. Medical Biological Lab.); Bootsma, D. (Erasmus Universiteit, Rotterdam (Netherlands). Dept. of Cell Biology and Genetics)

    1982-01-01

    In cultured cells from normal individuals and from patients having ataxia telangiectasia (AT) the rate of unscheduled DNA synthesis (UDS) induced by UV light was investigated by autoradiography. The number of grains in 6 different AT cell strains was similar to that observed in normal cells. Exposure of normal cells to doses of X-rays up to 20 krad had no influence on the rate of UV-induced UDS. In contrast, the UV-induced UDS was significantly modified in AT cells by treatment with X-rays. In AT cell strains that were reported to have reduced levels of ..gamma..-ray-induced repair DNA synthesis ('excision-deficient' AT cells) the effect of X-rays on UV-induced UDS was inhibitory, whereas UV-induced UDS was stimulated by X-ray exposure in 'excision-proficient' AT cell strains. Different UV and X-ray dose-response relationships were seen in the two categories of AT cell strains.

  7. 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

  8. 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.

  9. Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

    Science.gov (United States)

    Holden, Matthew T; Carter, Matthew C D; Wu, Cheng-Hsien; Wolfer, Jamison; Codner, Eric; Sussman, Michael R; Lynn, David M; Smith, Lloyd M

    2015-11-17

    The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.

  10. 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.

  11. A simple and accurate two-step long DNA sequences synthesis strategy to improve heterologous gene expression in pichia.

    Science.gov (United States)

    Yang, Jiang-Ke; Chen, Fang-Yuan; Yan, Xiang-Xiang; Miao, Li-Hong; Dai, Jiang-Hong

    2012-01-01

    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. 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. The Effects of Magnesium Ions on the Enzymatic Synthesis of Ligand-Bearing Artificial DNA by Template-Independent Polymerase

    Directory of Open Access Journals (Sweden)

    Yusuke Takezawa

    2016-06-01

    Full Text Available A metal-mediated base pair, composed of two ligand-bearing nucleotides and a bridging metal ion, is one of the most promising components for developing DNA-based functional molecules. We have recently reported an enzymatic method to synthesize hydroxypyridone (H-type ligand-bearing artificial DNA strands. Terminal deoxynucleotidyl transferase (TdT, a template-independent DNA polymerase, was found to oligomerize H nucleotides to afford ligand-bearing DNAs, which were subsequently hybridized through copper-mediated base pairing (H–CuII–H. In this study, we investigated the effects of a metal cofactor, MgII ion, on the TdT-catalyzed polymerization of H nucleotides. At a high MgII concentration (10 mM, the reaction was halted after several H nucleotides were appended. In contrast, at lower MgII concentrations, H nucleotides were further appended to the H-tailed product to afford longer ligand-bearing DNA strands. An electrophoresis mobility shift assay revealed that the binding affinity of TdT to the H-tailed DNAs depends on the MgII concentration. In the presence of excess MgII ions, TdT did not bind to the H-tailed strands; thus, further elongation was impeded. This is possibly because the interaction with MgII ions caused folding of the H-tailed strands into unfavorable secondary structures. This finding provides an insight into the enzymatic synthesis of longer ligand-bearing DNA strands.

  14. DNA-surfactant complexes : preparation, self-assembly properties and applications in synthesis and bioelectronics

    NARCIS (Netherlands)

    Liu, Kai

    2015-01-01

    The powerful ionic self-assembly behavior of DNA-surfactant complexes make it a unique material for various applications from optoelectronics to biomedicine. Three types of DNA-surfactant assemblies, including bulk films, lyotropic liquid crystals (LCs) and hydrogels have been investigated extensive

  15. Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

    NARCIS (Netherlands)

    Hamdan, Samir M.; Loparo, Joseph J.; Takahashi, Masateru; Richardson, Charles C.; Oijen, Antoine M. van

    2009-01-01

    In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesiz

  16. Synthesis of Distamycin Analogs and Their Interactions withCalf Thymus DNA

    Institute of Scientific and Technical Information of China (English)

    肖军华; 袁谷; 黄伟强; 杜卫红; 王保怀; 李芝芬

    2001-01-01

    Two distamycin analogs (PyPyPy-γ-Dp and PyPyPyPy-γ- Dp)were synthesized by a halform reaction and the DCC/HOBT coupling reaction in a simple and fast way without amino protection.By using calf thymus DNA,the interaction between the analogs and DNA duplex was studied by CD, and ITC.

  17. Synthesis of trimethoprim metal complexes: Spectral, electrochemical, thermal, DNA-binding and surface morphology studies.

    Science.gov (United States)

    Demirezen, Nihat; Tarınç, Derya; Polat, Duygu; Ceşme, Mustafa; Gölcü, Ayşegül; Tümer, Mehmet

    2012-08-01

    Complexes of trimethoprim (TMP), with Cu(II), Zn(II), Pt(II), Ru(III) and Fe(III) have been synthesized. Then, these complexes have been characterized by spectroscopic techniques involving UV-vis, IR, mass and (1)H NMR. CHN elemental analysis, electrochemical and thermal behavior of complexes have also been investigated. The electrochemical properties of all complexes have been investigated by cyclic voltammetry (CV) using glassy carbon electrode. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV spectroscopy and cyclic voltammetry. UV studies of the interaction of the complexes with DNA have shown that these compounds can bind to CT DNA. The binding constants of the complexes with CT DNA have also been calculated. The cyclic voltammograms of the complexes in the presence of CT DNA have shown that the complexes can bind to CT DNA by both the intercalative and the electrostatic binding mode. The antimicrobial activity of these complexes has been evaluated against three Gram-positive and four Gram-negative bacteria. Antifungal activity against two different fungi has been evaluated and compared with the reference drug TMP. Almost all types of complexes show excellent activity against all type of bacteria and fungi. The morphology of the CT DNA, TMP, metal ions and metal complexes has been investigated by scanning electron microscopy (SEM). To get the SEM images, the interaction of compounds with CT DNA has been studied by means of differential pulse voltammetry (DPV) at CT DNA modified pencil graphite electrode (PGE). The decrease in intensity of the guanine oxidation signals has been used as an indicator for the interaction mechanism.

  18. 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.

  19. 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.

  20. In vitro tuberization of Chlorophytum Borivilianum Sant & Fern (Safed musli) as influenced by sucrose, CCC and culture systems.

    Science.gov (United States)

    Farshad Ashraf, Mehdi; Abd Aziz, Maheran; Abdul Kadir, Mihdzar; Stanslas, Johnson; Farokhian, Elmira

    2013-08-01

    This study focuses on the establishment of in vitro tuberization of Chlorophytum borivilianum using solid and liquid culture systems. A high in vitro tuberization rate on solid and stationary liquid Murashige and Skoog media was observed in the presence of 60 g l⁻¹ sucrose with 950, 1,265 and 1,580 µM 2-chloroethyl-trimethylammonium chloride (CCC). Application of a higher sucrose concentration of 90 g l⁻¹ showed a negative interaction with CCC on in vitro tuber number and days to in vitro tuber induction. For economic feasibility, 950 µM CCC with 60 g l⁻¹ sucrose was chosen as the best combination for in vitro tuberization in both solid and stationary liquid media. For optimization of in vitro tuber production,a comparison between solid, stationary liquid and shake liquid culture was carried out. Liquid culture with shaking at 80 r.p.m. resulted in a >2.5-fold increase in in vitro tuber production compared with solid culture.

  1. 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

  2. Gas-phase synthesis of solid state DNA nanoparticles stabilized by l-leucine.

    Science.gov (United States)

    Raula, Janne; Hanzlíková, Martina; Rahikkala, Antti; Hautala, Juho; Kauppinen, Esko I; Urtti, Arto; Yliperttula, Marjo

    2013-02-28

    Aerosol flow reactor is used to generate solid-state nanoparticles in a one-step process that is based on drying of aerosol droplets in continuous flow. We investigated the applicability of aerosol flow reactor method to prepare solid state DNA nanoparticles. Precursor solutions of plasmid DNA with or without complexing agent (polyethylenimine), coating material (l-leucine) and mannitol (bulking material) were dispersed to nanosized droplets and instantly dried in laminar heat flow. Particle morphology, integrity and stability were studied by scanning electron microscopy. The stability of DNA was studied by gel electrophoresis. Plasmid DNA as such degraded in the aerosol flow process. Complexing agent protected DNA from degradation and coating material enabled production of dispersed, non-aggregated, nanoparticles. The resulting nanoparticles were spherical and their mean diameter ranged from 65 to 125nm. The nanoparticles were structurally stable at room temperature and their DNA content was about 10%. We present herein the proof of principle for the production of dispersed solid state nanoparticles with relevant size and intact plasmid DNA.

  3. 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.

  4. Mouse polyoma virus and adenovirus replication in mouse cells temperature-sensitive in DNA synthesis.

    Science.gov (United States)

    Sheinin, R; Fabbro, J; Dubsky, M

    1985-01-01

    Mouse adenovirus multiplies, apparently without impediment, in temperature-inactivated ts A1S9, tsC1 and ts2 mouse fibroblasts. Thus, the DNA of mouse adenovirus can replicate in the absence of functional DNA topoisomerase II, a DNA-chain-elongation factor, and a protein required for traverse of the G1/S interface, respectively, encoded in the ts A1S9, tsC1 and ts2 genetic loci. These results are compared with those obtained with polyoma virus.

  5. Synthesis of copper nanoparticles by electrolysis of DNA utilizing copper as sacrificial anode.

    Science.gov (United States)

    Singh, Dinesh Pratap; Srivastava, Onkar Nath

    2007-06-01

    Copper nanoparticles have been synthesized by anodic oxidation through a simple electrolysis process employing de-oxy ribonucleic acid (DNA) as electrolyte. Platinum was taken as cathode and copper as anode. The applied voltage was 4 V and the electrolysis was performed for duration of 1 h. The copper nanoparticles were prepared in situ from the electron beam irradiation on residues of electrolyte consisting of DNA and copper particles: DNA (Cu) complexes. The size of the nanoparticles ranges between 5-50 nm. A tentative explanation has been given for the formation of copper nanoparticles.

  6. Structurally Diverse Polyamines: Solid-Phase Synthesis and Interaction with DNA.

    Science.gov (United States)

    Umezawa, Naoki; Horai, Yuhei; Imamura, Yuki; Kawakubo, Makoto; Nakahira, Mariko; Kato, Nobuki; Muramatsu, Akira; Yoshikawa, Yuko; Yoshikawa, Kenichi; Higuchi, Tsunehiko

    2015-08-17

    A versatile solid-phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher-order structures of DNA.

  7. 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...

  8. 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 ...... in HL-60 and K-562 cells was measured simultaneous with CD13 expression....

  9. 8-Methoxypsoralen DNA interstrand cross-linking of the ribosomal RNA genes in Tetrahymena thermophila. Distribution, repair and effect on rRNA synthesis

    DEFF Research Database (Denmark)

    Fengquin, X; Nielsen, Henrik; Zhen, W;

    1993-01-01

    between three domains (terminal spacer, transcribed region and central spacer) as defined by restriction enzyme analysis (BamHI and ClaI). It is furthermore shown that a dosage resulting in approximately one cross-link per rDNA molecule (21 kbp, two genes) is sufficient to block RNA synthesis. Finally...

  10. DNA Synthesis during Endomitosis Is Stimulated by Insulin via the PI3K/Akt and TOR Signaling Pathways in the Silk Gland Cells of Bombyx mori

    Directory of Open Access Journals (Sweden)

    Yaofeng Li

    2015-03-01

    Full Text Available Silk gland cells undergo multiple endomitotic cell cycles during silkworm larval ontogeny. Our previous study demonstrated that feeding is required for continued endomitosis in the silk gland cells of silkworm larvae. Furthermore, the insulin signaling pathway is closely related to nutritional signals. To investigate whether the insulin signaling pathway is involved in endomitosis in silk gland cells, in this study, we initially analyzed the effects of bovine insulin on DNA synthesis in endomitotic silk gland cells using 5-bromo-2'-deoxyuridine (BrdU labeling technology, and found that bovine insulin can stimulate DNA synthesis. Insulin signal transduction is mainly mediated via phosphoinositide 3-kinase (PI3K/Akt, the target of rapamycin (TOR and the extracellular signal-regulated kinase (ERK pathways in vertebrates. We ascertained that these three pathways are involved in DNA synthesis in endomitotic silk gland cells using specific inhibitors against each pathway. Moreover, we investigated whether these three pathways are involved in insulin-stimulated DNA synthesis in endomitotic silk gland cells, and found that the PI3K/Akt and TOR pathways, but not the ERK pathway, are involved in this process. These results provide an important theoretical foundation for the further investigations of the mechanism underlying efficient endomitosis in silk gland cells.

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

    NARCIS (Netherlands)

    A.J.R. de Jonge; W. Vermeulen (Wim); W. Keijzer; J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1985-01-01

    textabstractThe UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of

  12. One-pot synthesis of dibenzo[b,h][1,6]naphthyridines from 2-acetylaminobenzaldehyde: application to a fluorescent DNA-binding compound.

    Science.gov (United States)

    Okuma, Kentaro; Koga, Tomohiro; Ozaki, Saori; Suzuki, Yutaro; Horigami, Kenta; Nagahora, Noriyoshi; Shioji, Kosei; Fukuda, Masatora; Deshimaru, Masanobu

    2014-12-21

    Dibenzo[b,h][1,6]naphthyridines were synthesized in one pot by reacting 2-acetylaminobenzaldehyde with methyl ketones under basic conditions via four sequential condensation reactions. This method was also applied to the synthesis of 1,2-dihydroquinolines. 6-Methyl-1,6-dibenzonaphthyridinium triflates showed strong fluorescence, and the fluorescence intensities were changed upon intercalation into double-stranded DNA.

  13. Jak1/STAT3 pathway mediates the inhibition of lipoxin A4 on TNF-α-induced DNA synthesis of glomerular mesangial cells in rats

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective: To examine whether lipoxin A4 (LXA4) has an inhibitory effect on tumor necrosis factor-α(TNF-α)-induced DNA synthesis of glomerular mesangial cells of rat, and explore the molecular mechanisms of LXA4 action. Methods: Glomerular mesangial cells of rat were cultured and preincubated with LXA4 at different concentrations, and then treated with TNF-α( 10 ng/ml). DNA synthesis was assessed by the incorporation of [3H]-thymidine in mesangial cells. Expression of cyclin E protein was determined by Western blotting analysis. Activities of signal transducers and activators of transcription-3 (STAT3) were analyzed by electrophoretic mobility shift assay (EMSA). Results: TNF-α-stimulated DNA synthesis of mesangial cells, upregulation of cyclin E protein and STAT3 activities were inhibited by LXA4 in a dose-dependent manner. Conclusion: TNF-α-induced DNA synthesis of mesangial cells can be inhibited by TXA4probably through the mechanism of Jak1/STAT3 pathway-dependent signal transduction.

  14. Cell division and subsequent radicle protrusion in tomato seeds are inhibited by osmotic stress but DNA synthesis and formation of microtubular cytoskeleton are not

    NARCIS (Netherlands)

    Castro, de R.D.; Lammeren, van A.A.M.; Groot, S.P.C.; Bino, R.J.; Hilhorst, H.W.M.

    2000-01-01

    We studied cell cycle events in embryos of tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds during imbibition in water and during osmoconditioning ("priming") using both quantitative and cytological analysis of DNA synthesis and -tubulin accumulation. Most embryonic nuclei of dry, untreate

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

    Science.gov (United States)

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

    2014-01-01

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

  16. Synthesis of dihydromyricetin-manganese (II) complex and interaction with DNA

    Science.gov (United States)

    Guo, Qingquan; Yuan, Juan; Zeng, Jinhua; He, Xiangzhu; Li, Daguang

    2012-11-01

    Dihydromyricetin has many physiological functions and its metal complex could have better effects. DNA is very important in biological body, but little attention has been devoted to the relationship between dihydromyricetin-metal complex and the DNA. In this paper, dihydromyricetin-Mn (II) complex has been prepared and characterized using UV-vis absorption spectrophotometry, IR spectroscopy, elemental analysis, and thermal gravimetric analysis (TG-DTA Analysis). The interaction of dihydromyricetin-Mn (II) complex with DNA was investigated using UV-vis spectra, fluorescence measurements and viscosity measurements. The results indicate that the dihydromyricetin-manganese (II) complex can intercalate into the stacked base pairs of DNA with binding constant Kb = 5.64 × 104 M and compete with the strong intercalator ethidium bromide for the intercalative binding sites with Stern-Volmer quenching constant, Ksq = 1.16.

  17. Synthesis, characterization, thermal and DNA-binding properties of new zinc complexes with 2-hydroxyphenones.

    Science.gov (United States)

    Mrkalić, Emina; Zianna, Ariadni; Psomas, George; Gdaniec, Maria; Czapik, Agnieszka; Coutouli-Argyropoulou, Evdoxia; Lalia-Kantouri, Maria

    2014-05-01

    The neutral mononuclear zinc complexes with 2-hydroxyphenones (ketoH) having the formula [Zn(keto)2(H2O)2] and [Zn(keto)2(enR)], where enR stands for a N,N'-donor heterocyclic ligand such as 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen) or 2,2'-dipyridylamine (dpamH), have been synthesized and characterized by IR, UV and (1)H NMR spectroscopies. The 2-hydroxyphenones are chelated to the metal ion through the phenolate and carbonyl oxygen atoms. The crystal structures of [bis(2-hydroxy-4-methoxy-benzophenone)(2,2'-bipyridine)zinc(II)] dimethanol solvate and [bis(2-hydroxy-benzophenone)(2,2'-bipyridine)zinc(II)] dimethanol solvate have been determined by X-ray crystallography. The thermal stability of the zinc complexes has been investigated by simultaneous TG/DTG-DTA technique. The ability of the complexes to bind to calf-thymus DNA (CT DNA) has been studied by UV-absorption and fluorescence emission spectroscopy as well as viscosity measurements. UV studies of the interaction of the complexes with DNA have shown that they can bind to CT DNA and the corresponding binding constants to DNA have been calculated and evaluated. The complexes most probably bind to CT DNA via intercalation as concluded by studying the viscosity of a DNA solution in the presence of the complexes. Competitive studies with ethidium bromide (EB) have shown that the reported complexes can displace the DNA-bound EB, suggesting strong competition with EB for the intercalation site.

  18. Recent developments in the chemistry of deoxyribonucleic acid (DNA) intercalators: principles, design, synthesis, applications and trends.

    Science.gov (United States)

    Neto, Brenno A D; Lapis, Alexandre A M

    2009-05-07

    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.

  19. Design, synthesis, physicochemical studies, solvation, and DNA damage of quinoline-appended chalcone derivative: comprehensive spectroscopic approach toward drug discovery.

    Science.gov (United States)

    Kumar, Himank; Chattopadhyay, Anjan; Prasath, R; Devaraji, Vinod; Joshi, Ritika; Bhavana, P; Saini, Praveen; Ghosh, Sujit Kumar

    2014-07-01

    The present study epitomizes the design, synthesis, photophysics, solvation, and interaction with calf-thymus DNA of a potential antitumor, anticancer quinoline-appended chalcone derivative, (E)-3-(anthracen-10-yl)-1-(6,8-dibromo-2-methylquinolin-3-yl)prop-2-en-1-one (ADMQ) using steady state absorption and fluorescence spectroscopy, molecular modeling, molecular docking, Fourier-transform infrared spectroscopy (FTIR), molecular dynamics (MD) simulation, and gel electrophoresis studies. ADMQ shows an unusual photophysical behavior in a variety of solvents of different polarity. The dual emission has been observed along with the formation of twisted intramolecular charge transfer (TICT) excited state. The radiationless deactivation of the TICT state is found to be promoted strongly by hydrogen bonding. Quantum mechanical (DFT, TDDFT, and ZINDO-CI) calculations show that the ADMQ is sort of molecular rotor which undergoes intramolecular twist followed by a complete charge transfer in the optimized excited state. FTIR studies reveals that ADMQ undergoes important structural change from its native structure to a β-hydroxy keto form in water at physiological pH. The concentration-dependent DNA cleavage has been identified in agarose gel DNA electrophoresis experiment and has been further supported by MD simulation. ADMQ forms hydrogen bond with the deoxyribose sugar attached with the nucleobase adenine DA-17 (chain A) and result in significant structural changes which potentially cleave DNA double helix. The compound does not exhibit any deleterious effect or toxicity to the E. coli strain in cytotoxicity studies. The consolidated spectroscopic research described herein can provide enormous information to open up new avenues for designing and synthesizing chalcone derivatives with low systematic toxicity for medicinal chemistry research.

  20. Synthesis, structure, DNA binding and cleavage activity of a new copper(Ⅱ) complex of bispyridylpyrrolide

    Institute of Scientific and Technical Information of China (English)

    MIN Rui; HU Xiao-hui; YI Xiao-yi; ZHANG Shou-chun

    2015-01-01

    A copper-bispyridylpyrrolide complex [Cu(PDPH)Cl] (PDPH = 2,5-bis(2′-pyridyl)pyrrole) was synthesized and characterized. The complex crystallizes in the orthorhombic system with space groupPccn,a = 0.9016(3) nm,b = 1.0931(4) nm,c = 2.5319(8) nm, andV = 2.4951(15) nm3. The copper center is situated in a square planar geometry. The interaction of the copper(Ⅱ) complexwith calf thymus DNA (CT-DNA) was investigated by electronic absorption, circular dichroism (CD) and fluorescence spectra. It is proposed that the complex binds to CT-DNA through groove binding mode. Nuclease activity of the complex was also studied by gel electrophoresis method. The complex can efficiently cleave supercoiled pBR322 DNA in the presence of ascorbate (H2A) via oxidative pathway. The preliminary mechanism of DNA cleavage by the complex with different inhibiting reagents indicates that the hydroxyl radicals were involved as the active species in the DNA cleavage process.

  1. 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.

  2. 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

  3. Simulated Screens of DNA Encoded Libraries: The Potential Influence of Chemical Synthesis Fidelity on Interpretation of Structure-Activity Relationships.

    Science.gov (United States)

    Satz, Alexander L

    2016-07-11

    Simulated screening of DNA encoded libraries indicates that the presence of truncated byproducts complicates the relationship between library member enrichment and equilibrium association constant (these truncates result from incomplete chemical reactions during library synthesis). Further, simulations indicate that some patterns observed in reported experimental data may result from the presence of truncated byproducts in the library mixture and not structure-activity relationships. Potential experimental methods of minimizing the presence of truncates are assessed via simulation; the relationship between enrichment and equilibrium association constant for libraries of differing purities is investigated. Data aggregation techniques are demonstrated that allow for more accurate analysis of screening results, in particular when the screened library contains significant quantities of truncates.

  4. Organometallic B12-DNA conjugate: synthesis, structure analysis, and studies of binding to human B12-transporter proteins.

    Science.gov (United States)

    Hunger, Miriam; Mutti, Elena; Rieder, Alexander; Enders, Barbara; Nexo, Ebba; Kräutler, Bernhard

    2014-10-06

    Design, synthesis, and structural characterization of a B12-octadecanucleotide are presented herein, a new organometallic B12-DNA conjugate. In such covalent conjugates, the natural B12 moiety may be a versatile vector for controlled in vivo delivery of oligonucleotides to cellular targets in humans and animals, through the endogenous B12 transport systems. Binding of the organometallic B12 octadecanucleotide to the three important human proteins of B12 transport was studied, to examine its structural suitability for the task of eventual in vivo oligonucleotide delivery. Binding was efficient with transcobalamin (TC), but not so efficient with the homologous glycoproteins intrinsic factor and haptocorrin. Binding of the B12 octadecanucleotide to TC suggests the capacity of the B12 moiety to serve as a natural vector for specific transport of single stranded, organometallic oligonucleotide loads from the blood stream into cells.

  5. Further studies on the ability of different metal salts to influence the DNA synthesis of human lymphoid cells.

    Science.gov (United States)

    Nordlind, K

    1986-01-01

    In a further study on the ability of different metal salts to influence the DNA synthesis of human lymphoid cells, aluminum chloride, beryllium chloride, cadmium chloride, cupric sulfate, ferric chloride, manganese chloride, palladium chloride, platinum chloride and silver nitrate, were tested regarding effect on thymocytes and peripheral blood lymphocytes in children. At certain concentrations in the range of 10(-4)-10(-5)M, all tested compounds but aluminum chloride and ferric chloride, were inhibitory, the latter compounds inhibited at 4.8 X 10(-3)M. A slight stimulation mainly on the thymocytes was obtained with beryllium chloride, cadmium chloride, palladium chloride, platinum chloride and silver nitrate, at certain concentrations in the range of 10(-5)-10(-6)M, while ferric chloride gave a slight stimulation at 1.2 X 10(-3)M. Thus, the tested metal salts should be suitable for use in lymphocyte transformation tests for diagnosis of contact allergy.

  6. Purification of a Factor from Human Placenta That Stimulates Capillary Endothelial Cell Protease Production, DNA Synthesis, and Migration

    Science.gov (United States)

    Moscatelli, David; Presta, Marco; Rifkin, Daniel B.

    1986-04-01

    A protein that stimulates the production of plasminogen activator and latent collagenase in cultured bovine capillary endothelial cells has been purified 106-fold from term human placenta by using a combination of heparin affinity chromatography, ion-exchange chromatography, and gel chromatography. The purified molecule has a molecular weight of 18,700 as determined by NaDodSO4/PAGE under both reducing and nonreducing conditions. The purified molecule stimulates the production of plasminogen activator and latent collagenase in a dose-dependent manner between 0.1 and 10 ng of protein/ml. The purified protein also stimulates DNA synthesis and chemotaxis in capillary endothelial cells in the same concentration range. Thus, this molecule has all of the properties predicted for an angiogenic factor.

  7. A microenvironment-sensitive fluorescent pyrimidine ribonucleoside analogue: synthesis, enzymatic incorporation, and fluorescence detection of a DNA abasic site.

    Science.gov (United States)

    Tanpure, Arun A; Srivatsan, Seergazhi G

    2011-11-04

    Base-modified fluorescent ribonucleoside-analogue probes are valuable tools in monitoring RNA structure and function because they closely resemble the structure of natural nucleobases. Especially, 2-aminopurine, a highly environment-sensitive adenosine analogue, is the most extensively utilized fluorescent nucleoside analogue. However, only a few isosteric pyrimidine ribonucleoside analogues that are suitable for probing the structure and recognition properties of RNA molecules are available. Herein, we describe the synthesis and photophysical characterization of a small series of base-modified pyrimidine ribonucleoside analogues derived from tagging indole, N-methylindole, and benzofuran onto the 5-position of uracil. One of the analogues, based on a 5-(benzofuran-2-yl)pyrimidine core, shows emission in the visible region with a reasonable quantum yield and, importantly, displays excellent solvatochromism. The corresponding triphosphate substrate is effectively incorporated into oligoribonucleotides by T7 RNA polymerase to produce fluorescent oligoribonucleotide constructs. Steady-state and time-resolved spectroscopic studies with fluorescent oligoribonucleotide constructs demonstrate that the fluorescent ribonucleoside photophysically responds to subtle changes in its environment brought about by the interaction of the chromophore with neighboring bases. In particular, the emissive ribonucleoside, if incorporated into an oligoribonucleotide, positively reports the presence of a DNA abasic site with an appreciable enhancement in fluorescence intensity. The straightforward synthesis, amicability to enzymatic incorporation, and sensitivity to changes in the microenvironment highlight the potential of the benzofuran-conjugated pyrimidine ribonucleoside as an efficient fluorescent probe to investigate nucleic acid structure, dynamics, and recognition events.

  8. 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.

  9. 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

    2016-06-02

    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.

  10. Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells

    Science.gov (United States)

    Quinet, Annabel; Martins, Davi Jardim; Vessoni, Alexandre Teixeira; Biard, Denis; Sarasin, Alain; Stary, Anne; Menck, Carlos Frederico Martins

    2016-01-01

    Ultraviolet-induced 6-4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD) can be tolerated by translesion DNA polymerases (TLS Pols) at stalled replication forks or by gap-filling. Here, we investigated the involvement of Polη, Rev1 and Rev3L (Polζ catalytic subunit) in the specific bypass of 6-4PP and CPD in repair-deficient XP-C human cells. We combined DNA fiber assay and novel methodologies for detection and quantification of single-stranded DNA (ssDNA) gaps on ongoing replication forks and postreplication repair (PRR) tracts in the human genome. We demonstrated that Rev3L, but not Rev1, is required for postreplicative gap-filling, while Polη and Rev1 are responsible for TLS at stalled replication forks. Moreover, specific photolyases were employed to show that in XP-C cells, CPD arrest replication forks, while 6-4PP are responsible for the generation of ssDNA gaps and PRR tracts. On the other hand, in the absence of Polη or Rev1, both types of lesion block replication forks progression. Altogether, the data directly show that, in the human genome, Polη and Rev1 bypass CPD and 6-4PP at replication forks, while only 6-4PP are also tolerated by a Polζ-dependent gap-filling mechanism, independent of S phase. PMID:27095204

  11. Chitosan Microparticles Intended for Anti-caries DNA Vaccine Mucosal Delivery: Synthesis, Characterization and Transfection

    Institute of Scientific and Technical Information of China (English)

    LI Yuhong; FAN Mingwen; BIAN Zhuan; CHEN Zhi; Zhang Qi

    2005-01-01

    In order to enhance the mucosal immunity of anti-caries DNA vaccine, chitosan-DNA microparticles for musocal vaccination were prepared by a coacervation method. The physicochemical structure of microparticles was investigated by a scanning electron microscope (SEM) and a cofocal laser scanning microscope (CLSM). For in-vitro studies, Hela cell was transfected by chitosan-DNA microparticles.The expression of proteins was measured by the immunohistochemical methods, and the cytotocity of chitosan in Hela cell line was determined by the MTT assay. The experimental results show that the microparticles are about 2-6 μm in size and spherical in shape. The encapsulation efficiency is 99%, and the DNA is almost captured in the micropraticles. Plasmid loaded into chitosan microparticles is distributed throughout these particles. The number of positive staining cells of chitosan-pGJA-P transfected cell is more than that of naked plasmid transfect cells, but less than that of Lipofect-DNA complex group. Chitosan was found to be less cytotoxic compared with lipofectin (p<0.01).

  12. 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.

  13. 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.

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

    Science.gov (United States)

    Sukri, Shahratul Ain Mohd; Heng, Lee Yook; Karim, Nurul Huda Abd

    2014-09-01

    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.

  15. 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.

  16. 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)

  17. Gelatin-based nanoparticles as DNA delivery systems: Synthesis, physicochemical and biocompatible characterization.

    Science.gov (United States)

    Morán, M C; Rosell, N; Ruano, G; Busquets, M A; Vinardell, M P

    2015-10-01

    The rapidly rising demand for therapeutic grade DNA molecules requires associated improvements in encapsulation and delivery technologies. One of the challenges for the efficient intracellular delivery of therapeutic biomolecules after their cell internalization by endocytosis is to manipulate the non-productive trafficking from endosomes to lysosomes, where degradation may occur. The combination of the endosomal acidity with the endosomolytic capability of the nanocarrier can increase the intracellular delivery of many drugs, genes and proteins, which, therefore, might enhance their therapeutic efficacy. Among the suitable compounds, the gelification properties of gelatin as well as the strong dependence of gelatin ionization with pH makes this compound an interesting candidate to be used to the effective intracellular delivery of active biomacromolecules. In the present work, gelatin (either high or low gel strength) and protamine sulfate has been selected to form particles by interaction of oppositely charged compounds. Particles in the absence of DNA (binary system) and in the presence of DNA (ternary system) have been prepared. The physicochemical characterization (particle size, polydispersity index and degree of DNA entrapment) have been evaluated. Cytotoxicity experiments have shown that the isolated systems and the resulting gelatin-based nanoparticles are essentially non-toxic. The pH-dependent hemolysis assay and the response of the nanoparticles co-incubated in buffers at defined pHs that mimic extracellular, early endosomal and late endo-lysosomal environments demonstrated that the nanoparticles tend to destabilize and DNA can be successfully released. It was found that, in addition to the imposed compositions, the gel strength of gelatin is a controlling parameter of the final properties of these nanoparticles. The results indicate that these gelatin-based nanoparticles have excellent properties as highly potent and non-toxic intracellular delivery

  18. Synthesis of RNA probes by the direct in vitro transcription of PCR-generated DNA templates.

    Science.gov (United States)

    Urrutia, R; McNiven, M A; Kachar, B

    1993-05-01

    We describe a novel method for the generation of RNA probes based on the direct in vitro transcription of DNA templates amplified by polymerase chain reaction (PCR) using primers with sequence hybrids between the target gene and those of the T7 and T3 RNA polymerases promoters. This method circumvents the need for cloning and allows rapid generation of strand-specific RNA molecules that can be used for the identification of genes in hybridization experiments. We have successfully applied this method to the identification of DNA sequences by Southern blot analysis and library screening.

  19. Synthesis of spin-labeled riboswitch RNAs using convertible nucleosides and DNA-catalyzed RNA ligation.

    Science.gov (United States)

    Büttner, Lea; Seikowski, Jan; Wawrzyniak, Katarzyna; Ochmann, Anne; Höbartner, Claudia

    2013-10-15

    Chemically stable nitroxide radicals that can be monitored by electron paramagnetic resonance (EPR) spectroscopy can provide information on structural and dynamic properties of functional RNA such as riboswitches. The convertible nucleoside approach is used to install 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and 2,2,5,5-tetramethylpyrrolidin-1-oxyl (proxyl) labels at the exocyclic N(4)-amino group of cytidine and 2'-O-methylcytidine nucleotides in RNA. To obtain site-specifically labeled long riboswitch RNAs beyond the limit of solid-phase synthesis, we report the ligation of spin-labeled RNA using an in vitro selected deoxyribozyme as catalyst, and demonstrate the synthesis of TEMPO-labeled 53 nt SAM-III and 118 nt SAM-I riboswitch domains (SAM=S-adenosylmethionine).

  20. Thermolytic 4-methylthio-1-butyl group for phosphate/thiophosphate protection in solid-phase synthesis of DNA oligonucleotides.

    Science.gov (United States)

    Cieślak, Jacek; Grajkowski, Andrzej; Livengood, Victor; Beaucage, Serge L

    2004-04-02

    The thermolabile 4-methylthio-1-butyl phosphate/thiophosphate protecting group for DNA oligonucleotides has been investigated for its potential application to a "heat-driven" process for either oligonucleotide synthesis on diagnostic microarrays or, oppositely, to the large-scale preparation of therapeutic oligonucleotides. The preparation of phosphoramidites 10a-d is straightforward, and the incorporation of these amidites into oligonucleotides via solid-phase techniques proceeds as efficiently as that achieved with 2-cyanoethyl deoxyribonucleoside phosphoramidites. The versatility of the 4-methylthio-1-butyl phosphate/thiophosphate protecting group is exemplified by its facile removal from oligonucleotides upon heating for 30 min at 55 degrees C in an aqueous buffer under neutral conditions or within 2 h at 55 degrees C in concentrated NH(4)OH. The deprotection reaction occurs through an intramolecular cyclodeesterification mechanism leading to the formation of sulfonium salt 18. When mixed with deoxyribonucleosides and N-protected 2'-deoxyribonucleosides or with a model phosphorothioate diester under conditions approximating those of large-scale (>50 mmol) oligonucleotide deprotection reactions, the salt 18 did not significantly alter DNA nucleobases or desulfurize the phosphorothioate diester model to an appreciable extent.

  1. Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA.

    Science.gov (United States)

    Nakamura, Takahiro; Pluskal, Tomáš; Nakaseko, Yukinobu; Yanagida, Mitsuhiro

    2012-09-01

    Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.

  2. Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner.

    Directory of Open Access Journals (Sweden)

    Josef Köck

    Full Text Available Persistence of hepatitis B virus (HBV infection requires covalently closed circular (cccDNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.

  3. Synthesis and spectroscopic DNA binding investigations of dibutyltin N-(5-chlorosalicylidene)-leucinate

    Science.gov (United States)

    Zhang, Shufang; Yuan, Hongyu; Tian, Laijin

    2017-02-01

    A new dibutyltin N-(5-Chlorosalicylidene)-leucinate (DNCL) was synthesized by the reaction of dibutyltin dichloride with in situ formed potassium N-(5-chlorosalicylidene)-L-isoleucinate 3 characterized by elemental analysis, IR, 1H NMR (1H, 13C and 119Sn) spectra. The interaction between DNCL and calf thymus deoxyribonucleic acid (ctDNA) in a pH 7.4 Tris-HCl buffer solution was investigated by UV-Vis spectroscopy, fluorescence spectroscopy, and viscosity measurements. It was found that DNCL molecules could intercalate into the base pairs of DNA, forming a DNCL-DNA complex with a binding constant of Kf = 5.75 × 105 L mol-1 (310 K). The thermodynamic parameters enthalpy change (ΔH), entropy change (ΔS) and Gibbs free energy (ΔG) were calculated to be 1.16 × 105 J mol-1, 486.5 J K-1 mol -1 and -3.48 × 104 J mol-1 at 310 K, respectively. Hydrophobic interaction was the predominant intermolecular force in stabilizing the DNCL-DNA complex.

  4. 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.

  5. Synthesis, characterization, DNA binding, cleavage activity and cytotoxicity of copper(II) complexes.

    Science.gov (United States)

    Li, Mei-Jin; Lan, Tao-Yu; Cao, Xiu-Hui; Yang, Huang-Hao; Shi, Yupeng; Yi, Changqing; Chen, Guo-Nan

    2014-02-21

    Three new mononuclear copper(II) complexes, [Cu(L2)](2+) (1), [Cu(acac)(L)](+) (2), and [Cu(acac-Cl)(L)](+) (3) (L = 2-(4-pyridine)oxazo[4,5-f]1,10-phenanthroline (4-PDOP); acac = acetylacetone; acac-Cl = 3-chloroacetylacetone), have been synthesized and characterized by elemental analysis, high resolution mass spectrometry (Q-TOF), and IR spectroscopy. Two of the complexes were structurally characterized by single-crystal X-ray diffraction techniques. Their interactions with DNA were studied by UV-vis absorption and emission spectra, viscosity, thermal melting, DNA unwinding assay and CD spectroscopy. The nucleolytic cleavage activity of the compounds was carried out on double stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment in the presence and absence of an oxidant (H2O2). Active oxygen intermediates such as hydroxyl radicals and hydrogen peroxide generated in the presence of L and complexes 1-3 may act as active species for the DNA scission. The cytotoxicity of the complexes against HepG2 cancer cells was also studied.

  6. Synthesis and characteristics of biodegradable pyridinium amphiphiles used for in vitro DNA delivery

    NARCIS (Netherlands)

    Roosjen, Astrid; Smisterova, Jarmila; Driessen, Cecile; Anders, Joachim T.; Wagenaar, Anno; Hoekstra, Dirk; Hulst, Ron; Engberts, Jan B.F.N.

    2002-01-01

    Pyridinium amphiphiles have found practical application for the delivery of DNA into eukaryotic cells. A general synthetic method starting from (iso)nicotinoyl chloride has been devised for the preparation of pyridinium amphiphiles based on (bio)degradable esters, allowing structural variation both

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    , and subsequently be interpreted to enable characterization of the aDNA damage behind the observed phenotypes. Through comparative analyses on 390,965 bp of modern chloroplast and 131,474 bp of ancient woolly mammoth GS20 sequence data we conclusively demonstrate that in this sample at least, a permafrost preserved...

  8. 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

  9. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2002-01-01

    AIM: To investigate the effects of growth inhibition ofhuman gastric cancer SGC-7901 cell with RRR-α-tocopherylsuccinate (VES), a derivative of natural Vitamin E, viainducing apoptosis and DNA synthesis arrest.METHODS: Human gastric cancer SGC-7901 cells wereregularly incubated in the presence of VES at 5, 10 and20mg@ L 1(VES was dissolved in absolute ethanol anddiluted in RPMI 1640 complete condition mediacorrespondingly to a final concentration of VES and 1mL@L-1 ethanol), succinic acid and ethanol equivalents asvehicle (VEH) control andcondition media only asuntreated (UT) control. Trypan blue dye exclusionanalysis and MTT assay were applied to detect the cellproliferation. 37kBq of tritiated thymidine was added tocells and [3H] TdR uptake was measured to observe DNAsynthesis. Apoptotic morphology was observed byelectron microscopy and DAPI staining. Flow cytometryand terminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL) assay were performed to detectVES-triggered apoptosis.RESULTS: VES inhibited SGC-7901 cell growth in a dose-dependent manner. The growth curve showed suppressionby 24.7%, 49.2% and 68.7% following 24h of VEStreatment at 5, 10 and 20 mg@L 1, respectively, similar tothe findings from MTT assay. DNA synthesis wasevidently reduced by 35%, 45% and 98% after 24h VEStreatment at 20 mg@ L-1 and 48h at 10 and 20 mg@ L 1,respectively. VES induced SGC-7901 cells to undergoapoptosis with typically apoptotic characteristics,including morphological changes of chromatincondensation, chromatin crescent formation/margination,nucleus fragmentation and apoptotic body formation,typical apoptotic sub-G1 peak by flow cytometry andincrease of apoptotic cells by TUNEL assay in which 90%of cells underwent apoptosis after 48h of VES treatment at20 mcg@L-1.CONCLUSION: VES can inhibit human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesisarrest. Inhibition of SGC-7901 cell growth by VES is dose-and time

  11. 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

  12. (3H)-isoproterenol binding to subcellular fractions of mouse parotid: relationship to cyclic nucleotide formation and the stimulation of DNA synthesis.

    Science.gov (United States)

    Durham, J P; Galanti, N

    1976-12-01

    (3H) Isoproterenol binding to subcellular fractions of mouse parotid: Relationship to cyclic nucleotide formation and the stimulation of DNA synthesis. (Unión the (3H) Isoproterenol a fracciones subcelulares de parótida de ratón y su relacón con la formacón de nucleótidos cíclicos y la estimulación de la síntesis de DNA). Arch. Biol. Med. Exper. 10: 105-114, 1976. Tritiated isoproterenol binds to all subcellular fractions of mouse parotid but 70% of the binding is to the nuclear fraction. Binding to other mouse tissues was less than to the parotid. The patterns of binding did not correlate with the distribution of adenylate cyclase, guanylate cyclase or catechol-O-methyl transferase among the fractions or tissues nor with the extent of response in stimulation of DNA synthesis among the tissues. Inhibition of (3H) Isoproterenol binding to parotid fractions by catecholamine analogs was studied. There was no correlation between their ability to inhibit binding and the ability of the analogs themselves to raise cyclic AMP levels or stimulate DNA synthesis.

  13. 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.

  14. 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.

  15. Synthesis of silver nanoparticlesbyLactobaciluus acidophilus01 strain andevaluation ofitsin vitro genomicDNA toxicity

    Institute of Scientific and Technical Information of China (English)

    S.Karthick Raja Namasivayam; Gnanendra Kumar E; ReepikaR

    2010-01-01

    Silver nanoparticles synthesized by dried biomass of Lactobaciluus acidophilus 01 strain was evaluated against toxicity of genomic DNA isolated from bacteria (E. coli) fungi (Beauveria bassiana) algae (Seenedesmus acutus) and human blood adopting standard condition was discussed in the present study. Synthesized silver nanoparticles were characterized by UV-Vis spectroscopy and SEM. The UV-Vis spectroscopy revealed the formation of silver nanoparticles by yielding the typical silver plasmon absorption maxima at 430 nm and SEM micrograph indicates the uniform spherical particles within the size range of 45~60 nm. The energy dispersive X-ray spectroscopy (EDX) of the nanoparticle confirmed the presence of elemental silver signal as strong peak. The above synthesized silver nanoparticles didn’t cause any toxic effect on all the tested genomic DNA at all tested concentrations which reveals nil genomic nanoparticles induced toxicity.

  16. The Eukaryotic Mismatch Recognition Complexes Track with the Replisome during DNA Synthesis.

    Directory of Open Access Journals (Sweden)

    Joanna E Haye

    2015-12-01

    Full Text Available During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Polε using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip. The Polε signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Polε signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Polε and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutSα/β to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles.

  17. Homodinuclear lanthanide complexes of phenylthiopropionic acid: Synthesis, characterization, cytotoxicity, DNA cleavage, and antimicrobial activity

    Science.gov (United States)

    Shiju, C.; Arish, D.; Kumaresan, S.

    2013-03-01

    Lanthanide complexes of La(III), Pr(III), Nd(III), Sm(III), and Ho(III) with phenylthiopropionic acid were synthesized and characterized by elemental analysis, mass, IR, electronic spectra, molar conductance, TGA, and powder XRD. The results show that the lanthanide complexes are homodinuclear in nature. The two lanthanide ions are bridged by eight oxygen atoms from four carboxylate groups. Thermal decomposition profiles are consistent with the proposed formulations. Powder XRD studies show that all the complexes are amorphous in nature. Antimicrobial studies indicate that these complexes exhibit more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on Escherichia coli DNA using gel electrophoresis in the presence of H2O2. The result shows that the Pr(III) and Nd(III) complexes have completely cleaved the DNA. The anticancer activities of the complexes have also been studied towards human cervical cancer cell line (HeLa) and colon cancer cells (HCT116) and it was found that the La(III) and Nd(III) complexes are more active than the corresponding Pr(III), Sm(III), Ho(III) complexes, and the free ligand on both the cancer cells.

  18. Homodinuclear lanthanide complexes of phenylthiopropionic acid: synthesis, characterization, cytotoxicity, DNA cleavage, and antimicrobial activity.

    Science.gov (United States)

    Shiju, C; Arish, D; Kumaresan, S

    2013-03-15

    Lanthanide complexes of La(III), Pr(III), Nd(III), Sm(III), and Ho(III) with phenylthiopropionic acid were synthesized and characterized by elemental analysis, mass, IR, electronic spectra, molar conductance, TGA, and powder XRD. The results show that the lanthanide complexes are homodinuclear in nature. The two lanthanide ions are bridged by eight oxygen atoms from four carboxylate groups. Thermal decomposition profiles are consistent with the proposed formulations. Powder XRD studies show that all the complexes are amorphous in nature. Antimicrobial studies indicate that these complexes exhibit more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on Escherichia coli DNA using gel electrophoresis in the presence of H(2)O(2). The result shows that the Pr(III) and Nd(III) complexes have completely cleaved the DNA. The anticancer activities of the complexes have also been studied towards human cervical cancer cell line (HeLa) and colon cancer cells (HCT116) and it was found that the La(III) and Nd(III) complexes are more active than the corresponding Pr(III), Sm(III), Ho(III) complexes, and the free ligand on both the cancer cells.

  19. Synthesis, characterization, anti-microbial, DNA binding and cleavage studies of Schiff base metal complexes

    Directory of Open Access Journals (Sweden)

    Poomalai Jayaseelan

    2016-09-01

    Full Text Available A novel Schiff base ligand has been prepared by the condensation between butanedione monoxime with 3,3′-diaminobenzidine. The ligand and metal complexes have been characterized by elemental analysis, UV, IR, 1H NMR, conductivity measurements, EPR and magnetic studies. The molar conductance studies of Cu(II, Ni(II, Co(II and Mn(II complexes showed non-electrolyte in nature. The ligand acts as dibasic with two N4-tetradentate sites and can coordinate with two metal ions to form binuclear complexes. The spectroscopic data of metal complexes indicated that the metal ions are complexed with azomethine nitrogen and oxyimino nitrogen atoms. The binuclear metal complexes exhibit octahedral arrangements. DNA binding properties of copper(II metal complex have been investigated by electronic absorption spectroscopy. Results suggest that the copper(II complex bind to DNA via an intercalation binding mode. The nucleolytic cleavage activities of the ligand and their complexes were assayed on CT-DNA using gel electrophoresis in the presence and absence of H2O2. The ligand showed increased nuclease activity when administered as copper complex and copper(II complex behave as efficient chemical nucleases with hydrogen peroxide activation. The anti-microbial activities and thermal studies have also been studied. In anti-microbial activity all complexes showed good anti-microbial activity higher than ligand against gram positive, gram negative bacteria and fungi.

  20. Synthesis, characterization, cytotoxicity, DNA cleavage and antimicrobial activity of homodinuclear lanthanide complexes of phenylthioacetic acid

    Institute of Scientific and Technical Information of China (English)

    T. F. Abbs Fen Reji; A. Jeena Pearl; Bojaxa A. Rosy

    2013-01-01

    Lanthanide complexes of Eu(III), Gd(III), Nd(III), Sm(III), and Tb(III) with phenylthioacetic acid were synthesized and characterized by elemental analysis, mass, infrared radiation (IR), electronic spectra, molar conductance, thermogravimetric analysis (TGA), and powder X-ray diffraction (XRD). The results showed that the lanthanide complexes were homodinuclear in nature. The two lanthanide ions were bridged by eight oxygen atoms from four carboxylate groups. Thermal decomposition profiles were consis-tent with the proposed formulations. Powder XRD studies showed that all the complexes were amorphous in nature. Antimicrobial studies indicated that these complexes exhibited more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on CT DNA using gel electrophoresis in the presence of H2O2. The result showed that the Eu(III) and Nd(III) complexes completely cleaved the DNA. The anticancer activities of the complexes were also studied towards human cervical cancer cell line (HeLa) and colon cancer cells (HCT116) and it was found that the Eu(III) and Nd(III) complexes were more active than the corresponding Gd(III), Sm(III), Tb(III) complexes and the free ligand on both the cancer cells.

  1. Regiospecific Photocyclization of Mono- and Bis-Styryl-Substituted N-Heterocycles: A Synthesis of DNA-Binding Benzo[c]quinolizinium Derivatives.

    Science.gov (United States)

    Aliyeu, Tseimur M; Berdnikova, Daria V; Fedorova, Olga A; Gulakova, Elena N; Stremmel, Christopher; Ihmels, Heiko

    2016-10-07

    Regiospecific C-N photocyclization of mono- and bis-styryl-substituted N-heterocycles was investigated. We demonstrated that the C-N regiospecificity of the photoinduced electrocyclization is a general feature of ortho-styryl-substituted N-heterocycles comprising one and two nitrogen atoms. This phototransformation provides a straightforward synthesis of the pharmaceutically important benzo[c]quinolizinium cation and its aza-analogues. Noticeably, bis-styryl derivatives undergo only one-fold cyclization with the second styryl fragment remaining uninvolved in the cyclization process. Photocyclization products of monostyryl derivativatives intercalate into calf thymus DNA (ct DNA), whereas photocyclization products of bis-styryl derivativatives possess a mixed binding mechanism with ct DNA. The results can be used for development of novel DNA-targeting chemotherapeutics based on benzo[c]quinolizinium derivatives.

  2. 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.

  3. Systematic evaluation and optimization of modification reactions of oligonucleotides with amines and carboxylic acids for the synthesis of DNA-encoded chemical libraries.

    Science.gov (United States)

    Franzini, Raphael M; Samain, Florent; Abd Elrahman, Maaly; Mikutis, Gediminas; Nauer, Angela; Zimmermann, Mauro; Scheuermann, Jörg; Hall, Jonathan; Neri, Dario

    2014-08-20

    DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.

  4. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes.

    Science.gov (United States)

    Astakhova, I Kira; Wengel, Jesper

    2013-01-14

    Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2'-O-propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy-transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40-110 nm), quenched fluorescence of single-stranded probes accompanied by up to 7.7-fold light-up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single-nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5 nM).

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

    Energy Technology Data Exchange (ETDEWEB)

    Roza, L.; Vermeulen, W.; Bergen Henegouwen, J.B.; Eker, A.P.; Jaspers, N.G.; Lohman, P.H.; Hoeijmakers, J.H. (TNO Medical Biological Laboratory, Rijswijk (Netherlands))

    1990-03-15

    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.

  6. Regulatory substances produced by lymphocytes. V. Production of inhibitor of DNA synthesis (IDS) by proliferating T lymphocytes.

    Science.gov (United States)

    Namba, Y; Jegasothy, B V; Waksman, B H

    1977-04-01

    The conditions neccessary for production of inhibitor of DNA synthesis (IDS) by rat lymphocytes were investigated. In concanavalin A (Con A)-stimulated lymph node cell (LNC) cultures, IDS production was not detected in the culture supernatant during the first 24 hr, and it increased gradually after that to reach a maximum at 3 to 4 days. When the cells were pretreated with mitomycin C, IDS was not produced, suggesting that DNA synthesis of LNC or a LNC subpopulation is necessary for IDS production. In contrast, Con A-stimulated spleen cells priduced a high level of IDS within 24 hr, and its production fell off sharply thereafter. Con A-stimulated rat thymocytes also produced IDS reaching a maximum at 2 to 3 dyas. However, thymus cells from rats treated with hydrocortisone 48 hr previously did not produce IDS. This finding implies that cortisol-sensitive (cortical) thymocytes are capable of producing IDS and cortisol-resistant (medullary) thymocytes are not. IDS production by lymphoblasts was proportional to cell number and unaffected eith by cell density (1 to 10 x 106/ml) or by the concomitant presence of normal cells from spleen, lymph node, or thymus. Thus Con A-stimulated cells, after becoming blasts, appear to produce IDS automatically wihtout affecting or being affected by other cells. Both spleen and thymus cells from rats injected with a large dose of antigen (ovalbumin, 100 mg, i.p.) 24 hr in advance produced substantial amounts of IDS in culture within 24 hr in the absence of mitogen or additional antigen, but not the cells from rats injected with an immunizing dose (1 mg) of the same antigen. The cells producing IDS in the spleen were shown to be adherent to glass wool, and those in the thymus were partially so. IDS production by antigen-stimulated spleen cells was abrogated by injecting rats with bromodexyuridine (BUdR) at 0 and 12 hr after the ovalbumin. These findings suggest that a subpopulation ofadherent spleen cells (possibly resembling cortical

  7. 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.

  8. Polyurethane Molecular Stamps for the in situ Synthesis of DNA Microarray

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Fabrication of polyurethane molecular stamps (PU stamps) based on polypropylene glycol (PPG) and toluene diisocyanate (TDI), using 3, 3(-dichloro-4, 4(-methylenedianiline (MOCA) as the crosslinker, is reported. It was shown from the contact angle measurement that PU stamps surface has good affinity with acetonitrile, guaranteeing the well distribution of DNA monomers on patterned stamps. Laser confocal fluorescence microscopy images of oligonucleotide arrays after hybridization confirmed polyurethane is an excellent material for molecular stamps when transferring polar chemicals and conducting reactions on interfaces by stamping.

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

    Directory of Open Access Journals (Sweden)

    Claudia Addamiano

    2016-08-01

    Full Text Available 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.

  10. Phase I Investigations at the Former CCC/USDA Grain Storage Facility in Montgomery City, Missouri, in 2010-2011

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, Lorraine M. [Argonne National Lab. (ANL), Argonne, IL (United States). Environmental Science Division. Applied Geoscience and Environmental Restoration Program

    2012-11-01

    This report presents the technical findings of Phase I of Argonne’s studies. The Phase I field investigation was initiated on October 18, 2010. The work was conducted in accord with (1) the final site-specific Phase I Work Plan for Montgomery City (Argonne 2010; approved by the MDNR [2010]); (2) applicable Missouri regulations; and (3) the standard operating procedures, quality assurance/quality control (QA/QC) measures, and general health and safety policies outlined in the Master Work Plan (Argonne 2002) for operations in Kansas, which was reviewed by the MDNR and accepted for current use. A draft master plan specific to work in Missouri and a set of draft standard operating procedures are in review with the MDNR. The site-specific Work Plan for Montgomery City (Argonne 2010) (1) summarizes the pre-existing knowledge base for the Montgomery City investigation site compiled by Argonne and (2) describes the site-specific technical objectives and the intended scope of work developed for the first phase of the investigation. Three primary technical objectives were identified for the Phase I studies, as follows: 1. Update the presently identified inventory and status of private and public drinking water wells in the immediate vicinity of the former CCC/USDA grain storage facility, and sample the identified wells for volatile organic compounds (VOCs) and geochemical analyses. In conjunction with this effort, determine the present sources(s) of drinking water for all residents in an approximate 0.5-mi radius of the former CCC/USDA facility. 2. Investigate for possible evidence of a soil source of carbon tetrachloride contamination in the unconsolidated sediments beneath the former CCC/USDA facility that might affect the underlying bedrock aquifer units. 3. Obtain preliminary information on the site-specific lithologic and hydrologic characteristics of the unconsolidated sediments overlying bedrock at the former CCC/USDA grain storage location. Section 2 of this report

  11. Synthesis, Cytotoxic Activity, and DNA Binding Properties of Copper (II Complexes with Hesperetin, Naringenin, and Apigenin

    Directory of Open Access Journals (Sweden)

    Mingxiong Tan

    2009-01-01

    Full Text Available Complexes of copper (II with hesperetin, naringenin, and apigenin of general composition [CuL2(H2O2]⋅nH2O (1–3 have been synthesized and characterized by elemental analysis, UV-Vis, FT-IR, ESI-MS, and TG-DTG thermal analysis. The free ligands and the metal complexes have been tested in vitro against human cancer cell lines hepatocellular carcinoma (HepG-2, gastric carcinomas (SGC-7901, and cervical carcinoma (HeLa. Complexes 1 and 3 were found to exhibit growth inhibition of SGC-7901 and HepG2 cell lines with respect to the free ligands; the inhibitory rate of complex 1 is 43.2% and 43.8%, while complex 3 is 46% and 36%, respectively. The interactions of complex 1 and its ligand Hsp with calf thymus DNA were investigated by UV-Vis, fluorescence, and CD spectra. Both complex 1 and Hsp were found to bind DNA in intercalation modes, and the binding affinity of complex 1 was stronger than that of free ligand.

  12. A parallel synthesis scheme for generating libraries of DNA polymerase substrates and inhibitors.

    Science.gov (United States)

    Strobel, Heike; Dugué, Laurence; Marlière, Philippe; Pochet, Sylvie

    2002-12-02

    We report a combinatorial approach aimed at producing in a single step a large family of nucleoside triphosphate derivatives that could be tested for their ability to be substrates for DNA polymerases. We propose as a unique triphosphate building block a nucleotide with a hydrazine function anchored to an imidazole ring. Condensation between the 5'-triphosphate derivative of 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide (dY(NH(2))TP) and any aldehyde or ketone, followed by reduction of the intermediate hydrazones dXmTP, resulted in the corresponding hydrazides (dXnTP). Following this scheme, a series of aldehydes having various aromatic parts yielded a number of adducts dY(NHR)TP. Vent (exo-) DNA polymerase is found to be able to catalyse the single incorporation of these bulky triphosphate derivatives. Subsequent extensions of the modified pairs with canonical triphosphates resulted mainly in abortive elongations at primer+2, except after the incorporation of dY(NHben)TP and, to a lesser extent, dY(NHphe)TP opposite C. These results illustrate the potential of this parallel synthetic scheme for generating new substrates or inhibitors of replication in a single step.

  13. Exploration of DAPI analogues: Synthesis, antitrypanosomal activity, DNA binding and fluorescence properties.

    Science.gov (United States)

    Farahat, Abdelbasset A; Kumar, Arvind; Say, Martial; Wenzler, Tanja; Brun, Reto; Paul, Ananya; Wilson, W David; Boykin, David W

    2017-03-10

    The DAPI structure has been modified by replacing the phenyl group with substituted phenyl or heteroaryl rings. Twelve amidines were synthesized and their DNA binding, fluorescence properties, in vitro and in vivo activities were evaluated. These compounds are shown to bind in the DNA minor groove with high affinity, and exhibit superior in vitro antitrypanosomal activity to that of DAPI. Six new diamidines (5b, 5c, 5d, 5e, 5f and 5j) exhibit superior in vivo activity to that of DAPI and four of these compounds provide 100% animal cure at a low dose of 4 × 5 mg/kg i.p. in T. b. rhodesiense infected mice. Generally, the fluorescence properties of the new analogues are inferior to that of DAPI with the exception of compound 5i which shows a moderate increase in efficacy while compound 5k is comparable to DAPI.

  14. Synthesis of novel porous magnetic silica microspheres as adsorbents for isolation of genomic DNA.

    Science.gov (United States)

    Zhang, Zhichao; Zhang, Liming; Chen, Lei; Chen, Ligong; Wan, Qian-Hong

    2006-01-01

    An improved procedure is described for preparation of novel mesoporous microspheres consisting of magnetic nanoparticles homogeneously dispersed in a silica matrix. The method is based on a three-step process, involving (i) formation of hematite/silica composite microspheres by urea-formaldehyde polymerization, (ii) calcination of the composite particles to remove the organic constituents, and (iii) in situ transformation of the iron oxide in the composites by hydrogen reductive reaction. The as-synthesized magnetite/silica composite microspheres were nearly monodisperse, mesoporous, and magnetizable, with as typical values an average diameter of 3.5 microm, a surface area of 250 m(2)/g, a pore size of 6.03 nm, and a saturation magnetization of 9.82 emu/g. These magnetic particles were tested as adsorbents for isolation of genomic DNA from Saccharomyces cerevisiae cells and maize kernels. The results are quite encouraging as the magnetic particle based protocols lead to the extraction of genomic DNA with satisfactory integrity, yield, and purity. Being hydrophilic in nature, the porous magnetic silica microspheres are considered a good alternative to polystyrene-based magnetic particles for use in biomedical applications where nonspecific adsorption of biomolecules is to be minimized.

  15. Corrigendum to "Synthesis, crystal structure and electrochemical and DNA binding studies of oxygen bridged-copper(II) carboxylate" [J. Mol. Struct. 1093 (2015) 135-143

    Science.gov (United States)

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

    2017-04-01

    The authors regret to inform that Scheme 1 in the article titled 'Synthesis, crystal structure and electrochemical and DNA binding studies of oxygen bridged-copper(II) carboxylate' in vol. 1093 of the Journal of Molecular Structure is incorrect. The corrected scheme is as shown in this correction. This is purely a copy error. The error does not affect the conclusion in paper. The authors would like to apologize for any inconvenience caused.

  16. 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.

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

    OpenAIRE

    Guo Jingsheng; Li Dongye; Bai Shiru; Xu Tongda; Zhou Zhongmin; Zhang Yanbin

    2012-01-01

    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...

  18. Zmiany w składzie chemicznym ściany komórkowej źdźbła pszenicy traktomanej chlorkiem chlorocholiny (CCC [Changes in the cell walls of wheat culms treated utith 2-chloroethyl-trime-thyl-ammonium chloride (CCC

    Directory of Open Access Journals (Sweden)

    Maria Przeszlakowska

    2015-06-01

    Full Text Available The influence of 2-chloroethyl-trimethyl-ammonium chloride (CCC on the content of various constituents of the cell wall of the wheat culms var. Eka Nowa was investigated. Chromatographic analysis of the polysaccharides obtained from the cell wall of the wheat culms has been carried out. The content of pectic substances (pectins and protopectins in nodes and internodes of the wheat culms treated with CCC was determined.

  19. β-diketone-cobalt complexes inhibit DNA synthesis and induce S-phase arrest in rat C6 glioma cells.

    Science.gov (United States)

    Zhang, Kaizhi; Zhao, Xingli; Liu, Junzhi; Fang, Xiangyang; Wang, Xuepeng; Wang, Xiaohong; Li, Rui

    2014-03-01

    β-diketone-cobalt complexes, a family of newly synthesized non-platinum metal compounds, exhibit potential antitumor activity; however, the antitumor mechanism is unclear. The current study investigated the mechanism by which β-diketone-cobalt complexes inhibit rat C6 glioma cell proliferation. It was found that β-diketone-cobalt complexes suppress rat C6 glioma cell viability in a dose-dependent manner (3.125-100 μg/ml). In rat C6 glioma cells, the IC50 value of β-diketone-cobalt complexes was 24.7±3.395 μg/ml and the IC10 value was 4.37±1.53 μg/ml, indicating a strong inhibitory effect. Further investigation suggested that β-diketone-cobalt complexes inhibit rat C6 glioma cell proliferation, which is associated with S-phase arrest and DNA synthesis inhibition. During this process, β-diketone-cobalt complexes decreased cyclin A expression and increased cyclin E and p21 expression. In addition, β-diketone-cobalt complexes exhibit a stronger antitumor capability than the antineoplastic agent, 5-fluorouracil.

  20. 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, (1)H NMR, (13)C 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kleijer, W.J.; Kraan, M. van der; Los, F.J. [Erasmus Univ., Rotterdam (Netherlands). Dept. of Clinical Genetics; Jaspers, N.G.J. [Erasmus Univ., Rotterdam (Netherlands). Lab. of Cell Biology and Genetics

    1994-12-01

    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).

  2. Cell interactions in concanavalin A activated cation flux and DNA synthesis of mouse lymphocytes

    DEFF Research Database (Denmark)

    Owens, T; Kaplan, J G

    1980-01-01

    Co-culture at constant cell density of nude mouse spleen cells (by themselves unresponsive to the T-cell mitogen concanavalin A (Con A)), with congenic T-enriched lymphocyte suspensions and Con A caused anomalously high activation of K+ transport (measured by 86Rb uptake) and of incorporation...... of thymidine into DNA; the expected dilution of these two responses by nude spleen cells did not occur. However, if the nude splenocytes were added immediately prior to assay to the enriched T cells that had been precultured in presence of Con A, the expected dilution of the activated T-cell responses occurred......; both 86Rb uptake and thymidine incorporation were reduced proportionally to the degree of dilution of the T cells by the nonresponding cells. These data indicate that during co-culture in presence of Con A there is interaction between the T cells, capable of responding to mitogens, and the nude spleen...

  3. 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.

  4. An Analysis of Content in Comprehensive Cancer Control (CCC) Plans that Address Chronic Hepatitis B and C Virus Infections as Major Risk Factors for Liver Cancer

    Science.gov (United States)

    Momin, Behnoosh; Richardson, Lisa

    2017-01-01

    Chronic hepatitis B and C virus (HBV and HCV) infections are among the leading causes of preventable death worldwide. Chronic viral hepatitis is the cause of most primary liver cancer, which is the third leading cause of cancer deaths globally and the ninth leading cause of cancer deaths in the U.S. The extent to which Comprehensive Cancer Control (CCC) programs in states, tribal governments and organizations, territories, and Pacific Island jurisdictions address chronic hepatitis B and/or hepatitis C infections as risk factors for liver cancer or recommend interventions for liver cancer prevention in their CCC plans remains unknown. We searched CCC plans for this information using the search tool at http://www.cdc.gov/cancer/ncccp/ to access the content of plans for this information. A combination of key search terms including “liver cancer,” “hepatitis,” “chronic alcohol,” and “alcohol abuse” were used to identify potential content regarding liver cancer risk factors and prevention. Relevant content was abstracted for further review and classification. Of 66* CCC plans, 27% (n=18) addressed liver cancer using the above-mentioned search terms. In the 23 plans that addressed HBV and/or HCV, there were 25 goals, objectives, strategies, and outcomes aimed at reducing the incidence or prevalence of HBV and/or HCV infection. While nearly a third of CCC programs identify at least one goal, objective, strategy, outcome, or prevention program to reduce cancer burden in their CCC plans, few plans discuss specific actions needed to reduce the burden of liver cancer. PMID:22160788

  5. Inhibition of HIV-1 reverse transcriptase-catalyzed synthesis by intercalated DNA Benzo[a]Pyrene 7,8-Dihydrodiol-9,10-Epoxide adducts.

    Directory of Open Access Journals (Sweden)

    Parvathi Chary

    Full Text Available To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT, this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N (6-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.

  6. 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).

  7. 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

  8. Synthesis, DNA cleavage and antimicrobial activity of 4-thiazolidinones-benzothiazole conjugates.

    Science.gov (United States)

    Singh, Meenakshi; Gangwar, Mayank; Nath, Gopal; Singh, Sushil K

    2014-11-01

    Antimicrobial screening of several novel 4-thiazolidinones with benzothiazole moiety has been performed. These compounds were evaluated for antimicrobial activity against a panel of bacterial and fungal strains. The strains were treated with these benzothiazole derivatives at varying concentrations, and MIC's were calculated. Structures of these compounds have been determined by spectroscopic studies viz., FT-IR, 1H NMR, 13C NMR and elemental analysis. Significant antimicrobial activity was observed for some members of the series, and compounds viz. 3-(4-(benzo[d]thiazol-2-yl) phenyl-2-(4-methoxyphenyl)thiazolidin-4-one and 3-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-hydroxy phenyl)thiazolidin-4-one were found to be the most active against E.coli and C. albicans with MIC values in the range of 15.6-125 microg/ml. Preliminary study of the structure-activity relationship revealed that electron donating groups associated with thiazolidine bearing benzothiazole rings had a great effect on the antimicrobial activity of these compounds and contributes positively for the action. DNA cleavage experiments gave valuable hints with supporting evidence for describing the mechanism of action and hence showed a good correlation between their calculated MIC's and its lethality.

  9. 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.

  10. Antitumor polycyclic acridines. 7. Synthesis and biological properties of DNA affinic tetra- and pentacyclic acridines.

    Science.gov (United States)

    Stanslas, J; Hagan, D J; Ellis, M J; Turner, C; Carmichael, J; Ward, W; Hammonds, T R; Stevens, M F

    2000-04-20

    New synthetic routes to a series of tetra- and pentacyclic acridines related in structure to marine natural products are reported. The novel water-soluble agent dihydroindolizino[7,6,5-kl]acridinium chloride 14 has inhibitory activity in a panel of non-small-cell lung and breast tumor cell lines exceeding that of m-AMSA. The salt inhibited the release of minicircle products of kDNA confirming that disorganization of topoisomerase II partly underlies the activity of the compound. COMPARE analysis of the NCI mean graph profile of compound 14 at the GI(50) level corroborates this conclusion with Pearson correlation coefficients (>0.6) to clinical agents of the topoisomerase II class: however, this correlation was not seen at the LC(50) level. The inhibitory action of 14 on Saccharomyces cerevisiae transfected with human topoisomerase II isoforms showed a 3-fold selectivity against the IIalpha isoform over the IIbeta isoform. Unlike m-AMSA, 14 is not susceptible to P-glycoprotein-mediated drug efflux and retains activity in lung cells with derived resistance to the topoisomerase II inhibitor etoposide.

  11. Synthesis and DNA Cleavage Studies of 2,6-Dimethoxyhydroquinone- 3-Mercaptoacetic Acid Conjugates

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In an effort to investigate the use of short peptide chains as carriers of new anti-tumor agents, we synthesized four tripeptide-cytotoxic agent conjugates: DMQ-MA-Lys(DMQ-MA)-Phe -Arg-Ome, DMQ-MA-Lys(DMQ-MA)-Ile-Arg-Ome, DMQ-MA-Lys(DMQ-MA)-Val-Arg-Ome, DMQ-MA-Lys(DMQ-MA)-Lys(Cbz)-Arg-Ome. The cytotoxic agent conjugated to the N-terminal and the ξ-amino group of Lysine of the tripeptide is 2,6-dimethoxyhydroquinone-3- mercaptoacetic acid (DMQ-MA). The tripeptides were synthesized by coupling protected amino acid residues according to Pfp/DCC methods (Pfp: pentafluorophenol, DCC:N,N'-dicyclohexyl- carbodiimide) in solution. Agarose gel electrophoresis showed that these compounds can cleave supercoiled DNA into open-circular form in drug concentration as low as 4-50 μM without H2O2 and UV irradiation. Further studies on their cytotoxicity for these conjugates are ongoing.

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

    Science.gov (United States)

    Burger, Amanda; Raymer, Jenny; Bockrath, R

    2002-10-01

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

  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. 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.

  15. Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy.

    Science.gov (United States)

    Pierzyńska-Mach, Agnieszka; Szczurek, Aleksander; Cella Zanacchi, Francesca; Pennacchietti, Francesca; Drukała, Justyna; Diaspro, Alberto; Cremer, Christoph; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

    2016-01-01

    Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2'-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts.

  16. A model for triplet mutation formation based on error-prone translesional DNA synthesis opposite UV photolesions.

    Science.gov (United States)

    Ikehata, Hironobu; Ono, Tetsuya; Tanaka, Kiyoji; Todo, Takeshi

    2007-05-01

    A triplet mutation is defined as multiple base substitutions or frameshifts within a three-nucleotide sequence which includes a dipyrimidine sequence. Triplet mutations have recently been identified as a new type of UV-specific mutation, although the mechanism of their formation is unknown. A total of 163 triplet mutations were identified through an extensive search of previously published data on UV-induced mutations, including mutations from skin, skin cancer, and cultured mammalian cells. Seven common patterns of sequence changes were found: Type I, NTC-->TTT; Type IIa, NCC-->PyTT or PyCT (Py, pyrimidine); Type IIb, TCC-->PuTT or PuCT (Pu, purine); Type III, NCC-->NAT or NTA; Type IV, NTT-->AAT; Type Va, NCT-->NTX; and Type Vb, PuCT-->XTT (N and X, independent anonymous bases). Furthermore, it is suggested that the type of UV lesion responsible for each of these triplet mutation classes are (a) pyrimidine(6-4)pyrimidone photoproducts for Types I, IIb, III, IV and Vb, (b) cyclobutane pyrimidine dimers for Type Va, and (c) Dewar valence isomers for Types IIa and IIb. These estimations are based primarily on results from previous studies using photolyases specific for each type of UV lesion. A model is proposed to explain the formation of each type of triplet mutation, based on error-prone translesional DNA synthesis opposite UV-specific photolesions. The model is largely consistent with the 'A-rule', and predicts error-prone insertions not only opposite photolesions but also opposite the undamaged template base one-nucleotide downstream from the lesions.

  17. 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.

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

    Science.gov (United States)

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

    2005-01-01

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

  19. 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.

  20. Pyridine-3-carboxamide-6-yl-ureas as novel inhibitors of bacterial DNA gyrase: structure based design, synthesis, SAR and antimicrobial activity.

    Science.gov (United States)

    Yule, Ian A; Czaplewski, Lloyd G; Pommier, Stephanie; Davies, David T; Narramore, Sarah K; Fishwick, Colin W G

    2014-10-30

    The development of antibacterial drugs based on novel chemotypes is essential to the future management of serious drug resistant infections. We herein report the design, synthesis and SAR of a novel series of N-ethylurea inhibitors based on a pyridine-3-carboxamide scaffold targeting the ATPase sub-unit of DNA gyrase. Consideration of structural aspects of the GyrB ATPase site has aided the development of this series resulting in derivatives that demonstrate excellent enzyme inhibitory activity coupled to potent Gram positive antibacterial efficacy.

  1. Chromatographic evidence that the AAA-coding isoacceptor of lysine tRNA primes DNA synthesis in murine mammary tumor virus

    Energy Technology Data Exchange (ETDEWEB)

    Waters, L.C.

    1981-07-30

    Most of the tRNA encapsulated within the murine mammary tumor virus is tRNA/sup LYS/. The reversed-phase chromatographic pattern of tRNA/sup LYS/ isoacceptors in the viral free tRNA and in the 70 S-associated tRNA that is released at 65/sup 0/ is similar to the pattern in virus-producing cells. However, the more tightly bound 70 S-associated tRNA/sup LYS/ is significantly enriched in the AAA-coding isoacceptor. This isoacceptor, but not the AAG-coding one, primes MuMTV 35 S RNA-directed DNA synthesis in vitro.

  2. Description of a PCR-based technique for DNA splicing and mutagenesis by producing 5' overhangs with run through stop DNA synthesis utilizing Ara-C

    Directory of Open Access Journals (Sweden)

    Silverman Mel

    2005-09-01

    Full Text Available Abstract Background Splicing of DNA molecules is an important task in molecular biology that facilitates cloning, mutagenesis and creation of chimeric genes. Mutagenesis and DNA splicing techniques exist, some requiring restriction enzymes, and others utilize staggered reannealing approaches. Results A method for DNA splicing and mutagenesis without restriction enzymes is described. The method is based on mild template-dependent polymerization arrest with two molecules of cytosine arabinose (Ara-C incorporated into PCR primers. Two rounds of PCR are employed: the first PCR produces 5' overhangs that are utilized for DNA splicing. The second PCR is based on polymerization running through the Ara-C molecules to produce the desired final product. To illustrate application of the run through stop mutagenesis and DNA splicing technique, we have carried out splicing of two segments of the human cofilin 1 gene and introduced a mutational deletion into the product. Conclusion We have demonstrated the utility of a new PCR-based method for carrying out DNA splicing and mutagenesis by incorporating Ara-C into the PCR primers.

  3. Transformation of isolated barley (Hordeum vulgare L.) microspores: I. the influence of pretreatments and osmotic treatment on the time of DNA synthesis.

    Science.gov (United States)

    Shim, Youn-Seb; Pauls, K Peter; Kasha, Ken J

    2009-02-01

    The objective of this study was to determine when DNA synthesis occurred during pretreatments of cultured barley (Hordeum vulgare L.) microspores and during their preparation for particle bombardment. Based on this information, an investigation of the influence of cell cycle stage on the ability to obtain homozygous transgenic plants by particle bombardment will be presented in paper II of this series. It was hypothesized that the introduction of foreign genes at the G1 cell cycle stage in cultured uninucleate microspores would produce homozygous transgenic plants. Experiments were conducted with two different commonly used pretreatments to induce microspore embryogenesis: cold (4 degrees C) for 21days and cold plus 0.3 mol/L mannitol for 4 days. After pretreatment, the microspores were placed in a higher osmotic medium for 4 h prior to and for 18 h following bombardment. It was confirmed that during the cold plus mannitol pretreatment, there was no apparent change in the cell cycle stage, with the majority of the microspores remaining at the G1 stage. While in the cold for 21 days, the microspores progressed slowly through to G2, with a few progressing further into the mitosis and binucleate stages. Hourly DNA density measurements that were taken during the 4 h osmotic adjustment period following the cold plus mannitol pretreatment indicated that DNA synthesis began during this period at 25 degrees C, while at 4 degrees C, there was no apparent change in cell cycle stage or in DNA density. Thus, one might expect to find a higher frequency of homozygous doubled haploids by maintaining the temperature low during the 4 h osmotic adjustment period following the cold plus mannitol pretreatment than following the 21 day cold pretreatment. However, it is also not known what effect the temperatures during the whole high-osmotic treatments will have on the rate and time of incorporation of the transgene.

  4. Final work plan : Phase I investigation of potential contamination at the former CCC/USDA grain storage facility in Savannah, Missouri.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M.; Environmental Science Division

    2007-10-12

    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. 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 occupied by the Missouri Department of Transportation [MoDOT]), approximately 400 ft east of the former CCC/USDA facility. Carbon tetrachloride concentrations in the Morgan well have ranged from the initial value of 29 {micro}g/L in 1998, up to a maximum of 61 {micro}g/L in 1999, and back down to 22 {micro}g/L in 2005. The carbon tetrachloride concentration in the MoDOT well in 2000 (the only time it was sampled) was 321 {micro}g/L. The concentrations for the two wells are above the EPA maximum contaminant level (MCL) of 5 {micro}g/L for carbon tetrachloride (EPA 1999; MoDNR 2000a,b). Because the observed contamination in the Morgan and MoDOT wells might be linked to the past use of carbon tetrachloride-based grain fumigants at its former grain storage facility, the CCC/USDA will conduct investigations to (1) characterize the source(s), extent, and factors controlling the subsurface distribution and movement of carbon tetrachloride at Savannah and (2) evaluate the health and environmental threats potentially posed by the contamination

  5. 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....

  6. Development of the Children's Communication Checklist (CCC): a method for assessing qualitative aspects of communicative impairment in children.

    Science.gov (United States)

    Bishop, D V

    1998-09-01

    The Children's Communication Checklist (CCC) was developed to assess aspects of communicative impairment that are not adequately evaluated by contemporary standardised language tests. These are predominantly pragmatic abnormalities seen in social communication, although other qualitative aspects of speech and language were also included. Some items covering social relationships and restricted interests were incorporated, so that the relationship between pragmatic difficulties and other characteristics of pervasive developmental disorders could be explored. Checklist ratings were obtained for 76 children aged 7 to 9 years, all of whom had received special education for language impairment. In 71 cases, 2 raters (usually a teacher and speech-language therapist) independently completed the checklist, making it possible to establish inter-rater reliability. From an initial pool of 93 items, 70 items, grouped into 9 scales, were retained. Five of the subscales were concerned with pragmatic aspects of communication. A composite pragmatic impairment scale formed from these subscales had inter-rater reliability and internal consistency of around .80. This composite discriminated between children with a school diagnosis of semantic-pragmatic disorder and those with other types of specific language impairment (SLI). The majority of children with pragmatic language impairments did not have any evidence of restricted interests or significant difficulties in the domains of social relationships.

  7. The effect of vitamin A on the migration and DNA synthesis of rat bladder tumor cell line NBT II in culture.

    Science.gov (United States)

    Tchao, R; Leighton, J

    1979-05-01

    In the presence of vitamin A, NBT II cells, derived from a carcinoma of rat bladder, grew as a monolayer with diminished piling up. Keratinization, which normally appeared within stratified cells in postconfluent cultures, was inhibited. A "wounding" technique suitable for quantitative analysis of cell migration was developed for confluent cultures grown on glass coverslips. Vitamin A treatment enhanced the migration of cells from the wound edge. In dense postconfluent monolayer cultures, vitamin A treatment maintained a higher percentage of cells in DNA synthesis than in the control cultures, as determined by 3H-TdR uptake and autoradiography. In contrast, in sparse cultures vitamin A did not stimulate DNA synthesis or increase the mitotic index. This stimulatory effect, limited to dense cultures, may be attributable to vitamin A causing viable cells to be shed into the medium, thereby maintaining the monolayer just at confluence. Thus vitamin A inhibits squamous cell differentiation, enhances migration, and maintains the culture in the proliferative phase. In a different system of high cell density, NBT II aggregates cultured in a combined matrix of chick plasma clot and collagen-coated sponge, vitamin A also enhanced the migration of cells. These results may explain, in part, the failure of vitamin A to inhibit completely the growth of some established tumors.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sahni, Abha [Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 (United States); Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555 (United States); Wang, Nadan [Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 (United States); Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Alexis, Jeffrey, E-mail: jeffrey_alexis@urmc.rochester.edu [Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 (United States)

    2013-02-15

    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.

  9. Investigation of DNA synthesis in experimentally induced Long-Evans rat myeloschisis by the BrdU/antiBrdU technique.

    Science.gov (United States)

    Chono, Y; Abe, H; Iwasaki, Y; Nagashima, K

    1994-04-01

    The volume and DNA synthesis of the neuroepithelium in induced myeloschisis in Long-Evans rats as shown by hematoxylin-eosin and BrdU/antiBrdU immunohistochemical staining patterns were examined at different stages of embryonal development. On day 14 of gestation, control animals contained BrdU-incorporating cells mainly at the alar plate of the closed neural tube. On the same day, the everted neural plate of rats with myeloschisis showed active, diffuse uptake of BrdU in the cells of the matrix layer, although an increase in the volume of the everted neural plate was not yet identifiable. On day 21 of gestation, rats with myeloschisis showed a marked increase in the volume of the neuroepithelium compared with controls. Our investigations suggest that, in myeloschisis, more neuroepithelial cells than normal retain their capability for DNA synthesis on day 14 of gestation, and the overgrowth of the neuroepithelium found on day 21 is possibly a secondary effect of failure of neural tube closure.

  10. 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

  11. 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.

  12. Final work plan : phase I investigation of potential contamination at the former CCC/USDA grain storage facility in Montgomery City, Missouri.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M.; Environmental Science Division

    2010-08-16

    From September 1949 until September 1966, the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) leased property at the southeastern end of Montgomery City, Missouri, for the operation of a grain storage facility. 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 January 2000, carbon tetrachloride was detected in a soil sample (220 {micro}g/kg) and two soil gas samples (58 {micro}g/m{sup 3} and 550 {micro}g/m{sup 3}) collected at the former CCC/USDA facility, as a result of a pre-CERCLIS site screening investigation (SSI) performed by TN & Associates, Inc., on behalf of the U.S. Environmental Protection Agency (EPA), Region VII (MoDNR 2001). In June 2001, the Missouri Department of Natural Resources (MoDNR) conducted further sampling of the soils and groundwater at the former CCC/USDA facility as part of a preliminary assessment/site inspection (PA/SI). The MoDNR confirmed the presence of carbon tetrachloride (at a maximum identified concentration of 2,810 {micro}g/kg) and chloroform (maximum 82 {micro}g/kg) in the soils and also detected carbon tetrachloride and chloroform (42.2 {micro}g/L and 58.4 {micro}g/L, respectively) in a groundwater sample collected at the former facility (MoDNR 2001). The carbon tetrachloride levels identified in the soils and groundwater are above the default target level (DTL) values established by the MoDNR for this contaminant in soils of all types (79.6 {micro}g/kg) and in groundwater (5.0 {micro}g/L), as outlined in Missouri Risk-Based Corrective Action (MRBCA): Departmental Technical Guidance (MoDNR 2006a). The corresponding MRBCA DTL values for chloroform are 76.6 {micro}g/kg in soils of all types and 80 {micro}g/L in groundwater. Because the observed contamination at Montgomery City might be linked to the past use of carbon tetrachloride-based fumigants at its

  13. Correlation between serum hepatitis B virus core-related antigen and intrahepatic covalently closed circular DNA in chronic hepatitis B patients.

    Science.gov (United States)

    Suzuki, Fumitaka; Miyakoshi, Hideo; Kobayashi, Mariko; Kumada, Hiromitsu

    2009-01-01

    Nucleos(t)ide analogues are utilized for the treatment of chronic HBV infection, and HBe seroconversion and HBV DNA levels are commonly used as markers of viral status and as primary treatment endpoints. Recently, a new assay was prepared for the detection of serum HBV core-related antigen (HBcrAg), consisting of HBcAg, HBeAg, and p22cr, which is a precore protein from amino acid -28 to at least amino acid 150, by coding the precore/core region. In this study, we examined the correlation between serum HBcrAg concentration and viral status by the analysis of serum HBeAg, HBsAg, peripheral HBV DNA, and intrahepatic covalently closed circular DNA (cccDNA) in 57 chronic hepatitis B patients. Intrahepatic cccDNA was detected in all 57 patients, 42 patients were HBcrAg-positive, and serum HBcrAg concentration level was closely correlated with cccDNA. Additionally, positive HBcrAg concentration level results were observed in 6 out of 13 HBsAg seroclearance patients and 20 out of 31 HBV DNA-negative patients. Moreover, the correlation between HBcrAg and cccDNA in these 31 HBV DNA-negative patients was statistically significant (r = 0.482, P = 0.006). These data suggest that serum HBcrAg concentration is well correlated with intrahepatic cccDNA level, and that the measurement of serum HBcrAg may be clinically useful for monitoring intrahepatic HBV viral status, especially in patients under treatment with nucleos(t)ide analogues.

  14. Synthesis, characterization, DNA interactions, DNA cleavage, radical scavenging activity, antibacterial, anti-proliferative and docking studies of new transition metal complexes.

    Science.gov (United States)

    Chennam, Kishan Prasad; Ravi, Mudavath; Ushaiah, B; Srinu, V; Eslavath, Ravi Kumar; Devi, Ch Sarala

    2016-01-01

    The compound N-(2-hydroxybenzylidene)-1-ethyl-1, 4-dihydro-7-methyl-4-oxo-1, 8 naphthyridine-3-carbohydrazide (LH) and its Cu (II), Co (II) and Zn (II) complexes were synthesized and characterized. The absorption spectral titrations and competitive DNA binding studies depicted those complexes of title compound bind to CT-DNA through intercalation. Interestingly [Cu (II)-(L2)] showed relatively high binding constant value (6.61 x 10(5) M(-1)) compared to [Co (II)-(L2)] (4.378× 10(5) M(-1)) and [Zn (II)-(L2)] (3.1x10(5) M(-1)). Ligand and its complexes were also examined for DNA nuclease activity against pBR-322 plasmid DNA, which showed that [Cu (II)-(L2)] had the best hydrolytic cleavage property displaying prominent double-strand DNA cleavage. In addition, antioxidant activities of the ligand and its metal complexes investigated through scavenging effects for DPPH radical in- vitro, indicated their potentiality as good antioxidants. The in vitro anti-bacterial study inferred the better anti-bacterial activity of [Cu (II)-(L2)] and this was also correlated theoretically by employing docking studies wherein [Cu (II)-(L2)] displayed good Gold score and Chem score. Finally the in vitro anti- proliferative activity of studied compounds was tested against HeLa and MCF-7 cell lines. Interestingly [Cu (II)-(L2)] displayed lower IC50 value and lower percentage of viability in both HeLa and MCF-7 cell lines.

  15. Protein kinase D1 mediates stimulation of DNA synthesis and proliferation in intestinal epithelial IEC-18 cells and in mouse intestinal crypts.

    Science.gov (United States)

    Sinnett-Smith, James; Rozengurt, Nora; Kui, Robert; Huang, Carlos; Rozengurt, Enrique

    2011-01-07

    We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p < 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p < 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.

  16. 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.

  17. 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.

  18. Synthesis, characterization, DNA binding properties, fluorescence studies and toxic activity of cobalt(III) and ruthenium(II) polypyridyl complexes.

    Science.gov (United States)

    Nagababu, Penumaka; Shilpa, Mynam; Latha, J Naveena Lavanya; Bhatnagar, Ira; Srinivas, P N B S; Kumar, Yata Praveen; Reddy, Kotha Laxma; Satyanarayana, Sirasani

    2011-03-01

    The new ligand 4-(isopropylbenzaldehyde)imidazo[4,5-f ][1,10]phenanthroline (ippip) and its complexes [Ru(phen)(2)(ippip)](2+)(1),[Co(phen)(2)(ippip)](3+)(2),[Ru(bpy)(2)(ippip)](2+)(3),[Co(bpy)(2)(ippip)](3+)(4)(bpy=2,2-bipyridine) and (phen=1,10-phenanthroline) were synthesized and characterized by ES(+)-MS, (1)H and (13)C NMR. The DNA binding properties of the four complexes were investigated by different spectrophotometric methods and viscosity measurements. The results suggest that complexes bind to calf thymus DNA (CT-DNA) through intercalation. When irradiated at 365 nm, the complexes promote the photocleavage of pBR322 DNA, and complex 1 cleaves DNA more effectively than 2, 3, 4 complexes under comparable experimental conditions. Furthermore, photocleavage studies reveal that singlet oxygen ((1)O(2)) plays a significant role in the photocleavage.

  19. Synthesis and Structure of a Ternary Copper(II) Complex with Mixed Ligands of Diethylenetriamine and Picrate: DNA/Protein-Binding Property and In Vitro Anticancer Activity Studies.

    Science.gov (United States)

    Shi, Ya-Ning; Zheng, Kang; Zhu, Ling; Li, Yan-Tuan; Wu, Zhi-Yong; Yan, Cui-Wei

    2015-05-01

    Based on the importance of the design and synthesis of transition metal complexes with noncovalent DNA/protein-binding abilities in the field of metallo pharmaceuticals, a new mononuclear ternary copper(II) complex with mixed ligands of diethylenetriamine (dien) and picrate anion (pic), identified as [Cu(dien)(pic)](pic), was synthesized and characterized by elemental analysis, molar conductivity measurement, infrared spectrum, electronic spectral studies, and single-crystal X-ray diffractometry. The structure analysis reveals that the copper(II) complex crystallizes in the monoclinic space group P21 /c, and the copper(II) ion has a distorted square pyramidal coordination geometry. A two-dimensional supramolecular structure is formed through hydrogen bonds. The DNA/bovine serum albumin (BSA)-binding properties of the complex are explored, indicating that the complex can interact with herring sperm DNA via intercalation mode and bind to BSA responsible for quenching of tryptophan fluorescence by static quenching mechanism. The in vitro anticancer activity shows that the copper(II) complex is active against the selected tumor cell lines.

  20. Mutation of the mouse Rad17 gene leads to embryonic lethality and reveals a role in DNA damage-dependent recombination.

    Science.gov (United States)

    Budzowska, Magda; Jaspers, Iris; Essers, Jeroen; de Waard, Harm; van Drunen, Ellen; Hanada, Katsuhiro; Beverloo, Berna; Hendriks, Rudolf W; de Klein, Annelies; Kanaar, Roland; Hoeijmakers, Jan H; Maas, Alex

    2004-09-01

    Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition. Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals. However, the role of many CCC genes in higher eukaryotes remains elusive. Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation. In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad17(5'Delta/5'Delta) embryos, produced truncated mRad17 and were viable. These cells displayed hypersensitivity to various DNA-damaging agents. Surprisingly, mRad17(5'Delta/5'Delta) ES cells were able to arrest cell cycle progression upon induction of DNA damage. However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency. In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents.

  1. Eating behaviours in preadolescence are associated with body dissatisfaction and mental disorders - Results of the CCC2000 study.

    Science.gov (United States)

    Munkholm, Anja; Olsen, Else Marie; Rask, Charlotte Ulrikka; Clemmensen, Lars; Rimvall, Martin K; Jeppesen, Pia; Micali, Nadia; Skovgaard, Anne Mette

    2016-06-01

    Preadolescence is a key period in the early stages of eating disorder development. The aim of the present study was, firstly, to investigate restrained, emotional and external eating in a general population-based sample of 11-12 year olds. Secondly, we sought to explore how these eating behaviours are associated with possible predictors of eating disorders, such as body dissatisfaction, weight status and mental disorders. A subsample of 1567 children (47.7% boys; 52.3% girls) from the Copenhagen Child Cohort (CCC2000) completed web-based questionnaires on eating behaviours and body dissatisfaction using The Eating Pattern Inventory for Children (EPI-C) and The Children's Figure Rating Scale. Mental disorders were assessed using the online version of the Development and Well-Being Assessment (DAWBA) based on parental replies with final DSM-IV diagnoses determined by experienced child- and adolescent psychiatrists. Height and weight were measured at a face-to-face assessment. The results showed that restrained eating was significantly associated with overweight, body dissatisfaction and emotional disorders in both genders. Emotional eating showed similar associations with overweight and body dissatisfaction in both genders, but was only associated with mental disorders in girls. External eating was significantly associated with body dissatisfaction and neurodevelopmental disorders in both genders, but was only associated with overweight in girls. Our findings show that problematic eating behaviours can be identified in preadolescence, and co-exist with weight problems and mental disorders. Thus restrained, emotional and external eating was, in different ways, associated with overweight, body dissatisfaction and mental disorders. Our findings point to significant eating behaviours in preadolescence, which could constitute potential predictors of later eating disorder risk.

  2. 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.

  3. Computational Evaluation of Nucleotide Insertion Opposite Expanded and Widened DNA by the Translesion Synthesis Polymerase Dpo4.

    Science.gov (United States)

    Albrecht, Laura; Wilson, Katie A; Wetmore, Stacey D

    2016-06-23

    Expanded (x) and widened (y) deoxyribose nucleic acids (DNA) have an extra benzene ring incorporated either horizontally (xDNA) or vertically (yDNA) between a natural pyrimidine base and the deoxyribose, or between the 5- and 6-membered rings of a natural purine. Far-reaching applications for (x,y)DNA include nucleic acid probes and extending the natural genetic code. Since modified nucleobases must encode information that can be passed to the next generation in order to be a useful extension of the genetic code, the ability of translesion (bypass) polymerases to replicate modified bases is an active area of research. The common model bypass polymerase DNA polymerase IV (Dpo4) has been previously shown to successfully replicate and extend past a single modified nucleobase on a template DNA strand. In the current study, molecular dynamics (MD) simulations are used to evaluate the accommodation of expanded/widened nucleobases in the Dpo4 active site, providing the first structural information on the replication of (x,y)DNA. Our results indicate that the Dpo4 catalytic (palm) domain is not significantly impacted by the (x,y)DNA bases. Instead, the template strand is displaced to accommodate the increased C1'-C1' base-pair distance. The structural insights unveiled in the present work not only increase our fundamental understanding of Dpo4 replication, but also reveal the process by which Dpo4 replicates (x,y)DNA, and thereby will contribute to the optimization of high fidelity and efficient polymerases for the replication of modified nucleobases.

  4. DNA nanostructure immobilization to lithographic DNA arrays

    Science.gov (United States)

    Negrete, Omar D.

    Although DNA is well known for its genetic role in biology, DNA has also been sought-after as a material for the self-assembly of biological and electronic devices. Examples of DNA nanostructure construction include DNA tiled self-assembly and DNA Origami, where by controlling the sequence and concentration of DNA molecules, the rational design of geometric DNA nanostructures is possible. The assembly of DNA nanostructures takes place in solution and thus they are in disorder and require further organization to construct circuitry or devices. Hence, it is essential for future applications of this technology to develop methods to direct the placement of DNA nanostructures on a surface. To address this challenge my research examines the use of DNA microarrays to capture DNA nanostructures via DNA hybridization. Modern DNA arrays offer a high-density of sequence-specific molecular recognition sites where the addressable placement of DNA nanostructures can be achieved. Using Maskless Array Synthesizer (MAS) technology, I have characterized photolithographic DNA arrays for the hybridization of DNA complexes like large DNA molecules (> 1 kb), DNA-gold nanoparticle conjugates, and DNA Origami. Although modern photolithographic DNA arrays can possess a high-density of sequence (106/cm2), the printed DNA areas are on the order of tens of microns. Thus, I have also developed a method to reduce the DNA array spot size to nanoscale dimensions through the combined use of electron beam lithography with photolithographic DNA synthesis. This work addresses the key elements towards developing a surface patterning technology that takes advantage of DNA base-pairing for both molecular sub-assembly and surface patterning.

  5. Final work plan : phase I investigation of potential contamination at the former CCC/USDA grain storage facility in Montgomery City, Missouri.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M.; Environmental Science Division

    2010-08-16

    From September 1949 until September 1966, the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) leased property at the southeastern end of Montgomery City, Missouri, for the operation of a grain storage facility. 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 January 2000, carbon tetrachloride was detected in a soil sample (220 {micro}g/kg) and two soil gas samples (58 {micro}g/m{sup 3} and 550 {micro}g/m{sup 3}) collected at the former CCC/USDA facility, as a result of a pre-CERCLIS site screening investigation (SSI) performed by TN & Associates, Inc., on behalf of the U.S. Environmental Protection Agency (EPA), Region VII (MoDNR 2001). In June 2001, the Missouri Department of Natural Resources (MoDNR) conducted further sampling of the soils and groundwater at the former CCC/USDA facility as part of a preliminary assessment/site inspection (PA/SI). The MoDNR confirmed the presence of carbon tetrachloride (at a maximum identified concentration of 2,810 {micro}g/kg) and chloroform (maximum 82 {micro}g/kg) in the soils and also detected carbon tetrachloride and chloroform (42.2 {micro}g/L and 58.4 {micro}g/L, respectively) in a groundwater sample collected at the former facility (MoDNR 2001). The carbon tetrachloride levels identified in the soils and groundwater are above the default target level (DTL) values established by the MoDNR for this contaminant in soils of all types (79.6 {micro}g/kg) and in groundwater (5.0 {micro}g/L), as outlined in Missouri Risk-Based Corrective Action (MRBCA): Departmental Technical Guidance (MoDNR 2006a). The corresponding MRBCA DTL values for chloroform are 76.6 {micro}g/kg in soils of all types and 80 {micro}g/L in groundwater. Because the observed contamination at Montgomery City might be linked to the past use of carbon tetrachloride-based fumigants at its

  6. Microinjection of Escherichia coli UvrA, B, C and D proteins into fibroblasts of xeroderma pigmentosum complementation groups A and C does not result in restoration of UV-induced DNA synthesis.

    NARCIS (Netherlands)

    J.C.M. Zwetsloot; A.P. Barbeiro; W. Vermeulen (Wim); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude)

    1986-01-01

    textabstractThe UV-induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts of repair-deficient xeroderma pigmentosum complementation groups A and C was assayed after injection of identical activities of either Uvr excinuclease (UvrA, B, C and D) from Escherichia coli or endonuclease V

  7. Synthesis of 5,10,15,20-Tetra[4-(N-ethylpiperazinyl)phenyl]-porphyrin and Its Interaction with DNA

    Institute of Scientific and Technical Information of China (English)

    郭灿城; 李和平; 张晓兵

    2005-01-01

    Piperazinyl-porphyrin, 5,10,15,20-tetra[4-(N-ethylpiperazinyl)phenyl]porphyrin (TEPPH2), was synthesized based on the special affinity of porphyrin to cancer cells and the antitumor activity of piperazine compounds. Its structure was characterized by UV-vis and 1H NMR spectra and elemental analysis. A model for the interaction between TEPPH2 and calf thymus DNA was built, and the binding mechanism was investigated by W-vis and fluorescence spectra. The results indicated that TEPPH2 could intercalate into the base pairs of DNA strongly. One calf thymus DNA molecule could bind 88 TEPPH2 molecules, and the binding constant K is 8.4×106 L-mol-1. The binding number and binding constant of TEPPH2 with DNA are higher than those of the known anti-tumor drugs,tetrakis(4-N-methylpyridyl)porphine and the Schiff bases Ca/sal-his and Ni/sal-aln.

  8. 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.

  9. Low Cost DNA Molecular Weight Marker: Primer-Directed Synthesis from pGEM-T Easy Vector

    Directory of Open Access Journals (Sweden)

    Siriporn RIYAJAN

    2011-06-01

    Full Text Available A low cost DNA molecular weight marker was produced by a marker primer-directed synthetic method using pGEM-T Easy vector as the DNA template. Seven primers were used to amplify eight different DNA fragments, which were 150, 300, 375, 500, 700, 1,000, 1,200 and 1,625 bp, from bacterial culture containing pGEM-T Easy vector. Polymerase chain reactions (PCR for all marker loci required the same optimal annealing temperature, which allowed all the PCR to be completed in a single run. To obtain the molecular weight marker, the PCR product of each locus was mixed together and directly used as marker without any further purification. This custom made molecular weight marker was found to be approximately 17 to 49 times less expensive than other commercial 100 bp DNA ladder markers.Graphical abstract

  10. 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.

  11. Synthesis of triazole-linked oligonucleotides with high affinity to DNA complements and an analysis of their compatibility with biosystems.

    Science.gov (United States)

    Varizhuk, Anna M; Kaluzhny, Dmitry N; Novikov, Roman A; Chizhov, Alexandr O; Smirnov, Igor P; Chuvilin, Andrey N; Tatarinova, Olga N; Fisunov, Gleb Y; Pozmogova, Galina E; Florentiev, Vladimir L

    2013-06-21

    New oligonucleotide analogues with triazole internucleotide linkages were synthesized, and their hybridization properties were studied. The analogues demonstrated DNA binding affinities similar to those of unmodified oligonucleotides. The modification was shown to protect the oligonucleotides from nuclease hydrolysis. The modified oligonucleotides were tested as PCR primers. Modifications remote from the 3'-terminus were tolerated by polymerases. Our results suggest that these new oligonucleotide analogues are among the most promising triazole DNA mimics characterized to date.

  12. 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.

  13. 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.

  14. Synthesis, characterization and DNA interaction of new copper(II) complexes of Schiff base-aroylhydrazones bearing naphthalene ring.

    Science.gov (United States)

    Gökçe, Cansu; Gup, Ramazan

    2013-05-05

    Two new copper(II) complexes with the condensation products of methyl 2-naphthyl ketone with 4-hydroxybenzohydrazide, 4-hydroxy-N'-[(1Z)-1-(naphthalen-2-yl)ethylidene]benzohydrazide [HL(1)] and (Z)-ethyl 2-(4-(2-(1-(naphthalen-2-yl)ethylidene)hydrazinecarbonyl)phenoxy)acetate (HL(2)) were synthesized and characterized by elemental analysis, infrared spectra, UV-Vis electronic absorption spectra, magnetic susceptibility measurements, TGA, powder XRD and SEM-EDS. The binding properties of the copper(II) complexes with calf thymus DNA were studied by using the absorption titration method. DNA cleavage activities of the synthesized copper complexes were examined by using agarose gel electrophoresis. The effect of complex concentration on the DNA cleavage reactions in the absence and presence of H2O2 was also investigated. The experimental results suggest that the copper complexes bind significantly to calf thymus DNA by both groove binding and intercalation modes and cleavage effectively pBR322 DNA. The mechanistic studies demonstrate that a hydrogen peroxide-derived species and singlet oxygen ((1)O2) are the active oxidative species for DNA cleavage.

  15. 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.

  16. dRecQ4 is required for DNA synthesis and essential for cell proliferation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Yanjuan Xu

    Full Text Available BACKGROUND: The family of RecQ DNA helicases plays an important role in the maintenance of genomic integrity. Mutations in three of the five known RecQ family members in humans, BLM, WRN and RecQ4, lead to disorders that are characterized by predisposition to cancer and premature aging. METHODOLOGY/PRINCIPAL FINDINGS: To address the in vivo functions of Drosophila RecQ4 (dRecQ4, we generated mutant alleles of dRecQ4 using the targeted gene knock-out technique. Our data show that dRecQ4 mutants are homozygous lethal with defects in DNA replication, cell cycle progression and cell proliferation. Two sets of experiments suggest that dRecQ4 also plays a role in DNA double strand break repair. First, mutant animals exhibit sensitivity to gamma irradiation. Second, the efficiency of DsRed reconstitution via single strand annealing repair is significantly reduced in the dRecQ4 mutant animals. Rescue experiments further show that both the N-terminal domain and the helicase domain are essential to dRecQ4 function in vivo. The N-terminal domain is sufficient for the DNA repair function of dRecQ4. CONCLUSIONS/SIGNIFICANCE: Together, our results show that dRecQ4 is an essential gene that plays an important role in not only DNA replication but also DNA repair and cell cycle progression in vivo.

  17. DNA binding propensity and nuclease efficacy of biosensitive Schiff base complexes containing pyrazolone moiety: Synthesis and characterization

    Science.gov (United States)

    Paulpandiyan, Rajakkani; Raman, Natarajan

    2016-12-01

    A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes (1-8) were synthesized from pyrazolone precursor Schiff base(s), obtained by the condensation of 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one (4-aminoantipyrine) with cinnamaldehyde/benzaldehyde and respective metal(II) chloride. They have been characterized by elemental analysis, magnetic susceptibility, molar conductance measurements, UV-Vis., IR, NMR, ESI mass spectra and EPR studies. These complexes show lower conductance values, supporting their non-electrolytic nature. Spectroscopic and other analytical data of the complexes suggest octahedral geometry. The binding properties of these complexes with DNA have been explored by electronic absorption spectra, cyclic voltammetry and viscosity measurements which reveal that the complexes have the ability to interact with calf thymus DNA (CT DNA) by intercalative mode. The binding constant (Kb) values clearly signify that the complex 1 has more intercalating ability than other complexes. DNA cleavage efficacy of these complexes with pUC18 DNA has been investigated by gel electrophoresis technique. All the complexes have been found to promote cleavage of pUC18 DNA from the super coiled form I to the open circular form II in presence of hydrogen peroxide. The in vitro antibacterial and antifungal assay, investigated by Minimum Inhibitory Concentration (MIC) method indicates that these complexes are good antimicrobial agents against various pathogens.

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

    Science.gov (United States)

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

    2015-12-14

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

  19. 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.

  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.

  1. Ionic liquid-mediated three-component synthesis of fluorinated spiro-thiazine derivatives and their antimycobacterial and DNA cleavage activities

    Indian Academy of Sciences (India)

    Anshu Dandia; Ruby Singh; Deepti Saini

    2013-09-01

    A simple, green and catalyst-free novel protocol is developed for the synthesis of medicinally important spiro[indole-3,2'[1,3]-thiazine]-2,4'-dione and spiro[acenaphthylene-1,2'-[1,3]thiazine]dione libraries by the tandem reaction of readily available reagents in 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF6]. The ionic liquid has been used as a solvent as well as catalyst for this reaction. This reaction proceeded smoothly in good to excellent yields and offered several other advantages including short reaction time, simple experimental workup procedure and no by-products. The synthesized compounds were subjected to antimycobacterial efficacy against Mycobacterium tuberculosis H37Rv strain and DNA cleavage activity.

  2. Design and synthesis of dithiocarbamate linked β-carboline derivatives: DNA topoisomerase II inhibition with DNA binding and apoptosis inducing ability.

    Science.gov (United States)

    Kamal, Ahmed; Sathish, Manda; Nayak, V Lakshma; Srinivasulu, Vunnam; Kavitha, Botla; Tangella, Yellaiah; Thummuri, Dinesh; Bagul, Chandrakant; Shankaraiah, Nagula; Nagesh, Narayana

    2015-09-01

    A series of new β-carboline-dithiocarbamate derivatives bearing phenyl, dithiocarbamate and H/methyl substitutions at position-1, 3 and 9, respectively, were designed and synthesized. These derivatives 8a-l and 13a-l and their starting precursors (7 a-d and 12 a-d) have been evaluated for their in vitro cytotoxic activity on selected human cancer cell lines. Among the derivatives tested, 7 c, 12 c, 8 a, 8 d, 8 i, 8 j, 8 k, 8l and 13 d-l exhibited considerable cytotoxicity against most of the tested cancer cell lines (IC50dithiocarbamate with β-carboline enhances the cytotoxicity of 8 a-l and 13 a-l. Moreover, the derivatives 8 j and 13 g exhibited significant cytotoxic activity with IC50 values of 1.34 μM and 0.79 μM on DU-145 cancer cells, respectively. Further, the induction of apoptosis by these derivatives was confirmed by Annexin V-FITC and Hoechst staining assays. However, both biophysical as well as molecular docking studies suggested a combilexin-type of interaction between these derivatives and DNA, unlike simple β-carbolines. With a view to understand their mechanism of action, DNA topoisomerase II (topo II) inhibition assay was also performed. Overall, the present study emphasizes the importance of linking a dithiocarbamate moiety to the β-carboline scaffold for exhibiting profound activity.

  3. Green synthesis of anticancerous honeycomb PtNPs clusters: Their alteration effect on BSA and HsDNA using fluorescence probe.

    Science.gov (United States)

    Pansare, Amol V; Kulal, Dnyaneshwar K; Shedge, Amol A; Patil, Vishwanath R

    2016-09-01

    The screening and characterization of cancer cells has been challenging due to sample insufficiency and extravagant. In this article, we highlighted easy green synthesis of Platinum nanoparticles (PtNPs) in the honeycomb like clusters, and their optical properties (by HRTEM, XRD, DLS, Zeta potential, EDAX, and UV-Visible techniques). PtNPs were responsive of binding mechanisms with the bovine serum albumin (BSA), herring sperm deoxyribonucleic acid (HsDNA) and cytotoxicity of human carcinomas cell. We are able to elucidate the responses of various concentrations of PtNPs for the control of MDA-MB-468 cell and binding conformation of BSA and HsDNA by using multi-spectroscopic techniques under the physiological conditions. The extent of quenching was in agreement of PtNPs-BSA binding reaction was mainly a static. The Ksv, K, the number of binding sites at different temperatures and the thermodynamic parameters between BSA and PtNPs were calculated. The positive ΔS(0) and negative ΔH(0), ΔG(0) values indicated that the binding pattern was determined by spontaneous hydrogen bond electrostatic interaction of BSA with esterage like activity. The binding properties of the PtNPs with HsDNA have been investigated by thermal denaturation, competitive DNA-binding studies with ethidium bromide (EB), Hochest-33258 and relative viscosity. The negative ΔH(0), ΔS(0) and ΔG(0) values indicated that the hydrophilic interaction were main force in spontaneity in binding mechanism of PtNPs to HsDNA. GI50 value of PtNPs demonstrated that these nanoparticles showed cytotoxicity against MDA-MB-468 human breast cancer cell line. Our results also clarified that PtNPs bind to BSA and can be effectively transported in the body and eliminated. PtNPs showed minor groove binding with HsDNA, which could be a useful guideline for further versatile approach to develop biomedical coatings with different functions of drug design.

  4. Glucose-stimulated DNA synthesis through mammalian target of rapamycin (mTOR) is regulated by KATP channels: effects on cell cycle progression in rodent islets.

    Science.gov (United States)

    Kwon, Guim; Marshall, Connie A; Liu, Hui; Pappan, Kirk L; Remedi, Maria S; McDaniel, Michael L

    2006-02-10

    The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation. Since previous studies indicated that glucose-stimulated activation of mammalian target of rapamycin (mTOR) leads to [3H]thymidine incorporation and that mTOR activation is mediated, in part, through the K(ATP) channel and changes in cytosolic Ca2+, we determined whether glyburide, an inhibitor of K(ATP) channels that stimulates Ca2+ influx, modulates [3H]thymidine incorporation. Glyburide (10-100 nm) at basal glucose stimulated [3H]thymidine incorporation to the same magnitude as elevated glucose and further enhanced the ability of elevated glucose to increase [3H]thymidine incorporation. Diazoxide (250 microm), an activator of KATP channels, paradoxically potentiated glucose-stimulated [3H]thymidine incorporation 2-4-fold above elevated glucose alone. Cell cycle analysis demonstrated that chronic exposure of islets to basal glucose resulted in a typical cell cycle progression pattern that is consistent with a low level of proliferation. In contrast, chronic exposure to elevated glucose or glyburide resulted in progression from G0/G1 to an accumulation in S phase and a reduction in G2/M phase. Rapamycin (100 nm) resulted in an approximately 62% reduction of S phase accumulation. The enhanced [3H]thymidine incorporation with chronic elevated glucose or glyburide therefore appears to be associated with S phase accumulation. Since diazoxide significantly enhanced [3H]thymidine incorporation without altering S phase accumulation under chronic elevated glucose, this increase in DNA synthesis also appears to be primarily related to an arrest in S phase and not cell proliferation.

  5. Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: shutoff of host DNA and protein synthesis gene dosage experiments, identification of a restrictive host, and possible biological significance.

    Science.gov (United States)

    Snustad, D P; Bursch, C J; Parson, K A; Hefeneider, S H

    1976-04-01

    The shutoff of host DNA synthesis is delayed until about 8 to 10 min after infection when Escherichia coli B/5 cells were infected with bacteriophage T4 mutants deficient in the ability to induce nuclear disruption (ndd mutants). The host DNA synthesized after infection with ndd mutants is stable in the absence of T4 endonucleases II and IV, but is unstable in the presence of these nucleases. Host protein synthesis, as indicated by the inducibility of beta-galactosidase and sodium dodecyl sulfate-polyacrylamide gel patterns of isoptopically labeled proteins synthesize after infection, is shut off normally in ndd-infected cells, even in the absence of host DNA degradation. The Cal Tech wild-type strain of E. coli CT447 was found to restrict growth of the ndd mutants. Since T4D+ also has a very low efficiency of plating on CT447, we have isolated a nitrosoguanidine-induced derivative of CT447 which yields a high T4D+ efficiency of plating while still restricting the ndd mutants. Using this derivative, CT447 T4 plq+ (for T4 plaque+), we have shown that hos DNA degradation and shutoff of host DNA synthesis occur after infection with either ndd98 X 5 (shutoff delayed) or T4D+ (shutoff normal) with approximately the same kinetics as in E. coli strain B/5. Nuclear disruption occurs after infection of CT447 with ndd+ phage, but not after infection with ndd- phage. The rate of DNA synthesis after infection of CT447 T4 plq+ with ndd98 X 5 is about 75% of the rate observed after infection with T4D+ while the burst size of ndd98 X 5 is only 3.5% of that of T4D+. The results of gene dosage experiments using the ndd restrictive host C5447 suggest that the ndd gene product is required in stoichiometric amounts. The observation by thin-section electron microscopy of two distinct pools of DNA, one apparently phage DNA and the other host DNA, in cells infected with nuclear disruption may be a compartmentalization mechanism which separates the pathways of host DNA degradation and

  6. Synthesis of PLGA nanoparticles of tea polyphenols and their strong in vivo protective effect against chemically induced DNA damage

    Directory of Open Access Journals (Sweden)

    Srivastava AK

    2013-04-01

    Full Text Available Amit Kumar Srivastava,1 Priyanka Bhatnagar,2 Madhulika Singh,1 Sanjay Mishra,1 Pradeep Kumar,2 Yogeshwer Shukla,1 Kailash Chand Gupta1,2 1Proteomics Laboratory, Indian Institute of Toxicology Research (CSIR, Lucknow, India; 2Nucleic Acid Research Laboratory, Institute of Genomics and Integrative Biology (CSIR, Delhi University Campus, India Abstract: In spite of proficient results of several phytochemicals in preclinical settings, the conversion rate from bench to bedside is not very encouraging. Many reasons are attributed to this limited success, including inefficient systemic delivery and bioavailability under in vivo conditions. To achieve improved efficacy, polyphenolic constituents of black (theaflavin [TF] and green (epigallocatechin-3-gallate [EGCG] tea in poly(lactide-co-glycolide nanoparticles (PLGA-NPs were entrapped with entrapment efficacy of ~18% and 26%, respectively. Further, their preventive potential against 7,12-dimethylbenzanthracene (DMBA-induced DNA damage in mouse skin using DNA alkaline unwinding assay was evaluated. Pretreatment (topically of mouse skin with either TF or EGCG (100 µg/mouse doses exhibits protection of 45.34% and 28.32%, respectively, against DMBA-induced DNA damage. However, pretreatment with TF-loaded PLGA-NPs protects against DNA damage 64.41% by 1/20th dose of bulk, 71.79% by 1/10th dose of bulk, and 72.46% by 1/5th dose of bulk. Similarly, 51.28% (1/20th of bulk, 57.63% (1/10th of bulk, and 63.14% (1/5th of bulk prevention was noted using EGCG-loaded PLGA-NP doses. These results showed that tea polyphenol-loaded PLGA-NPs have ~30-fold dose-advantage than bulk TF or EGCG doses. Additionally, TF- or EGCG-loaded PLGA-NPs showed significant potential for induction of DNA repair genes (XRCC1, XRCC3, and ERCC3 and suppression of DNA damage responsive genes (p53, p21, MDM2, GADD45α, and COX-2 as compared with respective bulk TF or EGCG doses. Taken together, TF- or EGCG-loaded PLGA-NPs showed a superior

  7. Synthesis, characterization, biological studies (DNA binding, cleavage, antibacterial and topoisomerase I) and molecular docking of copper(II) benzimidazole complexes.

    Science.gov (United States)

    Arjmand, Farukh; Parveen, Shazia; Afzal, Mohd; Shahid, Mohd

    2012-09-03

    To explore the therapeutic potential of copper-based benzimidazole complexes, tetranuclear Cu(II) complex 1 and dinuclear ternary amino acid complexes 2 and 3 {L-trp and L-val, respectively} were synthesized and thoroughly characterized. In vitro DNA binding studies of complexes 1-3 were carried out employing UV-vis titrations, fluorescence, circular dichroic and viscosity measurements which revealed that the complexes 1-3 bind to CT DNA preferably via groove binding. Complex 1 cleaved pBR322 DNA via hydrolytic pathway (validated by T4 DNA ligase assay), accessible to major groove while 2 followed oxidative mechanism, binding to minor groove of DNA double helix; binding events were further validated by molecular docking studies. Additionally, the complexes 1 and 2 exhibit high Topo-I inhibitory activity at different concentrations. The complexes 1-3 were evaluated for antibacterial activity against Escherichia coli and Staphylococcus aureus, and 2 was found to be most effective against Gram-positive bacteria.

  8. Synthesis, characterization and multi-spectroscopic DNA interaction studies of a new platinum complex containing the drug metformin

    Science.gov (United States)

    Shahabadi, Nahid; Heidari, Leila

    2014-07-01

    A new platinum(II) complex; [Pt(Met)(DMSO)Cl]Cl in which Met = metformin and DMSO: dimethylsulfoxide, was synthesized and characterized by 1H NMR, IR, UV-Vis spectra, molar conductivity and computational methods. Binding interaction of this complex with calf thymus (CT) DNA has been investigated by using absorption, emission, circular dichroism, viscosity measurements, differential pulse voltammetry and cleavage studies by agarose gel electrophoresis. UV-Vis absorption studies showed hyperchromism. CD studies showed less perturbation on the base stacking and helicity bands in the CD spectrum of CT-DNA (B → C structural transition). In fluorimeteric studies, the Pt(II) complex can bind with DNA-NR complex and forms a new non-fluorescence adduct. The anodic peak current in the differential pulse voltammogram of the Pt(II) complex decreased gradually with the addition of DNA. Cleavage experiments showed that the Pt(II) complex does not induce any cleavage under the experimental setup. Finally all results indicated that Pt(II) complex interact with DNA via groove binding mode.

  9. Synthesis of novel fluorescent probe Tb(III)-7-carboxymethoxy-4-methylcoumarin complex for sensing of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Hussein, Belal H.M., E-mail: belalhussein102@yahoo.com [Department of Chemistry, Faculty of Science, Suez Canal University, Ismailia (Egypt); Azab, Hassan A. [Department of Chemistry, Faculty of Science, Suez Canal University, Ismailia (Egypt); Fathalla, Walid [Department of Mathematical and Physical Sciences, Faculty of Engineering, Port-Said University, Port-Said (Egypt); Ali, Sherin A.M. [Department of Mathematical and Physical Sciences, Faculty of Engineering, Suez Canal University, Ismailia (Egypt)

    2013-02-15

    New fluorescent probe Tb(III) (7-carboxymethoxy-4-methylcoumarin)2(SCN) (C2H5OH)(H2O) was synthesized and characterized by spectroscopy and thermal analysis. The absorption and fluorescence spectra of 7-carboxymethoxy-4-methylcoumarin (CMMC) and Tb(III)-CMMC complex have been measured in different solvents. The interactions of Tb(III)-CMMC complex with calf thymus nucleic acid (CT-DNA) have been investigated using steady state fluorescence measurements. The changes in the fluorescence intensity have been used for the quantitative determination of DNA with LOD of 3.45 ng in methanol-water (9:1, v/v). The association constants of DNA with Tb(III)-CMMC complex was found to be 2.62 Multiplication-Sign 1010 M{sup -1}. - Highlights: Black-Right-Pointing-Pointer New fluorescent probe Terbium (III)-7-carboxy methoxy-4-methylcoumarin complex has been synthesized and characterized. Black-Right-Pointing-Pointer FTIR spectrum of Tb(III)-complex shows a characteristic band for thiocyanate group. Black-Right-Pointing-Pointer DNA interaction with Terbium (III)-7-carboxy methoxy-4-methylcoumarin has been studied by fluorescence techniques. Black-Right-Pointing-Pointer The change in the fluorescence intensity has been used for the quantitative determination of DNA. Black-Right-Pointing-Pointer The result was better than most of the well-known methods including the ethidium bromide method.

  10. D-glucose derived novel gemini surfactants: synthesis and study of their surface properties, interaction with DNA, and cytotoxicity.

    Science.gov (United States)

    Kumar, Vikash; Chatterjee, Amrita; Kumar, Nupur; Ganguly, Anasuya; Chakraborty, Indranil; Banerjee, Mainak

    2014-10-09

    Four new D-glucose derived m-s-m type gemini surfactants with variable spacer and tail length have been synthesized by a simple and efficient synthetic methodology utilizing the free C-3 hydroxy group of diisopropylidene glucose. The synthetic route to these gemini surfactants with a quaternary ammonium group as polar head group involves a sequence of simple reactions including alkylation, imine formation, quaternization of amine etc. The surface properties of the new geminis were evaluated by surface tension and conductivity measurements. These gemini surfactants showed low cytotoxicity by MTT assay on HeLa cell line. The DNA binding capabilities of these surfactants were determined by agarose gel electrophoresis, fluorescence titration, and DLS experiments. The preliminary studies by agarose gel electrophoresis indicated chain length dependent DNA binding abilities, further supported by ethidium bromide exclusion experiments. Two of the D-glucose derived gemini surfactants showed effective binding with pET-28a plasmid DNA (pDNA) at relatively low N/P ratio (i.e., cationic nitrogen/DNA phosphate molar ratio).

  11. New mixed ligand complexes of ruthenium(II) that incorporate a modified phenanthroline ligand: Synthesis, spectral characterization and DNA binding

    Indian Academy of Sciences (India)

    S Murali; C V Sastri; Bhaskar G Maiya

    2002-08-01

    The hexafluorophosphate and chloride salts of two ruthenium(II) complexes, viz. [Ru(phen)(ptzo)2]2+ and [Ru(ptzo)3]2+, where ptzo = 1,10-phenanthrolino[5,6-]1,2,4-triazine-3-one (ptzo) - a new modified phenanthroline (phen) ligand, have been synthesised. These complexes have been characterised by infrared, UV-Vis, steady-state emission and 1H NMR spectroscopic methods. Results of absorption and fluorescence titration as well as thermal denaturation studies reveal that both the bis- and tris-complexes of ptzo show moderately strong affinity for binding with calf thymus (CT) DNA with the binding constants being close to 105M-1 in each case. An intercalative mode of DNA binding has been suggested for both the complexes. Emission studies carried out in non-aqueous solvents and in aqueous media without DNA reveal that both [Ru(phen)(ptzo)2]2+ and [Ru(ptzo)3]2+ are weakly luminescent under these solution conditions. Successive addition of CT DNA to buffered aqueous solutions containing [Ru(phen)(ptzo)2]2+ results in an enhancement of the emission. These results have been discussed in the light of the dependence of the structure-specific deactivation processes of the MLCT state of the metallointercalator with the characteristic features of its DNA interaction. In doing so, attempts have been made to compare and contrast its properties with those of the analogous phenanthroline-based complexes including the ones reported by us previously.

  12. Synthesis, characterization and multi-spectroscopic DNA interaction studies of a new platinum complex containing the drug metformin.

    Science.gov (United States)

    Shahabadi, Nahid; Heidari, Leila

    2014-07-15

    A new platinum(II) complex; [Pt(Met)(DMSO)Cl]Cl in which Met = metformin and DMSO: dimethylsulfoxide, was synthesized and characterized by (1)H NMR, IR, UV-Vis spectra, molar conductivity and computational methods. Binding interaction of this complex with calf thymus (CT) DNA has been investigated by using absorption, emission, circular dichroism, viscosity measurements, differential pulse voltammetry and cleavage studies by agarose gel electrophoresis. UV-Vis absorption studies showed hyperchromism. CD studies showed less perturbation on the base stacking and helicity bands in the CD spectrum of CT-DNA (B→C structural transition). In fluorimeteric studies, the Pt(II) complex can bind with DNA-NR complex and forms a new non-fluorescence adduct. The anodic peak current in the differential pulse voltammogram of the Pt(II) complex decreased gradually with the addition of DNA. Cleavage experiments showed that the Pt(II) complex does not induce any cleavage under the experimental setup. Finally all results indicated that Pt(II) complex interact with DNA via groove binding mode.

  13. Palladium(II) complexes as biologically potent metallo-drugs: Synthesis, spectral characterization, DNA interaction studies and antibacterial activity

    Science.gov (United States)

    Prasad, Kollur Shiva; Kumar, Linganna Shiva; Chandan, Shivamallu; Naveen Kumar, R. M.; Revanasiddappa, Hosakere D.

    2013-04-01

    Four novel mononuclear Pd(II) complexes have been synthesized with the biologically active Schiff base ligands (L1-L4) derived from 3-amino-2-methyl-4(3H)-quinazolinone. The structure of the complexes has been proposed by elemental analysis, molar conductance, IR, 1H NMR, mass, UV-Vis spectrometric and thermal studies. The investigation of interaction of the complexes with calf thymus DNA (CT-DNA) has been performed with absorption and fluorescence spectroscopic studies. The nuclease activity was done using pUC19 supercoiled DNA by gel-electrophoresis. All the ligands and their Pd(II) complexes have also been screened for their antibacterial activity by discolor diffusion technique.

  14. Design, synthesis, DNA-binding affinity, cytotoxicity, apoptosis, and cell cycle arrest of Ru(II) polypyridyl complexes.

    Science.gov (United States)

    Venkat Reddy, Putta; Reddy, Mallepally Rajender; Avudoddi, Srishailam; Praveen Kumar, Yata; Nagamani, Chintakuntla; Deepika, Nancherla; Nagasuryaprasad, K; Singh, Surya Satyanarayana; Satyanarayana, Sirasani

    2015-09-15

    A novel polypyridyl ligand CNPFIP (CNPFIP=2-(5(4-chloro-2-nitrophenyl)furan-2-yl)-1H-imidazo[4,5f][1,10]phenanthroline) and its mononuclear Ru(II) polypyridyl complexes of [Ru(phen)2CNPFIP](2+)(1) (phen=1,10-phenanthroline), [Ru(bpy)2CNPFIP](2+)(2) (bpy=2,2'-bipyridine), and [Ru(dmb)2CNPFIP](2+)(3) (dmb=4,4'-dimethyl-2,2'-bipyridine) have been synthesized successfully and characterized thoroughly by elemental analysis, UV/Vis, IR, NMR, and ESI-MS. The interaction of the Ru(II) complexes with calf thymus DNA (CT-DNA) was investigated by absorption titration, fluorescence, viscosity measurements. The experimental results suggest that three complexes bind to CT-DNA through an intercalative mode and the DNA-binding affinity of complex 1 is greater than that of complexes 2 and 3. The photocleavage of plasmid pBR322 DNA by ruthenium complexes 1, 2, and 3 was investigated. We have also tested three complexes for their antimicrobial activity against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria. The in vitro cytotoxicity of these complexes was evaluated by MTT assay, and complex 1 shows higher cytotoxicity than 2 and 3 on HeLa cells. The induced apoptosis and cell cycle arrest of HeLa cells were investigated by flow cytometry for 24h. The molecular docking of ruthenium complexes 1, 2, and 3 with the active site pocket residues of human DNA TOP1 was performed using LibDock.

  15. Two half-sandwiched ruthenium (II compounds containing 5-fluorouracil derivatives: synthesis and study of DNA intercalation.

    Directory of Open Access Journals (Sweden)

    Zhao-Jun Li

    Full Text Available Two novel coordination compounds of half-sandwiched ruthenium(II containing 2-(5-fluorouracil-yl-N-(pyridyl-acetamide were synthesized, and their intercalation binding modes with calf thymus DNA were revealed by hyperchromism of ultraviolet-visible spectroscopy; the binding constants were determined according to a Langmuir adsorption equation that was deduced on the base of careful cyclic voltammetry measurements. The two compounds exhibited DNA intercalation binding activities with the binding constants of 1.13×106 M-1 and 5.35 ×105 M-1, respectively.

  16. Two half-sandwiched ruthenium (II) compounds containing 5-fluorouracil derivatives: synthesis and study of DNA intercalation.

    Science.gov (United States)

    Li, Zhao-Jun; Hou, Yong; Qin, Da-An; Jin, Zhi-Min; Hu, Mao-Lin

    2015-01-01

    Two novel coordination compounds of half-sandwiched ruthenium(II) containing 2-(5-fluorouracil)-yl-N-(pyridyl)-acetamide were synthesized, and their intercalation binding modes with calf thymus DNA were revealed by hyperchromism of ultraviolet-visible spectroscopy; the binding constants were determined according to a Langmuir adsorption equation that was deduced on the base of careful cyclic voltammetry measurements. The two compounds exhibited DNA intercalation binding activities with the binding constants of 1.13×106 M-1 and 5.35 ×105 M-1, respectively.

  17. The synthesis of new amphiphilic p-tert-butylthiacalix[4]arenes containing peptide fragments and their interaction with DNA.

    Science.gov (United States)

    Padnya, Pavel L; Andreyko, Elena A; Mostovaya, Olga A; Rizvanov, Ildar Kh; Stoikov, Ivan I

    2015-06-01

    New water-soluble p-tert-butylthiacalix[4]arenes containing peptide and quaternary ammonium fragments in cone and 1,3-alternate conformations were synthesized and characterized. The interaction of the macrocycles with DNA was studied by UV-spectroscopy, DLS and TEM. It was shown that the interaction of the self-associates based on p-tert-butylthiacalix[4]arenes tetrasubstituted at the lower rim with glycine and quaternary ammonium fragments in cone and 1,3-alternate conformations with DNA led to the formation of particles of about 99-192 nm in size.

  18. Prolactin-like activity of anti-prolactin receptor antibodies on casein and DNA synthesis in the mammary gland.

    OpenAIRE

    Djiane, J; Houdebine, L M; Kelly, P A

    1981-01-01

    Prolactin receptors were partially purified from rabbit mammary gland membranes by using an affinity chromatography technique. Antibodies against this prolactin receptor preparation were obtained in guinea pig and sheep. Both antisera were able to inhibit the binding of 125I-labeled ovine prolactin to rabbit mammary gland membranes. When added to culture media of rabbit mammary explants, the anti-prolactin receptor antiserum inhibited the capacity of prolactin to initiate casein synthesis and...

  19. Solid-phase synthesis of thermolytic DNA oligonucleotides functionalized with a single 4-hydroxy-1-butyl or 4-phosphato-/thiophosphato-1-butyl thiophosphate protecting group.

    Science.gov (United States)

    Grajkowski, Andrzej; Ausín, Cristina; Kauffman, Jon S; Snyder, John; Hess, Sonja; Lloyd, John R; Beaucage, Serge L

    2007-02-02

    Several thermolytic CpG-containing DNA oligonucleotides analogous to 1 have been synthesized to serve as potential immunotherapeutic oligonucleotide prodrug formulations for the treatment of infectious diseases in animal models. Specifically, the CpG motif (GACGTT) of each DNA oligonucleotide has been functionalized with either the thermolabile 4-hydroxy-1-butyl or the 4-phosphato-/thiophosphato-1-butyl thiophosphate protecting group. This functionalization was achieved through incorporation of activated deoxyribonucleoside phosphoramidite 8b into the oligonucleotide chain during solid-phase synthesis and, optionally, through subsequent phosphorylation effected by phosphoramidite 9. Complete conversion of CpG ODNs hbu1555, psb1555, and pob1555 to CpG ODN 1555 (homologous to 2) occurred under elevated temperature conditions, thereby validating the function of these diastereomeric oligonucleotides as prodrugs in vitro. Noteworthy is the significant increase in solubility of CpG ODN psb1555 and CpG pob1555 in water when compared to that of neutral CpG ODN fma1555 (homologous to 1).

  20. ex vivo DNA assembly

    Directory of Open Access Journals (Sweden)

    Adam B Fisher

    2013-10-01

    Full Text Available Even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. Advances in cloning techniques have resulted in powerful in vitro and in vivo assembly of DNA. However, monetary and time costs have limited these approaches. Here, we report an ex vivo DNA assembly method that uses cellular lysates derived from a commonly used laboratory strain of Escherichia coli for joining double-stranded DNA with short end homologies embedded within inexpensive primers. This method concurrently shortens the time and decreases costs associated with current DNA assembly methods.

  1. DNA display I. Sequence-encoded routing of DNA populations.

    Directory of Open Access Journals (Sweden)

    David R Halpin

    2004-07-01

    Full Text Available Recently reported technologies for DNA-directed organic synthesis and for DNA computing rely on routing DNA populations through complex networks. The reduction of these ideas to practice has been limited by a lack of practical experimental tools. Here we describe a modular design for DNA routing genes, and routing machinery made from oligonucleotides and commercially available chromatography resins. The routing machinery partitions nanomole quantities of DNA into physically distinct subpools based on sequence. Partitioning steps can be iterated indefinitely, with worst-case yields of 85% per step. These techniques facilitate DNA-programmed chemical synthesis, and thus enable a materials biology that could revolutionize drug discovery.

  2. Synthesis, Molecular Structure, DNA/Protein Binding, Cytotoxicity, Apoptosis, Reactive Oxygen Species, and Mitochondrial Membrane Potential of Dibenzoxanthenes Derivatives.

    Science.gov (United States)

    Yang, Hui-Hui; Han, Bing-Jie; Li, Wei; Liu, Yun-Jun; Wang, Xiu-Zhen

    2015-12-01

    Two dibenzoxanthene isomers 3 and 4 were synthesized and characterized. The crystal structures of the two compounds were solved by single-crystal X-ray diffraction. Binding of two compounds with calf thymus DNA (CT DNA) and BSA (bovine serum albumin) has been thoroughly investigated by UV-Vis and fluorescence spectroscopy. The DNA-binding constants were determined to be 2.51 (± 0.09) × 10(3) for compound 3 and 4.55 (± 0.10) × 10(3) for compound 4. Two compounds can cleave pBR322 DNA upon irradiation. Significant nuclear damages of BEL-7402 cells were observed with compound treatment in a comet assay. The cytotoxicity in vitro was investigated by MTT method. These compounds have been found to induce nuclear condensation and fragmentation in BEL-7402 cells. The two compounds can enhance intracellular reactive oxygen species and decrease the mitochondrial membrane potential. The compounds activated caspase-3 and caspase-7, down-regulated the expression levels of anti-apoptotic protein Bcl-2, and up-regulated the expression levels of pro-apoptotic protein Bax. These compounds induce apoptosis of BEL-7402 cells through an ROS-mediated mitochondrial dysfunction pathway.

  3. Synthesis, physicochemical studies, embryos toxicity and DNA interaction of some new Iron(II) Schiff base amino acid complexes

    Science.gov (United States)

    Abdel-Rahman, Laila H.; El-Khatib, Rafat M.; Nassr, Lobna A. E.; Abu-Dief, Ahmed M.

    2013-05-01

    New Fe(II) Schiff base amino acid complexes derived from the condensation of o-hydroxynaphthaldehyde with L-alanine, L-phenylalanine, L-aspartic acid, L-histidine and L-arginine were synthesized and characterized by elemental analysis, IR, electronic spectra, and conductance measurements. The stoichiometry and the stability constants of the complexes were determined spectrophotometrically. The investigated Schiff bases exhibited tridentate coordination mode with the general formulae [Fe(HL)2]·nH2O for all amino acids except L-histidine. But in case of L-histidine, the ligand acts as tetradentate ([FeL(H2O)2]·2H2O), where HL = mono anion and L = dianion of the ligand. The structure of the prepared complexes is suggested to be octahedral. The prepared complexes were tested for their toxicity on chick embryos and found to be safe until a concentration of 100 μg/egg with full embryos formation. The interaction between CT-DNA and the investigated complexes were followed by spectrophotometry and viscosity measurements. It was found that, the prepared complexes bind to DNA via classical intercalative mode and showed a different DNA cleavage activity with the sequence: nhi > nari > nali > nasi > nphali. The thermodynamic Profile of the binding of nphali complex and CT-DNA was constructed by analyzing the experimental data of absorption titration and UV melting studies with the McGhee equation, van't Hoff's equation, and the Gibbs-Helmholtz equation.

  4. Cu(I) complexes of bis(methyl)(thia/selena) salen ligands: Synthesis, characterization, redox behavior and DNA binding studies

    Science.gov (United States)

    Asatkar, Ashish K.; Tripathi, Mamta; Panda, Snigdha; Pande, Rama; Zade, Sanjio S.

    2017-01-01

    Mononuclear cuprous complexes 1 and 2, [{CH3E(o-C6H4)CH = NCH2}2Cu]ClO4; E = S/Se, have been synthesized by the reaction of bis(methyl)(thia/selena) salen ligands and [Cu(CH3CN)4]ClO4. Both the products were characterized by elemental analysis, ESI-MS, FT-IR, 1H/13C/77Se NMR, and cyclic voltammetry. The complexes possess tetrahedral geometry around metal center with the N2S2/N2Se2 coordination core. Cyclic voltammograms of complexes 1 and 2 displayed reversible anodic waves at E1/2 = + 0.08 V and + 0.10 V, respectively, corresponding to the Cu(I)/Cu(II) redox couple. DNA binding studies of both the complexes were performed applying absorbance, fluorescence and molecular docking techniques. Competitive binding experiment of complexes with ct-DNA against ethidium bromide is performed to predict the mode of binding. The results indicate the groove binding mode of complexes 1 and 2 to DNA. The binding constants revealed the strong binding affinity of complexes towards ct-DNA.

  5. Synthesis, biological investigation, calf thymus DNA binding and docking studies of the sulfonyl hydrazides and their derivatives

    Science.gov (United States)

    Murtaza, Shahzad; Shamim, Saima; Kousar, Naghmana; Tahir, Muhammad Nawaz; Sirajuddin, Muhammad; Rana, Usman Ali

    2016-03-01

    The present study describes the syntheses and biological investigations of sulfonyl hydrazides and their novel derivatives. The detailed investigations involved the characterization of the newly synthesized compounds using FTIR, NMR, mass spectrometry and by single crystal X-Ray diffraction (XRD) analysis techniques. The binding tendencies of these compounds with CT-DNA (calf thymus DNA) have been explored by electronic absorption (UV) spectroscopy and viscosity measurement. The binding constant (K) and Gibb's free energy (ΔG) values were also calculated accordingly. In addition, we also investigated the biological activities such as antioxidant, antibacterial, enzyme inhibition and DNA interactions. The antioxidant activity was assayed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, while antibacterial activity was investigated against four bacterial strains (viz. Escherichia coli, Crynibacteria bovius, Staphylococcus auras and Bacillus antherasis) by employing the common disc diffusion method. Enzyme inhibition activity of the synthesized compounds was examined against butyrylcholinestrase. The results of enzyme inhibition activity and the DNA binding interaction studies were also collected through molecular docking program using computational analysis. Our study reveals that the newly synthesized compounds possess moderate to good biological activities.

  6. Synthesis, structural characterization and biological activity of a trinuclear zinc(II) complex: DNA interaction study and antimicrobial activity

    Indian Academy of Sciences (India)

    Bhaskar Biswas; Niranjan Kole; Moumita Patra; Shampa Dutta; Mousumi Ganguly

    2013-11-01

    A trinuclear zinc(II) complex [Zn3L2(-O2CCH3)2(H2O)2]·H2O·2CH3OH (1) was synthesized from an in situ reaction between zinc acetate and a Schiff base ligand (H2L = 2-((2-hydroxyphenylimino) methyl)-6-methoxyphenol). The ligand was prepared by (1:1) condensation of ortho-vanillin and ortho-aminophenol. The ligand and zinc(II) complex were characterized by elemental analysis, Fourier Transform Infrared (FTIR), 1H-Nuclear Magnetic Resonance (NMR), UV-Vis spectroscopy, Powder X-ray Diffraction (PXRD) and thermogravimetric analysis. 1 crystallizes in P-1 space group with = 11.9241(3) Å, = 12.19746 Å, = 20.47784 Å with unit cell volume is 2674.440 (Å)3. Binding property of the complex with calf thymus DNA (CT-DNA) has been investigated using absorption and emission studies. Thermal melting and viscosity experiments were further performed to determine the mode of binding of 1 with CT-DNA. Spectroscopic and viscosity investigations revealed an intercalative binding mode of 1 with CT-DNA. The ligand and its zinc complex were screened for their biological activity against bacterial species and fungi. Activity data show that the metal complex has more antibacterial and antifungal activity than the parent Schiff base ligand and against those bacterial or fungi species.

  7. Synthesis and studies of polypeptide materials: Self-assembled block copolypeptide amphiphiles, DNA-condensing block copolypeptides and membrane-interactive random copolypeptides

    Science.gov (United States)

    Wyrsta, Michael Dmytro

    A new class of transition metal initiators for the controlled polymerization of alpha-aminoacid-N-carboxyanhydrides (alpha-NCAs), has been developed by Deming et al. This discovery has allowed for the synthesis of well-defined "protein-like" polymers. Using this chemistry we have made distinct block/random copolypeptides for biomedical applications. Drug delivery, gene delivery, and antimicrobial polymers were the focus of our research efforts. The motivation for the synthesis and study of synthetic polypeptide based materials comes from proteins. Natural proteins are able to adopt a staggeringly large amount of uniquely well-defined folded structures. These structures account for the diversity in properties of proteins. As catalysts (enzymes) natural proteins perform some of the most difficult chemistry with ease and precision at ambient pressures and temperatures. They also exhibit incredible structural properties that directly result from formation of complex hierarchical assemblies. Self-assembling block copolymers were synthesized with various compositions and architectures. In general, di- and tri-block amphiphiles were studied for their self-assembling properties. Both spherical and tubular vesicles were found to assemble from di- and tri-block amphiphiles, respectively. In addition to self-assembly, pH responsiveness was engineered into these amphiphiles by the incorporation of basic residues (lysine) into the hydrophobic block. Another form of self-assembly studied was the condensation of DNA using cationic block copolymers. It was found that cationic block copolymers could condense DNA into compact, ordered, water-soluble aggregates on the nanoscale. These aggregates sufficiently protected DNA from nucleases and yet were susceptible to proteases. These studies form the basis of a gene delivery platform. The ease with which NCAs are polymerized renders them completely amenable to parallel synthetic methods. We have employed this technique to discover new

  8. A critical role for the cccA gene product, cytochrome c2, in diverting electrons from aerobic respiration to denitrification in Neisseria gonorrhoeae.

    Science.gov (United States)

    Hopper, Amanda C; Li, Ying; Cole, Jeffrey A

    2013-06-01

    Neisseria gonorrhoeae is a microaerophile that, when oxygen availability is limited, supplements aerobic respiration with a truncated denitrification pathway, nitrite reduction to nitrous oxide. We demonstrate that the cccA gene of Neisseria gonorrhoeae strain F62 (accession number NG0292) is expressed, but the product, cytochrome c2, accumulates to only low levels. Nevertheless, a cccA mutant reduced nitrite at about half the rate of the parent strain. We previously reported that cytochromes c4 and c5 transfer electrons to cytochrome oxidase cbb3 by two independent pathways and that the CcoP subunit of cytochrome oxidase cbb3 transfers electrons to nitrite. We show that mutants defective in either cytochrome c4 or c5 also reduce nitrite more slowly than the parent. By combining mutations in cccA (Δc2), cycA (Δc4), cycB (Δc5), and ccoP (ccoP-C368A), we demonstrate that cytochrome c2 is required for electron transfer from cytochrome c4 via the third heme group of CcoP to the nitrite reductase, AniA, and that cytochrome c5 transfers electrons to nitrite reductase by an independent pathway. We propose that cytochrome c2 forms a complex with cytochrome oxidase. If so, the redox state of cytochrome c2 might regulate electron transfer to nitrite or oxygen. However, our data are more consistent with a mechanism in which cytochrome c2 and the CcoQ subunit of cytochrome oxidase form alternative complexes that preferentially catalyze nitrite and oxygen reduction, respectively. Comparison with the much simpler electron transfer pathway for nitrite reduction in the meningococcus provides fascinating insights into niche adaptation within the pathogenic neisseriae.

  9. A custom correlation coefficient (CCC) approach for fast identification of multi-SNP association patterns in genome-wide SNPs data.

    Science.gov (United States)

    Climer, Sharlee; Yang, Wei; de las Fuentes, Lisa; Dávila-Román, Victor G; Gu, C Charles

    2014-11-01

    Complex diseases are often associated with sets of multiple interacting genetic factors and possibly with unique sets of the genetic factors in different groups of individuals (genetic heterogeneity). We introduce a novel concept of custom correlation coefficient (CCC) between single nucleotide polymorphisms (SNPs) that address genetic heterogeneity by measuring subset correlations autonomously. It is used to develop a 3-step process to identify candidate multi-SNP patterns: (1) pairwise (SNP-SNP) correlations are computed using CCC; (2) clusters of so-correlated SNPs identified; and (3) frequencies of these clusters in disease cases and controls compared to identify disease-associated multi-SNP patterns. This method identified 42 candidate multi-SNP associations with hypertensive heart disease (HHD), among which one cluster of 22 SNPs (six genes) included 13 in SLC8A1 (aka NCX1, an essential component of cardiac excitation-contraction coupling) and another of 32 SNPs had 29 from a different segment of SLC8A1. While allele frequencies show little difference between cases and controls, the cluster of 22 associated alleles were found in 20% of controls but no cases and the other in 3% of controls but 20% of cases. These suggest that both protective and risk effects on HHD could be exerted by combinations of variants in different regions of SLC8A1, modified by variants from other genes. The results demonstrate that this new correlation metric identifies disease-associated multi-SNP patterns overlooked by commonly used correlation measures. Furthermore, computation time using CCC is a small fraction of that required by other methods, thereby enabling the analyses of large GWAS datasets.

  10. Synthesis, chemical characterization, computational studies and biological activity of new DNA methyltransferases (DNMTs) specific inhibitor. Epigenetic regulation as a new and potential approach to cancer therapy.

    Science.gov (United States)

    Pellerito, C; Morana, O; Ferrante, F; Calvaruso, G; Notaro, A; Sabella, S; Fiore, T

    2015-09-01

    This work deals with the synthesis, the chemical characterization of dibutyltin(IV) complex of caffeic acid (Bu2Sn(IV)HCAF, caf1) and its cytotoxic action on tumor cells. The coordination environment at the tin center was investigated by FTIR, (119)Sn{(1)H} cross polarization magic angle spinning, electrospray ionization mass spectroscopy in the solid state and UV-vis, fluorescence and (1)H, (13)C and (119)Sn NMR spectroscopy in solution phases. Density functional theory study confirmed the proposed structures in solution phase and indicated the most probably stable conformation. The effects on viability of breast cancer MDA-MB231, colorectal cancer HCT116, hepatocellular carcinoma HepG2 and Chang liver cells, an immortalized non-tumor hepatic cell line, have been investigated. The effect of a variation in structure of caf1 was found to lead to a change in the respective antiproliferative properties: caf1 induces loss of viability in HCT116, MDA-MB-231, and HepG2; the complex shows only moderate effects in non-tumor Chang liver cells. caf1 exerts lower cytotoxic activity than Bu2SnCl2, suggesting that the binding with H3CAF modulates the marked cytotoxic activity exerted by Bu2SnCl2; caf1 displays a considerably more pronounced antitumoural effect towards cell lines than caffeic acid. It is known that caffeic acid can modulate DNA (cytosine-5)-methyltransferases 1 (DNMT1) mediated DNA methylation. In this paper we demonstrate that caf1 treatment was able to induce a time-dependent reduction of global DNA methylated status. This effect was also confirmed by a concomitant reduction DNMT1 expression level. The effect induced by caf1 was more evident not only with respect to untreated cells but also compared to H3CAF treated cells.

  11. Synthesis, micellization behavior, antimicrobial and intercalative DNA binding of some novel surfactant copper(II) complexes containing modified phenanthroline ligands.

    Science.gov (United States)

    Nagaraj, Karuppiah; Ambika, Subramanian; Rajasri, Shanmugasundaram; Sakthinathan, Subramanian; Arunachalam, Sankaralingam

    2014-10-01

    The novel surfactant copper(II) complexes, [Cu(ip)2DA](ClO4)21, [Cu(dpqc)2DA](ClO4)22, [Cu(dppn)2DA](ClO4)23, where ip=imidazo[4,5-f][1,10]phenanthroline, dpqc=dipyrido[3,2-a:2',4'-c](6,7,8,9-tetrahydro)phenazine, dppn=benzo[1]dipyrido[3,2-a':2',3'-c]phenazine and DA-dodecylamine, were synthesized and characterized by physico-chemical and spectroscopic methods. In these complexes 1-3, the geometry of copper metal ions was described as square pyramidal. The critical micelle concentration (CMC) value of these surfactant copper(II) complexes in aqueous solution was found out from conductance measurements. Specific conductivity data at different temperatures served for the evaluation of the temperature-dependent CMC and the thermodynamics of micellization (ΔGm°, ΔHm° and ΔSm°). The binding interaction of these complexes with DNA (calf thymus DNA) in Tris buffer was studied by physico-chemical techniques. In the presence of the DNA UV-vis spectrum of complexes showed red shift of the absorption band along with significant hypochromicity indicating intercalation of our complexes with nucleic acids. Competitive binding study with ethidium bromide (EB) shows that the complexes exhibit the ability to displace the nucleic acid-bound EB indicating that the complexes bind to nucleic acids in strong competition with EB for the intercalative binding site. Observed changes in the circular dichoric spectra of DNA in the presence of surfactant complexes support the strong binding of complexes with DNA. CV results also confirm this mode of binding. Some significant thermodynamic parameters of the binding of the titled complexes to DNA have also been determined. The results reveal that the extent of DNA binding of 3 was greater than that of 1 and 2. The antibacterial and antifungal screening tests of these complexes have shown good results compared to its precursor chloride complexes.

  12. Synthesis, spectroscopic characterisation, thermal analysis, DNA interaction and antibacterial activity of copper(I) complexes with N, N‧- disubstituted thiourea

    Science.gov (United States)

    Chetana, P. R.; Srinatha, B. S.; Somashekar, M. N.; Policegoudra, R. S.

    2016-02-01

    copper(I) complexes [Cu(4MTU)2Cl] (2), [Cu(4MTU) (B)Cl] (3), [Cu(6MTU)2Cl] (5) and [Cu(6MTU) (B)Cl] (6) where 4MTU = 1-Benzyl-3-(4-methyl-pyridin-2-yl)-thiourea (1) and 6MTU = 1-Benzyl-3-(6-methyl-pyridin-2-yl)-thiourea (4), B is a N,N-donor heterocyclic base, viz. 1,10-phenanthroline (phen 3, 6), were synthesized, characterized by various physico-chemical and spectroscopic techniques. The elemental analysis suggests that the stoichiometry to be 1:2 (metal:ligand) for 2, 5 1:1:1 (metal:ligand:B) for 3, 6. X-ray powder diffraction illustrates that the complexes have crystalline nature. IR data coupled with electronic spectra and molar conductance values suggest that the complex 2, 5 show the presence of a trigonal planar geometry and the complex 3, 6 show the presence of a tetrahedral geometry about the Cu(I) centre. The binding affinity towards calf thymus (CT) DNA was determined using UV-Vis, fluorescence spectroscopic titrations and viscosity studies. These studies showed that the tested phen complexes 3, 6 bind moderately (in the order of 105 M-1) to CT DNA. The complex 2, 5 does not show any apparent binding to the DNA and hence poor cleavage efficiency. Complex 3, 6 shows efficient oxidative cleavage of plasmid DNA in the presence of H2O2 involving hydroxyl radical species as evidenced from the control data showing inhibition of DNA cleavage in the presence of DMSO and KI. The in vitro antibacterial assay indicates that these complexes are good antimicrobial agents against various pathogens. Anti-bacterial activity is higher when thiourea coordinates to metal ion than the thiourea alone.

  13. Copper(II) complexes with new fluoroquinolones: Synthesis, structure, spectroscopic and theoretical study, DNA damage, cytotoxicity and antiviral activity.

    Science.gov (United States)

    Dorotíková, Sandra; Kožíšková, Júlia; Malček, Michal; Jomová, Klaudia; Herich, Peter; Plevová, Kristína; Briestenská, Katarína; Chalupková, Anna; Mistríková, Jela; Milata, Viktor; Dvoranová, Dana; Bučinský, Lukáš

    2015-09-01

    Copper(II) complexes with fluoroquinolones in the presence of the nitrogen donor heterocyclic ligands 1,10-phenanthroline have been considered in detail. The phenanthroline moiety was introduced into the ligand environment with the aim to determine whether the nuclease activity is feasible. All suitable X-ray structures of the complexes under study reveal a distorted square pyramidal coordination geometry for Cu(II) atom. The conformational and spectroscopic (FT-IR and UV-visible) behavior has been analyzed and has been interpreted with respect to B3LYP/6-311G* calculations including molecular dynamics. The ability of the complexes to cleave DNA was studied by agarose gel electrophoresis with plasmid DNA pBSK+. The results have confirmed that the complexes under study behave as the chemical nucleases. Nuclease like activity in the absence of hydrogen peroxide allows us to deduce an interaction of the complexes with the DNA resulting in the conversion of supercoiled circular DNA to the nicked form. The DNA cleavage activity enhanced by the presence of hydrogen peroxide demonstrates the participation of reactive oxygen species, such as superoxide radical anions and hydroxyl radicals which presence was confirmed independently using the standard radical scavenging agents. It has been suggested that the radical formation through the Fenton/Haber-Weiss reaction is mediated by the redox cycling mechanisms with the participation of cupric/cuprous ions. Cytotoxic activity was evaluated as the 50% cytotoxic concentration (CC50). The potential effects of tested compounds on replication of murine gammaherpesvirus 68 (MHV-68) under in vitro conditions were also evaluated. However, no antiviral activity against MHV-68 was observed.

  14. Dwarfing effects of paclobutrazol and CCC on hedge plant Carmona microphylla%多效唑与矮壮素对福建茶的矮化效果

    Institute of Scientific and Technical Information of China (English)

    黄旭光; 罗恩波; 陆仟; 黄晓红; 雷雯; 罗宇

    2012-01-01

    [目的]分析多效唑与矮壮素对福建茶的矮化效果和经济效益,为寻找节约型的化学修剪新途径、合理使用植物生长延缓剂、降低城市园林绿化养护成本提供参考依据.[方法]调查不同浓度多效唑与矮壮素及其混剂作用下福建茶的叶面积、叶厚度、叶绿素含量和新枝条生长量,研究其对福建茶矮化效果和经济效益的影响.[结果]600倍矮壮素+600倍多效唑混剂或400倍多效唑对福建茶的矮化效果较好,全年喷施3次能使福建茶绿篱保持良好的景观效果,节约成本约29.82元/m2.[结论]绿篱植物福建茶喷施一定剂量多效唑与矮壮素的混剂可以代替人工修剪,节约成本.%This research aimed to analyze the dwarfing effects of paclobutrazol and CCC on hedge plant Carmona microphylla and rclrvemt economic benefits in order to obtain an economical way of chemical pruning the hedge plant, reasonably using plant growth retardation, and decreasing maintenance cost of unban landscaping. [Method]The leaf area, leaf thickness, chlorophyll content, and new shoot increment of Carmona microphylla were analyzed after each subject was treated with different concentrations of paclobutrazol and CCC; their mixture was used to investigate the corresponding dwarfing effects and economic benefits. [Result]Trie optimum combinations to create the best dwrafting effects were determined to be as follows: 600 times of CCC+600 times of paclobutrazol or 400 times of paclobutrazol. According to the large-scale experiment, spraying the above combinations three times a year to Carmona microphylla could assure excellent landscaping effect. Meanwhile, the production cost could be lowered by 29.82 yuan/m2 as opposed to not using the chemical concentration. [ Conclusion ] Artificially pruning Carmona microphylla could be replaced by spraying certain concentrations of paclobutrazol and CCC, as well as their mixture, which could save the cost production.

  15. Final work plan : indoor air and ambient air sampling near the former CCC/USDA grain storage facility in Everest, Kansas.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M. (Environmental Science Division)

    2010-05-24

    The Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) operated a grain storage facility at the western edge of Everest, Kansas, from the early 1950s to the early 1970s. Sampling by the Kansas Department of Health and Environment (KDHE) in 1997 resulted in the detection of carbon tetrachloride in one domestic well (the Nigh well) northwest of the former facility. On behalf of the CCC/USDA, Argonne National Laboratory subsequently conducted a series of investigations to characterize the contamination (Argonne 2003, 2006a,b,c). Automatic, continuous monitoring of groundwater levels began in 2002 and is ongoing at six locations. The results have consistently indicated groundwater flow toward the north-northwest from the former CCC/USDA property to the Nigh property, then west-southwest from the Nigh property to the intermittent creek. Sitewide periodic groundwater and surface water sampling with analysis for volatile organic compounds (VOCs) began in 2008. Argonne's combined data indicate no significant downgradient extension of contamination since 2000. At present, the sampling is annual, as approved by the KDHE (2009) in response to a plan developed for the CCC/USDA (Argonne 2009). This document presents a plan for collecting indoor air samples in homes located along and adjacent to the defined extent of the carbon tetrachloride contamination. The plan was requested by the KDHE. Ambient air samples to represent the conditions along this pathway will also be taken. The purpose of the proposed work is to satisfy KDHE requirements and to collect additional data for assessing the risk to human health due to the potential upward migration of carbon tetrachloride and its primary degradation product (chloroform) into homes located in close proximity to the former grain storage facility, as well as along and within 100 ft laterally from the currently defined plume emanating from the former Everest facility. Investigation of the indoor air

  16. Replicon properties of chromosomal DNA fibers and the duration of DNA synthesis of sunflower root-tip meristem cells at different temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Van' t Hof, J.; Bjerknes, C.A.; Clinton, J.H.

    1978-01-01

    Chromosomal DNA fiber autoradiography was used to examine the replicon properties of root-tip meristem cells of Helianthus annuus intact seedlings grown at temperatures from 10 to 38/sup 0/C and those of root-tip cells grown in vitro at 23/sup 0/. The average replicon size was approximately 22 ..mu..m and it did not change with temperature nor when the roots were grown in culture. The average fork rate was 6 ..mu..m/h at 10/sup 0/ and it rose gradually to 12 ..mu..m/h at 38/sup 0/. The responses of replication fork movement and of the duration of S to temperature were of three types: those in which change in fork rate was primarily (more than 90%) responsible for change in the duration of S, those in which the fork rate remained constant while S increased nearly twofold, and those in which the duration of S increased even though the replication forks were moving faster. The first type of response listed was observed at temperatures from 20 to 35/sup 0/, the second type listed was observed at 10 to 15/sup 0/, and the third, was produced at 38/sup 0/.

  17. Recombinant covalently closed circular hepatitis B virus DNA induces prolonged viral persistence in immunocompetent mice.

    Science.gov (United States)

    Qi, Zhihua; Li, Gaiyun; Hu, Hao; Yang, Chunhui; Zhang, Xiaoming; Leng, Qibin; Xie, Youhua; Yu, Demin; Zhang, Xinxin; Gao, Yueqiu; Lan, Ke; Deng, Qiang

    2014-07-01

    It remains crucial to develop a laboratory model for studying hepatitis B virus (HBV) chronic infection. We hereby produced a recombinant covalently closed circular DNA (rcccDNA) in view of the key role of cccDNA in HBV persistence. A loxP-chimeric intron was engineered into a monomeric HBV genome in a precursor plasmid (prcccDNA), which was excised using Cre/loxP-mediated DNA recombination into a 3.3-kb rcccDNA in the nuclei of hepatocytes. The chimeric intron was spliced from RNA transcripts without interrupting the HBV life cycle. In cultured hepatoma cells, cotransfection of prcccDNA and pCMV-Cre (encoding Cre recombinase) resulted in accumulation of nuclear rcccDNA that was heat stable and epigenetically organized as a minichromosome. A mouse model of HBV infection was developed by hydrodynamic injection of prcccDNA. In the presence of Cre recombinase, rcccDNA was induced in the mouse liver with effective viral replication and expression, triggering a compromised T-cell response against HBV. Significant T-cell hyporesponsiveness occurred in mice receiving 4 μg prcccDNA, resulting in prolonged HBV antigenemia for up to 9 weeks. Persistent liver injury was observed as elevated alanine transaminase activity in serum and sustained inflammatory infiltration in the liver. Although a T-cell dysfunction was induced similarly, mice injected with a plasmid containing a linear HBV replicon showed rapid viral clearance within 2 weeks. Collectively, our study provides an innovative approach for producing a cccDNA surrogate that established HBV persistence in immunocompetent mice. It also represents a useful model system in vitro and in vivo for evaluating antiviral treatments against HBV cccDNA. Importance: (i) Unlike plasmids that contain a linear HBV replicon, rcccDNA established HBV persistence with sustained liver injury in immunocompetent mice. This method could be a prototype for developing a mouse model of chronic HBV infection. (ii) An exogenous intron was

  18. Five-coordinated oxovanadium(IV) complexes derived from amino acids and ciprofloxacin: synthesis, spectral, antimicrobial, and DNA interaction approach.

    Science.gov (United States)

    Patel, M N; Patel, S H; Chhasatia, M R; Parmar, P A

    2008-12-15

    Five-coordinated oxovanadium(IV) complexes with ciprofloxacin and various uninegative bidentate amino acids have been prepared. The structure of complexes has been investigated using spectral, physicochemical, mass spectroscopy, and elemental analyses. The antimicrobial activities (MIC) of the complexes, ligands, metal salt, and some standard drugs have been evaluated using the doubling dilution technique against Staphylococcus aureus, Bacillus subtilis, Serratia marcescens (gram-positive), and Pseudomonas aeruginosa, and Escherichia coli (gram-negative) bacteria. The result shows the significant increase in the antibacterial activity of the ligand, metal, and ciprofloxacin on complexation. The interaction of the complexes with pBR322 DNA has been investigated using spectroscopic, gel electrophoresis, and viscometric techniques. This shows that the complexes can bind to pBR322 DNA by the intercalative mode. The superoxide dismutase-like activity of the complexes has been determined.

  19. Synthesis, characterization, antibacterial activity, SOD mimic and interaction with DNA of drug based copper(II) complexes

    Science.gov (United States)

    Patel, Mohan N.; Dosi, Promise A.; Bhatt, Bhupesh S.; Thakkar, Vasudev R.

    2011-02-01

    Novel metal complexes of the second-generation quinolone antibacterial agent enrofloxacin with copper(II) and neutral bidentate ligands have been prepared and characterized with elemental analysis reflectance, IR and mass spectroscopy. Complexes have been screened for their in-vitro antibacterial activity against two Gram (+ve)Staphylococcus aureus, Bacillus subtilis, and three Gram (-ve)Serratia marcescens, Escherichia coli and Pseudomonas aeruginosa organisms using the double dilution technique. The binding of this complex with CT-DNA has been investigated by absorption titration, salt effect and viscosity measurements. Binding constant is ranging from 1.3 × 10 4-3.7 × 10 4. The cleavage ability of complexes has been assessed by gel electrophoresis using pUC19 DNA. The catalytic activity of the copper(II) complexes towards the superoxide anion (O 2rad -) dismutation was assayed by their ability to inhibit the reduction of nitroblue tetrazolium (NBT).

  20. 2,3-Bifunctionalized Quinoxalines: Synthesis, DNA Interactions and Evaluation of Anticancer, Anti-tuberculosis and Antifungal Activity

    Directory of Open Access Journals (Sweden)

    Tom Ellis

    2002-08-01

    Full Text Available A variety of 2,3-bifunctionalized quinoxalines (6-14 have been prepared by the condensation of 1,6-disubstituted-hexan-1,3,4,6-tetraones (1-4 with o-phenylenediamine, (R,R-1,2-diaminocyclohexane and p-nitro-o-phenylenediamine. It is concluded that strong intramolecular N-H----O bonds in the favoured keto-enamine form may be responsible for the minimal biological activities observed in DNA footprinting, antitubercular, anti-fungal and anticancer tests with these hyper π-conjugated quinoxaline derivatives. However, subtle alteration by addition of a nitro group affecting the charge distribution confers significant improvements in biological effects and binding to DNA.

  1. Synthesis, characterization, crystal structure and DNA-binding study of four cadmium(II) pyridine-carboxamide complexes

    Indian Academy of Sciences (India)

    BIPLAB MONDAL; BUDDHADEB SEN; SANDIPAN SARKAR; ENNIO ZANGRANDO; PABITRA CHATTOPADHYAY

    2017-01-01

    Treatment of perchlorate or nitrate salt of cadmium(II) with carboxamide derivatives (L) generated four novel mononuclear metal complexes, represented as [Cd(L)₄](ClO₄)₂ (1a and 1b) and [Cd(L)₂(ONO₂)₂] (2a and 2b) in appreciable yields (L = L¹ = N-(furan-2-ylmethyl)-2-pyridine carboxamide and L = L² = N-(thiophen-2-ylmethyl)-2-pyridine carboxamide). The complexes have been characterized by FT-IR, UVVisible, elemental analysis and single crystal X-ray crystallographic analysis which revealed eight coordinated cadmium ions, but in different coordination environments, depending on the counter anion used. In addition,electronic absorption, fluorescence spectroscopy and viscosity measurements revealed a significant interaction of the four complexes with CT-DNA via intercalative/groove binding mode. The intrinsic binding constant Kbobtained varies from 0.4 × 10⁴ to 1.11 × 10⁵ M⁻¹. The results suggest that neutral complexes 2a and 2b bind to DNA in an intercalative mode. On the other hand, cationic complexes 1a and 1b bind with DNA via weak electrostatic/covalent interaction.

  2. Novel complexes of Co(III) and Ni(II) containing peptide ligands: synthesis, DNA binding and photonuclease activity.

    Science.gov (United States)

    Sudhamani, C N; Bhojya Naik, H S; Girija, D; Sangeetha Gowda, K R; Giridhar, M; Arvinda, T

    2014-01-24

    The new cobalt(III) and nickel(II) complexes of the type [M(L)2(H2O)2](n)(+) (where M = Co(III) or Ni(II) ion, n = 3 for Co and 2 for Ni, L = peptides Fmoc. Ala-val-OH (F-AVOH), Fmoc-Phe-Leu-Ome (F-PLOMe) and Z-Ala-Phe-CONH2 (Z-APCONH2)) were synthesized and structurally characterized by FTIR, (1)H NMR, elemental analysis and electronic spectral data. An octahedral geometry has been proposed for all the synthesized Co(III) and Ni(II) metal complexes. The binding property of the complexes with CT-DNA was studied by absorption spectral analysis, followed by viscosity measurement and thermal denaturation studies. Detailed analysis revealed that the metal complexes intercalates into the DNA base stack as intercalator. The photo induced cleavage studies shows that the complexes possess photonuclease property against pUC19 DNA under UV-Visible irradiation.

  3. Synthesis, DNA/HSA Interaction Spectroscopic Studies and In Vitro Cytotoxicity of a New Mixed Ligand Cu(II) Complex.

    Science.gov (United States)

    Gan, Qian; Fu, Xiabing; Chen, Weijiang; Xiong, Yahong; Fu, Yinlian; Chen, Shi; Le, Xueyi

    2016-05-01

    A new mixed ligand copper(II)-dipeptide complex with 2-(2'-pyridyl)benzothiazole (pbt), [Cu(Gly-L-leu)(pbt)(H2O)]·ClO4 (Gly-L-leu = Glycyl-L-leucine anion) was synthesized and characterized by various physico-chemical means. The DNA binding and cleavage properties of the complex investigated by viscosity, agarose gel electrophoresis and multi-spectroscopic techniques (UV, circular dichroism (CD) and fluorescence) showed that the complex was bound to CT-DNA through intercalation mode with moderate binding constant (K b = 3.132 × 10(4) M(-1)), and cleaved pBR322 DNA efficiently (~ 5 μM) in the presence of Vc, probably via an oxidative mechanism induced by •OH. Additionally, the interaction of the complex with human serum albumin (HSA) was explored by UV-visible, CD, fluorescence, synchronous fluorescence and 3D fluorescence spectroscopy. The complex exhibits desired affinity to HSA through hydrophobic interaction. Moreover, the cytotoxicity of the complex against three human carcinoma cell lines (HeLa, HepG2 and A549) was evaluated by MTT assay, which showed that the complex had effective cytotoxicity and higher inhibition toward A549 cell lines with IC50 of 38.0 ± 3.2 μM.

  4. Synthesis of novel naphthoquinone-spermidine conjugates and their effects on DNA-topoisomerases I and II-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Cunha, Andrea S.; Lima, Edson L.S.; Pinto, Angelo C.; Esteves-Souza, Andressa; Torrese, Jose C. [Universidade Federal, Rio de Janeiro, RJ (Brazil). Inst. de Quimica; Echevarria, Aurea [Universidade Federal Rural do Rio de Janeiro, RJ (Brazil). Dept. de Quimica; Camara, Celso A. [Paraiba Univ., Joao Pessoa, PB (Brazil). Lab. de Tecnologia Farmaceutica; Vargas, Maria D. [Universidade Federal Fluminense, Niteroi, RJ (Brazil). Inst. de Quimica]. E-mail: mdvargas@vm.uff.br

    2006-05-15

    Novel derivatives of lapachol 2, nor-lapachol 3 and lawsone 4 have been synthesized by nucleophilic displacement of the methoxynaphthoquinones 2a, 3a and 4a with the polyamine (PA) N{sup 1}-Boc-N{sup 5}-Bn-spermidine 1a. The respective products 2b-4b were obtained in good yields and characterized by spectroscopic and analytical methods. The inhibitory action of these naphthoquinone-PA conjugates on DNA-topoisomerases (topo) I and II-{alpha} was evaluated by relaxation assay of supercoiled DNA plasmid. All compounds (1a 2b, 3b and 4b) presented significant inhibition of topo II-{alpha} catalytic activity at the 2 {mu}M dose. Considering that only PA 1a did not inhibit the enzyme catalytic activity at the 0.2 {mu}M dose, the appended naphthoquinone moiety acts as a 'value added' fragment. Compounds 1a 2b, 3b and 4b did not inhibit the enzyme DNA-topo I at the 200 {mu}M dose. (author)

  5. Synthesis, molecular structure, theoretical calculation, DNA/protein interaction and cytotoxic activity of manganese(III) complex with 8-hydroxyquinoline.

    Science.gov (United States)

    Thamilarasan, V; Sengottuvelan, N; Sudha, A; Srinivasan, P; Siva, A

    2015-01-01

    Manganese(III) complex (1) [Mn(8-hq)3] (where 8-hq=8-hydroxyquinoline) has been synthesized and characterized by elemental, spectral (UV-vis, FT-IR) and thermal analysis. The structure of complex (1) has been determined by single crystal X-ray diffraction studies and the configuration around manganese(III) ion was elongated octahedral coordination geometry. Density functional theory calculations were performed for ligand and its complex. Binding studies of ligand and complex 1 with calf thymus DNA (CT-DNA) was investigated by absorption, fluorescence, circular dichroic (CD) spectroscopy and viscosity measurements. Absorption spectral studies revealed that ligand and complex 1 binds to DNA groove and its intrinsic binding strength has been found to be 2.57×10(4) and 2.91×10(4)M(-1). A molecular docking study confirm that the complex 1 is a minor groove binder and was stabilized through hydrogen bonding interactions. Complex 1 exhibits a good binding propensity to bovine serum albumin (BSA) protein. The in vitro cytotoxicity study of complex 1 on breast cancer cell line (MCF-7) indicate that it has the potential to act as effective anticancer drug, with IC50 values of 3.25μM. The ligand and its complex have been screened for antimicrobial activities and the complex showed better antimicrobial activity than the free ligand.

  6. Synthesis, interaction with DNA and antiproliferative activities of two novel Cu(II) complexes with Schiff base of benzimidazole

    Science.gov (United States)

    Song, Wen-Ji; Cheng, Jian-Ping; Jiang, Dong-Hua; Guo, Li; Cai, Meng-Fei; Yang, Hu-Bin; Lin, Qiu-Yue

    2014-03-01

    Two novel copper(II) complexes with Schiff base of benzimidazole [Cu(L)Cl]2·CH3OH have been synthesized. HL1 (N-(benzimidazol-2-ymethyl)-5-chlorosalicylideneimine, C15H11ClN3O) and HL2 (N-(benzimidazol-2-ymethyl)-salicylideneimine, C15H12N3O) are ligands of complex (1) and complex (2), respectively. The complexes were characterized by elemental analysis, IR, UV-Vis, TGA and X-ray diffraction. Within the complexes, Cu(II) ions were four coordinated by two nitrogen atom of azomethine and imine, one phenolic oxygen atom from HL and one chloride atom. A distorted quadrilateral structure was formed. Complex (1) crystallized in the triclinic crystal system. Results showed that π-π stacking effect occurred due to the existence of aromatic ring from Schiff base and hydrogen bonding between methanol and adjacent atoms. The DNA binding properties of the complexes were investigated by electronic absorption spectra, fluorescence spectra and viscosity measurements. Results indicated that complexes bound to DNA via partial intercalation mode. The DNA binding constants Kb/(L mol-1) were 1.81 × 104 (1), 1.37 × 104 (2), 6.27 × 103 (HL1) and 3.14 × 103 (HL2) at 298 K. The title complexes could quench the emission intensities of EB-DNA system significantly. The results of agarose gel electrophoresis indicated complex (1) could cleave supercoiled DNA through the oxidative mechanism. The inhibition ratios revealed that complex (1) and HL1 had strong antiproliferative activities against human breast cancer cells (MCF-7) lines and human colorectal cancer cells (COLO205) lines in vitro. The antiproliferative activities of complex (1) against MCF-7 lines (IC50 = 16.9 ± 1.5 μmol L-1) and against COLO205 lines (IC50 = 16.5 ± 3.4 μmol L-1) is much stronger than that of HL1, which had the potential to develop anti-cancer drug.

  7. Application of hydrostatic CCC-TLC-HPLC-ESI-TOF-MS for the bioguided fractionation of anticholinesterase alkaloids from Argemone mexicana L. roots.

    Science.gov (United States)

    Kukula-Koch, Wirginia; Mroczek, Tomasz

    2015-03-01

    A rapid hydrostatic counter-current chromatography-thin-layer chromatography-electrospray-ionization time-of-flight mass spectrometry (CCC-TLC-ESI-TOF-MS) technique was established for use in seeking potent anti-Alzheimer's drugs among the acethylcholinesterase inhibitors in Argemone mexicana L. underground parts, with no need to isolate components in pure form. The dichloromethane extract from the roots of Mexican prickly poppy that was most rich in secondary metabolites was subjected to hydrostatic-CCC-based fractionation in descending mode, using a biphasic system composed of petroleum ether-ethyl acetate-methanol-water at the ratio of 1.5:3:2.1:2 (v/v). The obtained fractions were analyzed in a TLC-based AChE-inhibition "Fast Blue B" test. All active components in the fractions, including berberine, protopine, chelerithrine, sanguinarine, coptisine, palmatine, magnoflorine, and galanthamine, were identified in a direct TLC-HPLC-ESI-TOF-MS assay with high accuracy. This is the first time galanthamine has been reported in the extract of Mexican prickly poppy and the first time it has been identified in any member of the Papaveraceae family, in the significant quantity of 0.77%.

  8. Application of pH-zone refining hydrostatic countercurrent chromatography (hCCC) for the recovery of antioxidant phenolics and the isolation of alkaloids from Siberian barberry herb.

    Science.gov (United States)

    Kukula-Koch, Wirginia; Koch, Wojciech; Angelis, Apostolis; Halabalaki, Maria; Aligiannis, Nektarios

    2016-07-15

    The development of a fast hCCC method tailored to recover phenolics of Siberian barberry (Berberis sibirica, Berberidaceae) responsible for the observed strong antioxidant activity was performed. Initially, the optimization of extraction procedure was evaluated based on the antiradical potential assessment (DPPH and Folin-Ciocalteu assays). 100 °C methanol ASE extract exhibited the highest antiradical activity (IC50=60 ± 4 μg/mL), and a significant TPC (159 ± 2 mgGAE/g). Thorough determination of phenolic content by UHPLC-DAD-ESI(-)HRMS revealed the presence of 10 phenolics as major constituents, and several groups of alkaloids. pH-zone refining hCCC was chosen as the most promising method for the extract's fractionation due to the ionizable character of its constituents. For this purpose a MtBE-H2O (1:1) system with 10mM TEA and HCl was applied leading to a phenolic fraction, free of alkaloids, with higher antioxidant capacity (IC50=25 μg/mL, TPC=178 mg GAE/g). Additionally, fractionation of alkaloids was achieved resulting isolation of pharmacologically important alkaloids: magnoflorine and berberine.

  9. Novel substituted benzothiophene and thienothiophene carboxanilides and quinolones: synthesis, photochemical synthesis, DNA-binding properties, antitumor evaluation and 3D-derived QSAR analysis.

    Science.gov (United States)

    Aleksić, Maja; Bertoša, Branimir; Nhili, Raja; Uzelac, Lidija; Jarak, Ivana; Depauw, Sabine; David-Cordonnier, Marie-Hélène; Kralj, Marijeta; Tomić, Sanja; Karminski-Zamola, Grace

    2012-06-14

    A series of new N,N-dimethylaminopropyl- and 2-imidazolinyl-substituted derivatives of benzo[b]thienyl- and thieno[2,3-b]thienylcarboxanilides and benzo[b]thieno[2,3-c]- and thieno[3',2':4,5]thieno[2,3-c]quinolones were prepared. Quinolones were prepared by the reaction of photochemical dehydrohalogenation of corresponding anilides. Carboxanilides and quinolones were tested for the antiproliferative activity. 2-Imidazolinyl-substituted derivatives showed very prominent activity. By use of the experimentally obtained antitumor measurements, 3D-derived QSAR analysis was performed for the set of compounds. Highly predictive 3D-derived QSAR models were obtained, and molecular properties that have the highest impact on antitumor activity were identified. Carboxanilides 6a-c and quinolones 9a-c and 11a were evaluated for DNA binding propensities and topoisomerases I and II inhibition as part of their mechanism of action assessment. The evaluated differences in the mode of action nicely correlate with the results of the 3D-QSAR analysis. Taken together, the results indicate which modifications of the compounds from the series should further improve their anticancer properties.

  10. DNA sequences encoding erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Lin, F.K.

    1987-10-27

    A purified and isolated DNA sequence is described consisting essentially of a DNA sequence encoding a polypeptide having an amino acid sequence sufficiently duplicative of that of erythropoietin to allow possession of the biological property of causing bone marrow cells to increase production of reticulocytes and red blood cells, and to increase hemoglobin synthesis or iron uptake.

  11. Progress Report on the ISCR Pilot Test Conducted at the Former CCC/USDA Grain Storage Facility in Montgomery City, Missouri, as of April 2013

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, Lorraine M. [Argonne National Lab. (ANL), Argonne, IL (United States). Environmental Science Division. Applied Geoscience and Environmental Restoration Program

    2013-06-01

    The Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) is conducting an environmental investigation at the former CCC/USDA grain storage facility on the county fairgrounds in Montgomery City, Missouri, to evaluate contamination associated with the former use of grain fumigants containing carbon tetrachloride at the site. The CCC/USDA studies have identified carbon tetrachloride in the soils (primarily unconsolidated glacial tills) at concentrations that exceed the U.S. Environmental Protection Agency (EPA) regional screening level (RSL) values for this compound in residential soils (610 μg/kg) but are below the corresponding RSL for industrial soils (3,000 μg/kg). Concentrations of carbon tetrachloride greater than the EPA maximum contaminant level (MCL; 5.0 μg/L) for this contaminant in drinking water were also identified in the shallow groundwater (Argonne 2012). On the basis of these findings, remedial actions are considered necessary to mitigate the present and potential future impacts of the contamination. In cooperation with the Missouri Department of Natural Resources (MDNR), the CCC/USDA has initiated a field-scale pilot test to evaluate an in situ technology for treatment of the carbon tetrachloride contamination. In this approach, a chemical amendment consisting primarily of slow-release organic matter and zero-valent iron is employed to induce oxygen-depleted, chemically reducing conditions in the subsurface. These conditions foster the in situ chemical reduction (ISCR) of carbon tetrachloride and its degradation products (chloroform, methylene chloride, and chloromethane) via both inorganic and biologically mediated processes. The chemical amendment being used, EHC™, was developed by the Adventus Group, Freeport, Illinois, and is now manufactured and distributed by FMC Environmental Solutions, Philadelphia, Pennsylvania. With the approval of the MDNR (2012), the ISCR technology is being tested in two target areas

  12. Synthesis and characterization of DNA fenced, self-assembled SnO2 nano-assemblies for supercapacitor applications.

    Science.gov (United States)

    Nithiyanantham, U; Ramadoss, Ananthakumar; Kundu, Subrata

    2016-02-28

    Self-assembled, aggregated, chain-like SnO2 nano-assemblies were synthesized at room temperature by a simple wet chemical route within an hour in the presence of DNA as a scaffold. The average size of the SnO2 particles and the chain diameter were controlled by tuning the DNA to Sn(ii) molar ratio and altering the other reaction parameters. A formation and growth mechanism of the SnO2 NPs on DNA is discussed. The SnO2 chain-like assemblies were utilized as potential anode materials in an electrochemical supercapacitor. From the supercapacitor study, it was found that the SnO2 nanomaterials showed different specific capacitance (Cs) values depending on varying chain-like morphologies and the order of Cs values was: chain-like (small size) > chain-like (large size). The highest Cs of 209 F g(-1) at a scan rate of 5 mV s(-1) was observed for SnO2 nano-assemblies having chain-like structure with a smaller size. The long term cycling stability study of a chain-like SnO2 electrode was found to be stable and retained ca. 71% of the initial specific capacitance, even after 5000 cycles. A supercapacitor study revealed that both morphologies can be used as a potential anode material and the best efficiency was observed for small sized chain-like morphology which is due to their higher BET surface area and specific structural orientation. The proposed route, by virtue of its simplicity and being environmentally benign, might become a future promising candidate for further processing, assembly, and practical application of other oxide based nanostructure materials.

  13. Mixed-ligand copper(II) phenolate complexes: Synthesis, spectral characterization, phosphate-hydrolysis, antioxidant, DNA interaction and cytotoxic studies

    Science.gov (United States)

    Gurumoorthy, Perumal; Mahendiran, Dharmasivam; Prabhu, Durai; Arulvasu, Chinnasamy; Rahiman, Aziz Kalilur

    2015-01-01

    A series of phenol-based mixed-ligand copper(II) complexes of the type [CuL1-4(diimine)] (1-8), where L1-4 = N1,N2-bis(5-substituted-2-hydroxybenzylidene)-1,2-ethylene/phenylenediimine and diimine = 2,2‧-bipyridyl (bpy) or 1,10-phenanthroline (phen), have been isolated and fully characterized by analytical and spectral techniques. Electronic spectra of complexes suggest Cu(II) cation has a d9 electronic configuration, adopting distorted octahedral geometry with axial elongation, due to Jahn-Teller effect. Electrochemical studies of complexes evidenced one-electron irreversible reduction wave in the cathodic region. The observed rate constant (k) values for the hydrolysis of 4-nitrophenylphosphate (4-NPP) are in the range of 0.25-3.82 × 10-2 min-1. The obtained room temperature magnetic moment values (1.79-1.90 BM) lies within the range observed for octahedral copper(II) complexes. Antioxidant studies revealed that these complexes possess considerable radical scavenging potency against DPPH. The binding studies of complexes with calf thymus DNA (CT-DNA) revealed intercalation with minor-groove binding, and the complex 4 exhibits highest binding activity than the other complexes. The cleavage activity on supercoiled pBR322 DNA revealed the involvement of hydroxyl radical and singlet-oxygen as reactive oxygen species, and complexes encourage binding to minor-groove. Further, the cytotoxicity of complex 4 on human hepatocellular liver carcinoma HepG2 cell line implies the cell death through apoptosis.

  14. Synthesis, X-ray crystal structure, DNA binding and Nuclease activity of lanthanide(III) complexes of 2-benzoylpyridine acetylhydrazone

    Indian Academy of Sciences (India)

    KARREDDULA RAJA; AKKILI SUSEELAMMA; KATREDDI HUSSAIN REDDY

    2016-08-01

    Lanthanide(III) complexes of general formula [La(BPAH)₂(NO₃)₃] and [Ce(BPAH)₂(NO₃)(H₂O)₂] 2NO₃.H₂O (where, BPAH = 2-benzoylpyridine acetyl hydrazone), were synthesized and characterized by elemental analysis, molar conductance, IR spectroscopy and single crystal X-ray diffraction and Hirschfeld studies. The central metal ion is 12-coordinate in lanthanum complex and 10-coordinated in the cerium complex. The coordination polyhedra around the lanthanum and cerium were found to have distorted icosahedron and distorted bicapped square antiprism respectively. DNA binding and nuclease activity of these complexes were also investigated in the present work.

  15. Synthesis and Photooxygenation of Furo[3,2-c]coumarin Derivatives as Antibacterial and DNA Intercalating Agent

    Institute of Scientific and Technical Information of China (English)

    Al-Sehemi, Abdullah G.; EI-Gogary, Sameh R.

    2012-01-01

    Syntheses of 2,3-dimethyl-4H-furo[3,2-c]coumarin and 3-phenyl-4H-furo[3,2-c]coumarin as angular furocou- marins were carried out through Williamson reaction of 4-hydroxycoumarin with a-haloketones followed by cycli- zation. Photooxygenation of the synthesized furocoumarin derivatives was performed and the photoproducts were isolated and characterized. The affinity of 2,3-dimethyl-4H-furo[3,2-c]coumarin towards DNA and the antibacterial activity were evaluated and compared with 8-methoxypsoralen (8-MOP).

  16. Initiation of adenovirus DNA replication.

    OpenAIRE

    Reiter, T; Fütterer, J; Weingärtner, B; Winnacker, E L

    1980-01-01

    In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared ...

  17. Chiral manganese (IV) complexes derived from Schiff base ligands: Synthesis, characterization, in vitro cytotoxicity and DNA/BSA interaction.

    Science.gov (United States)

    Li, Zhen; Niu, Meiju; Chang, Guoliang; Zhao, Changqiu

    2015-12-01

    Two new couples of chiral manganese (IV) complexes with Schiff-base ligands, Λ-[Mn(R-L(1))2]·2(CH3OH) (Λ-1) and Δ-[Mn(S-L(1))2]·2(CH3OH) (Δ-1), Λ-[Mn(R-L(2))2]·(H2O)2 (Λ-2) and Δ-[Mn(S-L(2))2]·(H2O)2 (Δ-2), {H2L(1)=(R/S)-(±)-1-[(1-hydroxymethyl-propylimino)-methyl]-naphthalen-2-ol, H2L(2)=(R/S)-(±)-1-[(1-Hydroxymethyl-2-phenyl-ethylimino)-methyl]-naphthalen-2-ol} have been synthesized, and fully characterized by elemental analyses, UV-Vis spectrum, circular dichroism spectrum, FT-IR spectrum, mass spectrum, and single crystal X-ray diffraction (SXRD). The interaction of the four chiral Mn (IV) complexes with CT-DNA and BSA were also investigated by various spectroscopic techniques (UV-visible, fluorescence spectroscopic). The results show that the Δ-complexes exhibit more efficient CT-DNA interaction with respect to the Λ-complexes. All the complexes could quench the intrinsic fluorescence of BSA by a static quenching process. In addition, the vitro cytotoxicity of these complexes toward four kinds of cancerous cell lines (A549, HeLa, HL-60, and Caco-2) was assayed by the MTT method, which exhibited to be selectively active against certain cell lines.

  18. Synthesis, characterization, biological activity and DNA cleavage studies of tridentate Schiff bases and their Co(II complexes

    Directory of Open Access Journals (Sweden)

    P. Kavitha

    2016-01-01

    Full Text Available In the present study a series of Co(II complexes of formyl chromone Schiff bases have been synthesized characterized by analytical, molar conductance, IR, electronic, magnetic susceptibility, thermal, fluorescence and powder XRD measurements and screened for various biological activities (antimicrobial, antioxidant, nematicidal, DNA cleavage and cytotoxicity. In all the Co(II complexes 1:2 metal to ligand molar ratio was obtained from analytical data. The molar conductance data confirm that all complexes are non-electrolytic in nature. Based on the electronic and magnetic data, an octahedral geometry is ascribed for all the Co(II complexes. Thermal behaviour of the synthesized complexes illustrates the general decomposition patterns of the complexes. The X-ray analysis data show that all the Co(II complexes have triclinic crystal system with different unit cell parameters. Metal complexes have greater antimicrobial activity than ligands. Antioxidant and nematicidal activities indicate that the ligands exhibit greater activity when compared to their respective Co(II complexes. All ligands and Co(II complexes of HL1 and HL2 showed considerable anticancer activity against Raw, MCF-7 and COLO 205 cell lines. All ligands and their Co(II complexes showed more pronounced DNA cleavage activity in the presence of H2O2.

  19. Synthesis, spectroscopic characterization, DNA cleavage and antibacterial studies of a novel tridentate Schiff base and some lanthanide(III) complexes

    Institute of Scientific and Technical Information of China (English)

    K. Mohanan; R. Aswathy; L.P. Nitha; Niecy Elsa Mathews; B. Sindhu Kumari

    2014-01-01

    A novel potential tridentate Schiff base was prepared by condensing equimolar quantities of 2-hydroxyacetophenone and 2-aminopyrimidine in methanol. This ligand was versatile in forming a series of complexes with lanthanide ions such as La(III), Pr(III), Nd(III), Sm(III), Gd(III), Dy(III) and Yb(III). The ligand and the metal complexes were characterized through elemental analysis, molar conductance, UV-Visible, IR, 1H NMR, and mass spectral studies. The spectral studies indicated that the ligand was coordinated to the metal ion in neutral tridentate fashion through the azomethine nitrogen, one of the nitrogen atoms in the pyrimidine ring and the phenolic oxygen without deprotonation. Thermal decomposition and luminescence property of lanthanum(III) complex were also examined. The X-ray diffraction patterns showed the crystalline nature of the ligand and its lanthanum(III) complex. The DNA cleavage studies of the ligand and the metal complexes were carried out and it was observed that the lanthanum(III) and neo-dymium(III) complexes cleaved the pUC19 DNA effectively. The ligand and the metal complexes were screened for their antibacte-rial activities. The metal complexes were found to be more potent bactericides than the ligand.

  20. Synthesis, Spectroscopic Characterisation, and Biopotential and DNA Cleavage Applications of Mixed Ligand 4-N,N-Dimethylaminopyridine Metal Complexes

    Directory of Open Access Journals (Sweden)

    C. Surendra Dilip

    2013-01-01

    Full Text Available The mixed ligand transition metal complexes of 4-N,N-dimethylaminopyridine (DP and chloride as primary and secondary ligands with the general formula [M(DP3Cl3]; M = Cr(III and Fe(III; [M′(DP4Cl2]M′ = Co(II, Ni(II, Cu(II, and Cd(II were synthesized in a microwave oven. The complexes were characterized by FT-IR and UV, 1HNMR, 13CNMR spectra, TG/DTG, and various physicoanalytical techniques. From the magnetic moment measurements and the electronic spectral data, a distorted octahedral geometry was proposed for the complexes. The complexes express similar trend of thermal behaviour such that they lose water of hydration initially with the subsequent emission of organic and inorganic fragments and leave left the metal oxides as residue. The activation thermodynamic parameters, such as , , , and of the metal complexes, illustrate the spontaneous formation of the complexes. The antimicrobial studies against various pathogenic bacterial and fungal serums insist on that the enhanced potential of the complexes over their ligand and their biopotential properties increases with concentration. The DNA interaction of the synthesized complexes on CT-DNA was investigated by UV-Vis spectroscopy, viscosity, thermal denaturation, and electroanalytical experiments and their binding constants ( were also calculated.

  1. Racemic DNA crystallography.

    Science.gov (United States)

    Mandal, Pradeep K; Collie, Gavin W; Kauffmann, Brice; Huc, Ivan

    2014-12-22

    Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L- and D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four-way junction, showing that the advantages of racemic crystallography should extend to DNA.

  2. Synthesis of circular double-stranded DNA having single-stranded recognition sequence as molecular-physical probe for nucleic acid hybridization detection based on atomic force microscopy imaging.

    Science.gov (United States)

    Nakano, Koji; Matsunaga, Hideshi; Murata, Masaharu; Soh, Nobuaki; Imato, Toshihiko

    2009-08-01

    A new class of DNA probes having a mechanically detectable tag is reported. The DNA probe, which consists of a single-stranded recognition sequence and a double-stranded circular DNA entity, was prepared by polymerase reaction. M13mp18 single strand and a 32mer oligodeoxynucleotide whose 5'-end is decorated with the recognition sequence were used in combination as template and primer, respectively. We have successfully demonstrated that the DNA probe is useful for bioanalytical purposes: by deliberately attaching target DNA molecules onto Au(111) substrates and by mechanically reading out the tag-entity using a high-resolution microscopy including atomic force microscopy, visualization/detection of the individual target/probe DNA conjugate was possible simply yet straightforwardly. The present DNA probe can be characterized as a 100%-nucleic acid product material. It is simply available by one-pod synthesis. A surface topology parameter, image roughness, has witnessed its importance as a quantitative analysis index with particular usability in the present visualization/detection method.

  3. Synthesis, antibacterial, antifungal activity and interaction of CT-DNA with a new benzimidazole derived Cu(II) complex.

    Science.gov (United States)

    Arjmand, Farukh; Mohani, Bhawana; Ahmad, Shamim

    2005-11-01

    The ligand [C(16)H(10)O(2)N(4)S(2)] L has been synthesized by the condensation reaction of 2-mercaptobenzimidazole and diethyloxalate. The ligand L was allowed to react with bis(ethylenediamine)Cu(II)/Ni(II) complexes to yield [C(20)H(22)N(8)S(2)Cu]Cl(2)1 and [C(20)H(22)N(8)S(2)Ni]Cl(2)2 complexes. The Ni(II) complex was synthesized only to elucidate the structure of the complex. The complexes 1 and 2 were characterized by elemental analyses, IR, NMR, EPR, UV-vis spectroscopy and molar conductance measurements. Both the complexes are ionic in nature and possess square-planar geometry. The binding of the complex 1 to calf thymus DNA was investigated spectrophotometrically. The absorption spectra of complex 1 exhibits a slight red shift with "hyperchromic effect" in presence of CTDNA. Electrochemical analysis and viscosity measurements were also carried out to ascertain the mode of binding. The complex 1 in the absence and in presence of CT DNA in aqueous solution exhibits one quasi-reversible redox wave corresponding to Cu(II)/Cu(I) redox couple at a scan rate of 0.2 V s(-1). The shift in DeltaE(p), E(1/2) and I(pa)/I(pc) values ascertain the interaction of calf thymus DNA with copper(II) complex. There is decrease in viscosity of CTDNA which indicates that the complex 1 binds to CTDNA through a partial intercalative mode. The antibacterial and antifungal studies of the [C(7)H(6)N(2)S], [C(4)H(16)N(4)Cu]Cl(2,) [C(16)H(10)N(4)S(2)O(2)] and [C(20)H(22)N(8)S(2)Cu]Cl(2) were carried out against S. aureus, E. coli and A. niger. All the results reveal that the complex 1 is highly active against the bacterial strains and also inhibits fungal growth.

  4. Effects of Nerve Growth Factor on Proliferation and DNA Synthesis of Cultured Human Fetal Retinal Pigment Epithelium Cells

    Institute of Scientific and Technical Information of China (English)

    Wensheng Li; Jun Wen; Deyong Jiang; Jianguang Ding

    2002-01-01

    Objective: To investigate the effects of nerve growth factor(NGF)on proliferation and DNAthesis of cultured human fetal retinal pigment epithelium (RPE)cells in vitro.Methods: Primary culture and subculture of human fetal retinal pigment epithelium cellswere established in vitro first. Cultured RPE cells were treated with NGF by variousconcentrations 0μg/L, 50μg/L, 100μg/L, 200μg/L and 300μg/L(final concentration)for 48 hs.After 48 hs, cells proliferation was measured with methyl thiazolyl tetrazolium(MTT)assay method and the amount of DNA was determined by the absorbance at 280nm of nucleic acid & protein analysis.Results: The A values of 100 μg/L, 200 μg/L, 300 μg/L NGF was(0. 213 7 ± 0. 23 3),(0. 218 8 ±0. 018 1), (0. 232 2 ±0. 016 4) as compared with(0. 189 7 ±0. 015 2) of Avalue of 0 μg/L NGF respectively, q value was 3.63,4.40, 6. 42 and P value was0. 015, 0. 000, 0. 000(q-test). The DNA concentrations of 100 μg/L, 200 μg/L, 300μg/L and 400 μg/L NGF was (981. 220 4 ± 123.535 7), (1 375. 848 4 ±244. 471 8),(1 658.707 1 ± 176. 938 1), (2 353.086 3 ±609. 906 4) μg/ml as compared with(666. 818 8 ± 141. 330 2) μg/ml of DNA concentration of 0 μg/L NGF respectively, qvalue was 3.63,8.20,11.47,19.46, P value was 0. 024,0. 000,0. 000,0. 000 (q-test).Conclusion: The data suggested that NGF could stimulate the proliferation and DNAsynthesis of cultured of hRPE cells in vitro in a dose-dependent manner.

  5. Neutral and ionic platinum compounds containing a cyclometallated chiral primary amine: synthesis, antitumor activity, DNA interaction and topoisomerase I-cathepsin B inhibition.

    Science.gov (United States)

    Albert, Joan; Bosque, Ramon; Crespo, Margarita; Granell, Jaume; López, Concepción; Martín, Raquel; González, Asensio; Jayaraman, Anusha; Quirante, Josefina; Calvis, Carme; Badía, Josefa; Baldomà, Laura; Font-Bardia, Mercè; Cascante, Marta; Messeguer, Ramon

    2015-08-14

    The synthesis and preliminary biological evaluation of neutral and cationic platinum derivatives of chiral 1-(1-naphthyl)ethylamine are reported, namely cycloplatinated neutral complexes [PtCl{(R or S)-NH(2)CH(CH(3))C(10)H(6)}(L)] [L = SOMe(2) ( 1-R or 1-S ), L = PPh(3) (2-R or 2-S), L = P(4-FC(6)H(4))(3) (3-R), L = P(CH(2))(3)N(3)(CH(2))(3) (4-R)], cycloplatinated cationic complexes [Pt{(R)-NH(2)CH(CH(3))C(10)H(6)}{L}]Cl [L = Ph(2)PCH(2)CH(2)PPh(2) (5-R), L = (C(6)F(5))(2)PCH(2)CH(2)P(C(6)F(5))(2) (6-R)] and the Pt(ii) coordination compound trans-[PtCl(2){(R)-NH(2)CH(CH(3))C(10)H(6)}(2)] (7-R). The X-ray molecular structure of 7-R is reported. The cytotoxic activity against a panel of human adenocarcinoma cell lines (A-549 lung, MDA-MB-231 and MCF-7 breast, and HCT-116 colon), cell cycle arrest and apoptosis, DNA interaction, topoisomerase I and cathepsin B inhibition, and Pt cell uptake of the studied compounds are presented. Remarkable cytotoxicity was observed for most of the synthesized Pt(ii) compounds regardless of (i) the absolute configuration R or S, and (ii) the coordinated/cyclometallated (neutral or cationic) nature of the complexes. The most potent compound 2-R (IC(50) = 270 nM) showed a 148-fold increase in potency with regard to cisplatin in HCT-116 colon cancer cells. Preliminary biological results point out to different biomolecular targets for the investigated compounds. Neutral cyclometallated complexes 1-R and 2-R, modify the DNA migration as cisplatin, cationic platinacycle 5-R was able to inhibit topoisomerase I-promoted DNA supercoiling, and Pt(ii) coordination compound 7-R turned out to be the most potent inhibitor of cathepsin B. Induction of G-1 phase ( 2-R and 5-R ), and S and G-2 phases (6-R) arrests are related to the antiproliferative activity of some representative compounds upon A-549 cells. Induction of apoptosis is also observed for 2-R and 6-R.

  6. Synthesis of Diphenyl Pyridazinone-based flexible system for conformational studies through weak noncovalent interactions: Application in DNA binding

    Indian Academy of Sciences (India)

    Ranjeet Kumar; Praveen Singh; Archana Gaurav; Pratima Yadav; Ranjana S Khanna; Ashish Kumar Tewari

    2016-04-01

    This paper reports conformational studies of pyridazinone-based flexible dimer connected through diethylamine linker. The conformational studies have been done by X-ray crystal structure and DFT calculation. Further, after crystallization, the compound has shown two types of crystals, one is hydrated and another one is non-hydrated. The hydrated and non-hydrated crystals showed difference in their conformation due to the presence of water in crystal lattice of hydrated crystal. The difference in their conformation has been proved by crystallographic studies, DSC curves and detailed analysis of Hirshfeld surfaces and fingerprint plots facilitating a comparison of intermolecular interactions. Along with conformational studies, this compound also showed DNA binding, as revealed in docking simulation studies.

  7. A major portion of DNA gyrase inhibitor microcin B17 undergoes an N,O-peptidyl shift during synthesis.

    Science.gov (United States)

    Ghilarov, Dmitry; Serebryakova, Marina; Shkundina, Irina; Severinov, Konstantin

    2011-07-29

    Microcin B17 (McB) is a 43-amino acid antibacterial peptide targeting the DNA gyrase. The McB precursor is ribosomally produced and then post-translationally modified by the McbBCD synthase. Active mature McB contains eight oxazole and thiazole heterocycles. Here, we show that a major portion of mature McB contains an additional unusual modification, a backbone ester bond connecting McB residues 51 and 52. The modification results from an N → O shift of the Ser(52) residue located immediately downstream of one of McB thiazole heterocycles. We speculate that the N,O-peptidyl shift undergone by Ser(52) is an intermediate of post-translational modification reactions catalyzed by the McbBCD synthase that normally lead to formation of McB heterocycles.

  8. Synthesis and thermal studies of tetraaza macrocylic ligand and its transition metal complexes. DNA binding affinity of copper complex.

    Science.gov (United States)

    Saif, M; Mashaly, Mahmoud M; Eid, Mohamed F; Fouad, R

    2011-09-01

    A Tetraaza Macrocylic Ligand (H2L) and its complexes, [Cd(H2L)(OH2)2](NO3)(2)·1/2OH2 (I), [Co(H2L)(OH2)](NO3)(2)·1/2OH2 (II), [Cu(H2L)(NO3)2]·3/2OH2 (III) and [Ni(H2L)(NO3)(OH2)]NO3·OH2 (IV), have been synthesized and characterized on the basis of elemental analysis, molar conductivity, 1H NMR, UV-vis, FT-IR and mass spectroscopy. All results confirm that the prepared compounds have 1:1 metal-to-ligand stoichiometry, octahedral configuration and the ligand behaves as a neutral tetradendate towards the metal ions. [CdL(OH2)2] (V), [CoL(OH2)2] (VI), [CuL(OH2)2] (VII) and [Ni(H2L)(NO3)2] (VIII) were synthesized pyrolytically in solid state from corresponding compounds (I-IV). Analytical results of complexes (V-VIII) show that the ligand behaves either as a neutral tetradendate or dianionic tetradentate ligand towards the metal ions. The binding of H2L and its copper complex (III) to DNA has been investigated by ultraviolet absorption spectroscopy. The experiments indicate that H2L and its copper complex (III) can bind to DNA through an intercalative mode. The H2L and its copper complex (III) exhibited anti-tumor activity against Ehrlich Acites Carcinoma (E.A.C) at the concentration of 100 μg/ml.

  9. Synthesis and thermal studies of tetraaza macrocylic ligand and its transition metal complexes. DNA binding affinity of copper complex

    Science.gov (United States)

    Saif, M.; Mashaly, Mahmoud M.; Eid, Mohamed F.; Fouad, R.

    2011-09-01

    A Tetraaza Macrocylic Ligand (H 2L) and its complexes, [Cd(H 2L)(OH 2) 2](NO 3) 2·1/2OH 2 (I), [Co(H 2L)(OH 2)](NO 3) 2·1/2OH 2 (II), [Cu(H 2L)(NO 3) 2]·3/2OH 2 (III) and [Ni(H 2L)(NO 3)(OH 2)]NO 3·OH 2 (IV), have been synthesized and characterized on the basis of elemental analysis, molar conductivity, 1H NMR, UV-vis, FT-IR and mass spectroscopy. All results confirm that the prepared compounds have 1:1 metal-to-ligand stoichiometry, octahedral configuration and the ligand behaves as a neutral tetradendate towards the metal ions. [CdL(OH 2) 2] (V), [CoL(OH 2) 2] (VI), [CuL(OH 2) 2] (VII) and [Ni(H 2L)(NO 3) 2] (VIII) were synthesized pyrolytically in solid state from corresponding compounds (I-IV). Analytical results of complexes (V-VIII) show that the ligand behaves either as a neutral tetradendate or dianionic tetradentate ligand towards the metal ions. The binding of H 2L and its copper complex (III) to DNA has been investigated by ultraviolet absorption spectroscopy. The experiments indicate that H 2L and its copper complex (III) can bind to DNA through an intercalative mode. The H 2L and its copper complex (III) exhibited anti-tumor activity against Ehrlich Acites Carcinoma (E.A.C) at the concentration of 100 μg/ml.

  10. Synthesis,Crystal Structure and DNA-binding Properties of a New Copper(Ⅱ) Schiff Base Complex

    Institute of Scientific and Technical Information of China (English)

    QIN Bei

    2012-01-01

    A new asymmetric bidentate copper(Ⅱ) complex,CuL 2(HL=2-((E)-(4-bromophenylimino)methyl)-6-bromo-4-chlorophenol),has been synthesized and characterized by elemental analyses and single-crystal X-ray diffraction.The complex crystallizes in the monoclinic space group P2 1 /c with a=11.218(3),b=9.355(3),c=13.449(4),β=108.722(4)°,V=1336.8(6)3,Z=2,Dc=2.008 g/cm 3,μ(MoKα)=7.024 mm-1,F(000)=806,S=0.999,the final R=0.0342 and wR=0.0641for2611observed reflections (I〉2σ(I)).The central copper(Ⅱ) is four-coordinate and bonds to two nitrogen and two oxygen atoms from two Schiff base ligands.The complex is linked into a two-dimensional supramolecular structure by weak intermolecular interactions.In addition,DNA-binding properties of the metal complex were investigated using spectrometric titrations and viscosity measurements.The results show that the complex binds with calf-thymus DNA(CT-DNA),presumably via a partial intercalative mode.The intrinsic binding constant of the Cu(Ⅱ) complex with DNA is 7.335×10 3 M-1.

  11. DNA ligase I, the replicative DNA ligase.

    Science.gov (United States)

    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 of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication.

  12. Tumorigenesis of mammary gland by 7,12-dimethylbenz(a)anthracene during pregnancy: relationship with DNA synthesis.

    Science.gov (United States)

    Sinha, D K; Pazik, J E

    1981-06-15

    The relationship between mammary cell proliferation during pregnancy and susceptibility to 7,12-dimethylbenz(a)anthracene (DMBA) was examined. DMBA was administered intravenously to Sprague-Dawley rats on the 5th, 10th or 15th day of pregnancy. [3H]thymidine labelling index (LI) of the mammary cells at the time of treatment with the carcinogen was determined and found to be higher in the pregnant rats than in age-matched virgin controls. In spite of the high proliferative index of the mammary cells, significant inhibition of tumorigenesis occurred in the pregnancy rats allowed to complete pregnancy and parturition following treatment with DMBA. However, when pregnancy was terminated by cesarian section shortly after treatment with DMBA, there was a significantly higher tumor incidence as compared to the "full-term" rats. It was observed that the earlier the pregnancy was terminated, the greater was the incidence of mammary tumors. This would indicate that the inhibitory effect of pregnancy is related to changes occurring during the later half of gestation. The differentiation of mammary cells for milk synthesis as pregnancy progresses is postulated to be a major reason for the observed refractoriness of the mammary cells to DMBA at that time.

  13. Progress report and technical evaluation of the ISCR pilot test conducted at the former CCC/USDA grain storage facility in Centralia, Kansas.

    Energy Technology Data Exchange (ETDEWEB)

    LaFreniere, L. M.; Environmental Science Division

    2009-01-14

    In October, 2007, the Commodity Credit Corporation of the U.S. Department of Agriculture (CCC/USDA) presented the document Interim Measure Conceptual Design (Argonne 2007a) to the Kansas Department of Health and Environment, Bureau of Environmental Remediation (KDHE/BER), for a proposed non-emergency Interim Measure (IM) at the site of the former CCC/USDA grain storage facility in Centralia, Kansas (Figure 1.1). The IM was recommended to mitigate existing levels of carbon tetrachloride contamination identified in the vadose zone soils beneath the former facility and in the groundwater beneath and in the vicinity of the former facility, as well as to moderate or decrease the potential future concentrations of carbon tetrachloride in the groundwater. The Interim Measure Conceptual Design (Argonne 2007a) was developed in accordance with the KDHE/BER Policy No.BERRS-029, Policy and Scope of Work: Interim Measures (KDHE 1996). The hydrogeologic, geochemical, and contaminant distribution characteristics of the Centralia site, as identified by the CCC/USDA, factored into the development of the nonemergency IM proposal. These characteristics were summarized in the Interim Measure Conceptual Design (Argonne 2007a) and were discussed in detail in previous Argonne reports (Argonne 2002a, 2003, 2004, 2005a,b,c, 2006a,b, 2007b). The identified remedial goals of the proposed IM were as follows: (1) To reduce the existing concentrations of carbon tetrachloride in groundwater in three 'hot spot' areas identified at the site (at SB01, SB05, and SB12-MW02; Figure 1.2) to levels acceptable to the KDHE. (2) To reduce carbon tetrachloride concentrations in the soils near the location of former soil boring SB12 and existing monitoring well MW02 (Figure 1.2) to levels below the KDHE Tier 2 Risk-Based Screening Level (RBSL) of 200 {micro}g/kg for this contaminant. To address these goals, the potential application of an in situ chemical reduction (ISCR) treatment technology

  14. The Biological Effect of Y-family DNA Polymerases on the Translesion Synthesis%DNA聚合酶Y家族在跨损伤复制中的作用

    Institute of Scientific and Technical Information of China (English)

    弓毅

    2013-01-01

    普通的DNA聚合酶可以对正常的DNA完成复制,但是当DNA发生损伤,损伤位置就会成为DNA复制的阻滞点,普通的DNA聚合酶就无法完成基因组的复制.为了应对这种情况,生物体内还拥有另一类DNA聚合酶:聚合酶Y家族,又被称为跨损伤复制(TLS)聚合酶,它们的主要功能就是跨越损伤位点,完成基因组复制,解救濒死细胞.本文主要对Y家族聚合酶的结构特点、功能效应、作用机制等方面做一综述.%A common DNA polymerase can replicate DNA which functions normally. However, if DNA suffers damage, the genome can not be replicated by a common DNA polymerase because DNA lesions will block the replication apparatus. Another kind of DNA polymerases in organism, Y-family DNA polymerases which is also called transle-sion synthesis (TLS) polymerases, can deal with this problem. Their main functions are bypassing the lesions in DNA, replicating the genome and saving the dying cells. This thesis presents a historical review of the literature pertinent to the structure, functions and roles of Y-family DNA polymerases.

  15. Synthesis, DNA and Photocleavage Studies of Ru(ll) Polypyridyl Complexes: [Ru(dppz)(pyz)4](ClO4)2 and [Ru(dppz)(dmpyz)4](ClO4)2 Complexes

    Institute of Scientific and Technical Information of China (English)

    Pallavi, Ponuganti; Nagababu, Penumaka; Laxmareddy, Kotha; Padmaja, Naishadham; Satyanarayana, Sirasani

    2012-01-01

    In view of the growing interest for the synthesis of metal complexes and their interaction with DNA, we have synthesized and characterized two complexes containing ruthenium as metal center. The complexes are of the type [Ru(dppz)L4](C104)2 where L are biologically important ligands such as pyrazole and dimethylpyrazole. The characterization of these complexes is done by 1 H NMR, 13C NMR, elemental analysis and mass spectroscopy. The interaction of these complexes with CT DNA was monitored and binding constants were determined using absorption and fluorescence spectroscopy. The mode of binding was found to be intercalative for both complexes and was determined using hydrodynamic viscosity studies. The complexes were further studied for photocleavage studies with supercoiled plasmid pBR322 DNA.

  16. 换流站无功功率补偿新概念-CCC%The New Concept of Inversion Reactive Power Compensation

    Institute of Scientific and Technical Information of China (English)

    郭天兴; 孙振权; 邱毓昌

    2003-01-01

    本文对直流输电用传统的补偿方式滤波器(filter)加并联电容器组(shunt capacitor bank)、晶闸管控制串联补偿技术(Thyristor controlled Series Capacitor简称TCSC)、以及一种新的补偿技术即换流电容器装置(Ca-pacitor commutated converters,简称CCC)进行了分析.并着重对CCC技术的性能进行了理论分析.应用CCC技术可使经过逆变的交流电网有较高的抗干扰能力,并可增强交流电网的传输能力,提高系统的稳定性.

  17. Synthesis, structural characterization, fluorescence, antimicrobial, antioxidant and DNA cleavage studies of Cu(II) complexes of formyl chromone Schiff bases.

    Science.gov (United States)

    Kavitha, P; Saritha, M; Laxma Reddy, K

    2013-02-01

    Cu(II) complexes have been synthesized from different Schiff bases, such as 3-((2-hydroxy phenylimino)methyl)-4H-chromen-4-one (HL(1)), 2-((4-oxo-4H-chromen-3-yl)methylneamino) benzoicacid (HL(2)), 3-((3-hydroxypyridin-2-ylimino)methyl)-4H-chromen-4-one (HL(3)) and 3-((2-mercaptophenylimino)methyl)-4H-chromen-4-one (HL(4)). The complexes were characterized by analytical, molar conductance, IR, electronic, magnetic, ESR, thermal, powder XRD and SEM studies. The analytical data reveal that metal to ligand molar ratio is 1:2 in all the complexes. Molar conductivity data indicates that all the Cu(II) complexes are neutral. On the basis of magnetic and electronic spectral data, distorted octahedral geometry is proposed for all the Cu(II) complexes. Thermal behaviour of the synthesized complexes illustrates the presence of lattice water molecules in the complexes. X-ray diffraction studies reveal that all the ligands and their Cu(II) complexes have triclinic system with different unit cell parameters. Antimicrobial, antioxidant and DNA cleavage activities indicate that metal complexes exhibited greater activity as compared with ligands.

  18. Designing, synthesis and spectral characterization of Schiff base transition metal complexes: DNA cleavage and antimicrobial activity studies

    Directory of Open Access Journals (Sweden)

    N. RAMAN

    2008-10-01

    Full Text Available A new series of transition metal complexes of Cu(II, Ni(II, Co(II and Zn(II have been designed and synthesized using a Schiff base (L derived from 4-aminoantipyrine, benzaldehyde and o-phenylenediamine. The structural features were derived from their elemental analyses, magnetic susceptibility and molar conductivity, as well as from mass, IR, UV–Vis, 1H-NMR and ESR spectral studies. The FAB mass spectral data and elemental analyses showed that the complexes had a composition of the ML type. The UV–Vis and ESR spectral data of the complexes suggested a square-planar geometry around the central metal ion. The magnetic susceptibility values of the complexes indicated that they were monomeric in nature. Antimicrobial screening tests were also performed against four bacteria, viz. Salmonella typhi, Staphylococcus aureus, Escherichia coli, and Bacillus subtilis and three fungi, viz. Aspergillus niger, Aspergillus flavus and Rhizoctonia bataicola. These data gave good results in the presence of metal ion in the ligand system. The nuclease activity of the above metal complexes shows that only the copper complex cleaves CT DNA in the presence of an oxidant.

  19. Synthesis, spectral characterisation, morphology, biological activity and DNA cleavage studies of metal complexes with chromone Schiff base

    Directory of Open Access Journals (Sweden)

    P. Kavitha

    2016-07-01

    Full Text Available Cu(II, Co(II, Ni(II and Zn(II complexes have been synthesized using 3-((pyridine-2-yliminomethyl-4H-chromen-4-one as a ligand derived from 3-formyl chromone and 2-amino pyridine. All the complexes were characterised by analytical, conductivity, IR, electronic, magnetic, ESR, thermal, powder XRD and SEM studies. The analytical data revealed that the metal to ligand molar ratio is 1:2 in all the complexes. Molar conductivity data indicates that all the complexes are neutral in nature. On the basis of magnetic and electronic spectral data, octahedral geometry is proposed for all the complexes. Thermal behaviour of the synthesized complexes indicates the coordinated and lattice water molecules are present in the complexes. The X-ray diffraction data suggest a triclinic system for all compounds. Different surface morphologies were identified from SEM micrographs. All metal complexes exhibit fluorescence. The antimicrobial and nematicidal activity data show that metal complexes are more potent than the parent l